Search results: MPI-CBG

Wen Yu, Yanrun Zhou, Leshan Yang, Xiao Yan, Samuel N Smukowski, Yuefeng Ma, Jiali Fan, Young Ah Goo, Anthony Hyman, Yifan Dai
Origins of the Intrinsic Redox Activity of Biomolecular Condensates.
J Am Chem Soc, Art. No. doi: 10.1021/jacs.6c01750 (2026)
Open Access DOI

How inherent redox activity arises in biomolecular condensates remains unclear. Unlike interfacial systems, such as water microdroplets, where water oxidation underpins redox chemistry, condensates comprise biomolecules that can potentially furnish alternative electron-transfer routes. Here, using electron paramagnetic resonance, electrochemical potentiometry, mass spectrometry, and confocal microscopy assays, we discovered that orthogonal to water oxidation, microenvironment-dependent spontaneous tyrosine oxidation encodes an alternative redox pathway. Through proton-coupled electron transfer, self-induced tyrosine autoxidation in condensates drives the formation of reactive carbon and oxygen species, providing a pathway in parallel to hydroxide oxidation for hydrogen peroxide formation in condensates. This self-induced redox pathway modulates nonequilibrium condensate behaviors, including responses to external chemical perturbations and evolution of the condensate interior microenvironment. By correlating condensate biomolecular composition with inherent redox activities, our work establishes a conceptual framework suggesting that condensate-dependent electron transfer can be critical to define the functions of condensates and deliver a new redox mechanism for cell biology.

Shweta Nandakumar, Jonas Bosche, Mirko Wieczorek, Constantin Matteo Albrecht, Belinda König, Mona Grünewald, Ludger Santen, Stefan Diez, Reza Shaebani^#, Laura Schaedel^#
Kinesin-Induced Buckling Reveals the Limits of Microtubule Self-Repair.
Adv Sci (Weinh), Art. No. doi: 10.1002/advs.202521721 (2026)
Open Access DOI

Microtubules are stiff cytoskeletal polymers whose ability to rapidly switch between growth and disassembly relies on a metastable lattice. This metastability is also reflected in their sensitivity to environmental conditions and in intrinsic lattice dynamics, where spontaneous tubulin loss is balanced by tubulin incorporation from solution-a process that also enables microtubules to self-repair when damaged. Whether such intrinsic self-repair is sufficient to preserve microtubule integrity during dynamic molecular motor-induced buckling, which frequently occurs in cells, remains unclear. Here, we show that kinesin-driven microtubule buckling in vitro induces severe lattice damage, leading to extensive tubulin incorporation. In many cases, however, the damage exceeds the microtubules' capacity for self-repair, resulting in breakage. In contrast, microtubules survive continuous buckling substantially longer in the presence of intracellular factors. Our results identify the limits of intrinsic microtubule self-repair and demonstrate that additional cellular mechanisms are essential to maintain microtubule integrity under sustained mechanical load.

Michael Hecht, Phil-Alexander Hofmann, Damar Wicaksono, Uwe Hernandez Acosta, Krzysztof Gonciarz, Jannik Kissinger, Vladimir Sivkin, Ivo F. Sbalzarini
Multivariate Newton interpolation in downward closed spaces reaches the optimal Bernstein-Walsh approximation rate.
IMA Journal of Numerical Anaysis, Art. No. doi: 10.1093/imanum/draf137 (2026)
Open Access DOI

Recent advances in Bernstein-Walsh theory have extended Bernstein's Theorem to multiple dimensions, stating that a multivariate function can be approximated with a geometric rate in a downward-closed polynomial space if and only if it is analytic in a generalized Bernstein polyellipse. To compute approximations of this class of functions-which we term Bos-Levenberg-Trefethen-(BLT) functions-we extend the classic univariate Newton interpolation algorithm to arbitrary multivariate downward-closed polynomial spaces, while maintaining its quadratic runtime and linear storage complexity. The present generalization supports any choice of (nontensorial) unisolvent interpolation nodes, whose number coincides with the dimension of the chosen downward-closed space. We prove that by selecting Leja nodes, the optimal geometric approximation rates for BLT-functions are achieved and that these rates extend to the derivatives of the interpolants. Choosing a Euclidean degree results in downward-closed spaces whose dimension only grows sub-exponentially with spatial dimension, while delivering approximation rates close to or matching those of the tensorial maximum-degree case, hence mitigating the curse of dimensionality. Importantly, our constructive proof directly inspires an algorithm for multivariate polynomial interpolation. We implemented this algorithm as a Python package and use it here to validate our theoretical findings in numerical experiments. These experiments corroborate the superiority of multivariate Newton interpolation over state-of-the-art alternatives, and they suggest that Leja-ordered Chebyshev-Lobatto nodes offer the same approximation power as Leja nodes.

C Darrin Hulsey, Paolo Franchini, Paul Masonick, Andreas F Kautt, Gonzalo Machado-Schiaffino, Martin Pippel, Francisco J García-de León, Eugene W Myers, Axel Meyer
Divergence at the IRX gene cluster underlies extreme trophic polymorphism in a cichlid fish (Herichthys minckleyi).
Commun Biol, Art. No. doi: 10.1038/s42003-026-09689-6 (2026)
Open Access DOI

The origin of the extensive phenotypic divergence characterizing adaptive radiation could often be geographically localized and genetically simple. In a classic case of a trophically polymorphic cichlid fish (Herichthys minckleyi), we investigated alternative genomic processes that could have produced its extreme within-population variation in pharyngeal jaw tooth size. First, we generated a high-quality reference genome for its close relative (H. cyanoguttatus) to dissect the genetic architecture of this dental polymorphism. Then, using whole genome resequencing across the small Cuatro Ciénegas valley where H. minckleyi is endemic, we found substantial micro-geographic subdivision and effectively no genetic structure due to pharyngeal morphotype. We also employed quantitative trait loci mapping and genome wide association to pinpoint a single peak in an Iroquois-related (IRX) gene cluster associated with H. minckleyi's dental divergence. Interspecific introgression in this genomic region appears negligible, suggesting the genomic basis of the polymorphism likely arose within cichlids confined to Cuatro Ciénegas. Because H. minckleyi tooth size disparity is comparable to that found in all Central American cichlids, this offers a striking example of how genomic divergence at a single locus could produce a punctuated burst of eco-morphological divergence that generates phenotypic breadth comparable to a highly diverse cichlid adaptive radiation.

Rashmiparvathi Keshara, Karolina Kuodyte, Antje Janosch, Cordula Andree, Marc Bickle, Martin Stöter, Rico Barsacchi, Yung Hae Kim, Anne Grapin-Botton
High-content screening of organoids reveals the mechanisms of human pancreas acinar specification.
Cell Stem Cell, 33(2) 325-339 (2026)
Open Access DOI

Organoids derived from pluripotent stem cells have emerged as powerful models to study human development. To investigate signaling pathways regulating human pancreas differentiation and morphogenesis, we developed a high-content, image-based screen and quantitative multivariate analysis pipelines robust to heterogeneity to extract single-cell and organoid features using pancreatic progenitor organoids. Here, we identified 54 compounds affecting cell identity and/or morphological landscape. Focusing on one family of compounds, we found that glycogen synthase kinase 3α/β (GSK3A/B) inhibition via wingless/int-1 (WNT) signaling has a reversible effect on cell identity, repressing pancreatic progenitor markers and inducing a poised state in progenitors transitioning to acinar cells. We show that additional fibroblast growth factor (FGF) repression enables further differentiation of acinar cells, recapitulating pancreatic acinar morphogenesis and function. The ability to produce acinar cells is valuable for future studies on pancreatic exocrine function and cancer initiation in humans, as acinar cells are thought to be an important cell of origin for pancreatic adenocarcinoma.

Gaelen Guzman, André Nadler, Frank Stein, Jeremy M Baskin, Carsten Schultz, Fikadu G Tafesse
The Lipid Interactome: an interactive and open access platform for exploring cellular lipid-protein interactions.
Bioinformatics, 42(2) Art. No. btaf651 (2026)
Open Access DOI

Lipid-protein interactions play essential roles in cellular signaling and membrane dynamics, yet their systematic characterization has long been hindered by the inherent biochemical properties of lipids. Recent advances in functionalized lipid probes-equipped with photoactivatable crosslinkers, affinity handles, and photocleavable protecting groups-have enabled proteomics-based identification of lipid interacting proteins with unprecedented specificity and resolution. Despite the growing number of published lipid interactomes, there remains no centralized effort to harmonize, compare, or integrate these datasets. The Lipid Interactome addresses this gap by providing a structured, interactive web portal that adheres to FAIR data principles-ensuring that lipid interactome studies are Findable, Accessible, Interoperable, and Reusable. Through standardized data formatting, interactive visualizations, and direct cross-study comparisons, this resource enables researchers to systematically explore the protein-binding partners of diverse bioactive lipids. By consolidating and curating lipid interactome proteomics data from multiple studies, the Lipid Interactome database serves as a critical tool for deciphering the biological functions of lipids in cellularsystems.

Javier Bregante^*, Flaminia Kaluthantrige Don^*, Fabian Rost, André Gohr, German Belenguer, Franziska Baenke, Dylan Liabeuf, Jessie Pöche, Clemens Schafmayer, Michaela Wilsch-Bräuninger, Sebastian Hinz, Kevin O' Holleran, Daniel E Stange, Meritxell Huch
Human liver cholangiocyte organoids capture the heterogeneity of in vivo liver ductal epithelium.
Cell Rep, 45(1) Art. No. 116786 (2026)
Open Access DOI

Human liver ductal epithelium is morphologically, functionally, and transcriptionally heterogeneous. Under- standing the impact of this heterogeneity has been challenging due to the absence of systems that recapit- ulate this heterogeneity in vitro. Here, we found that human liver cholangiocyte organoids do not retain the complex cellular heterogeneity of the native ductal epithelium. Inspired by the knowledge of the cellular niche, we refined our previous organoid medium to fully capture the in vivo cellular heterogeneity. We em- ployed this refined system to analyze the relationships between human biliary epithelial cell states. In our refined model, cholangiocytes transition toward hepatocyte-like states through a bipotent state. Additionally, inhibiting WNT signaling enhances the differentiation capacity of the cells toward hepatocyte-like states. By capturing the in vivo cholangiocyte heterogeneity, our improved organoid model represents a platform to investigate the impact of the different liver ductal cell states in cell plasticity, regeneration, and disease.

Fotini Papagavriil, Pablo Mateos-Gil, Janelle Lauer, Marino Zerial, Electra Gizeli
A QCM-D Study of the Interaction of Early Endosomal Antigen 1 (EEA1) Protein with Supported Lipid Bilayers Mimicking the Early Endosomal Lipid Composition.
Membranes (Basel), 16(2) Art. No. 49 (2026)
Open Access DOI

The combination of supported lipid bilayers (SLBs) with the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) has been proven to be a powerful tool to simultaneously monitor mass and viscoelastic changes related to membrane binding-events. In this work, the above methodology is employed for the study of the interaction of the Early Endosomal Antigen 1 (EEA1) to a model lipid bilayer that mimics the early endosome (EE) membrane, focusing on the membrane composition. Starting with the formation of a lipid bilayer through the vesicles fusion technique, we investigated the formation of SLBs that incorporate phosphatidylinositol 3-phosphate (PI(3)P), a key component for EEA1 binding, in combination with other lipids, e.g., (1,2-dioleoyl-sn-glycero-3)-phosphocholine (DOPC), -phosphoserine (DOPS), -phosphoethanolamine (DOPE), and cholesterol (Chol). The interaction of the full-length coiled-coil EEA1 to the formed SLBs was further studied in real time with the QCM-D and characterized with respect to the lipid composition and pH. Our findings confirm that PI(3)P is essential for the EEA1-membrane interaction, while it was shown that Chol and phosphatidylserine greatly influence the binding event. In fact, including 30% Chol in a PI(3)P (3%):PS (6%) SLB resulted in almost double EEA1 binding than in the absence of Chol. Moreover, we employed the QCM-viscoelastic model available to analyze the QCM-D data with emphasis on the study of the protein conformation. Our results showed that, in our in vitro system, EEA1 is not fully extended and/or highly packed, but is mainly in a bent, distorted conformation with an average size close to 100 nm. This study complements previous works employing in vitro assays, also demonstrating the ability to reconstitute more complex biomimetic EE membranes containing inositol phospholipids on a QCM surface for the study of EEA1 binding.

Madlen Matz-Soja^*, Christiane Körner^*, Fritzi Ott, Janett Fischer, Eugenia Marbach-Breitrück, Christian Bergmann, Ute Hofmann, Andrej Shevchenko, Iwona Wallach, Kathrin Textoris-Taube, Michael Mülleder, Rolf Gebhardt, Thomas Berg, Nikolaus Berndt
Modeling the dynamics of hepatic metabolism: the predominance of 12-hour rhythmicity in metabolic adaptation.
Cell Mol Life Sci, 83(1) Art. No. 55 (2026)
Open Access DOI

The liver continuously adjusts its metabolic activity to synchronize the nutrient supply with the body's demands. This synchronization involves the complex coordination of acute metabolic needs, nutrient availability, and activity levels, which is orchestrated according to cyclic internal rhythms governed by the circadian clock. This study aimed to decipher the role of circadian rhythms in liver metabolic functions, including mitochondrial activities that are critical for energy production and metabolic adaptation.

Mehrez Gammoudi, Bernhard Egger, Isabel L. Dittmann, Veronica N. Bulnes, Pavel Tomancak, Abdel Halim Harrath, Johannes Girstmair
The Embryonic Development of the Cotylean Polyclad Phrikoceros jannetae.
Acta Zool, Art. No. doi: 10.1111/azo.70019 (2026)
Open Access DOI

Polyclads exhibit distinct developmental modes ranging from direct to indirect development, with several transitional stages also recognised. The existence of an indirect developmental mode in polyclads with a planktonic life history stage in the form of a free-swimming larva is unique among all free-living flatworms and makes polyclads a compelling subject for comparative studies. Early development is robust across different polyclad species, including direct and indirect developers, stemming from the polyclad flatworms' retention of a stereotyped spiral cleavage pattern, a trait shared with other phyla grouped as the Spiralia. Using a live-imaging approach, we describe the embryonic development of the cotylean polyclad Phrikoceros jannetae (Pseudocerotidae), documenting all major developmental stages from meiosis to the formation of a three-eyed, six-lobed larva. We characterise key events such as cytoplasmic perturbations during meiosis, early cleavages in macromeres, and the appearance of larval morphological features including eyes and lobes. By aligning individual developmental trajectories to the 4-cell stage, we provide a coherent timeline that facilitates comparisons across embryos. This work expands the developmental knowledge of P. jannetae and contributes to a broader understanding of embryogenesis within Pseudocerotidae. Additionally, our observations reinforce previous findings on symmetry breaking in polyclads, particularly the pivotal role of micromere 4d, a process often overlooked in studies relying solely on static images.

Kerstin Howe, Arang Rhie, Sergey Koren, Elisabeth Busch-Nentwich, Shane A McCarthy, Jonathan Wood, Michelle Smith, Gene Myers, Karen Oliver
A trio-binned, haplotype-resolved genome sequence of the zebrafish, Danio rerio Hamilton 1822, SAT strain.
Wellcome Open Res, 10 Art. No. 682 (2025)
Open Access DOI

We present the trio-binned, haplotype-resolved genome assemblies (released in 2020) of both haplotypes of an individual of the Danio rerio SAT strain, a cross between Tuebingen (maternal) and AB (paternal) strains (zebrafish; Chordata; Actinopteri; Cypriniformes; Cyprinidae). The genome sequence of the paternal haplotype (fDreABH1) is 1,354.1 megabases long, while the genome sequence of the maternal haplotype (fDreTuH) is 1,360.5 megabases long. Most of the assembly is scaffolded into 25 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.6 kilobases in length.

Claudia Colasante, Jiangping Chen, Vannuruswamy Garikapati, Bernhard Spengler, Klaus-Dieter Schlüter, Eveline Baumgart-Vogt
The Knockout of PEX11a Results in Mild Peroxisomal Dysfunction and Lowered Cardiac Recovery Following Langendorff-Mediated Ischemia-Reperfusion in Mice.
Cells, 15(1) Art. No. 12 (2025)
Open Access DOI

Peroxisomal biogenesis defects frequently trigger processes of remodeling, increased oxidative stress and metabolic dysregulations that cause cellular dysfunction. Despite extensive research into cardiomyocyte ultrastructure and metabolism, knowledge on peroxisomal function in these cells is scarce. The objective of this study was therefore to investigate the impact of the purportedly asymptomatic (mild) deficiency of the peroxisomal biogenesis protein PEX11a on cardiomyocyte structure and cardiac function in mice. Langendorff-reperfusion experiments revealed diminished post-ischemic recovery following Pex11a knockout suggesting compromised cardiac response to ischemic stress. The suboptimal recovery might be attributable to increased ischemia-induced tissue deterioration consequent to morphological and metabolic abnormalities of the cardiomyocytes. Indeed, several alterations were observed in these cells in Pex11a knockout mice: (i) augmented size and number of peroxisomes and lipid droplets; (ii) increased sarcomere length; (iii) altered gene expression of peroxisome proliferator-activated receptors, organellar fission machinery proteins and cardiac markers; and (iv) a lipid composition shift. We hypothesized that peroxisomes contribute to the preservation of cardiomyocyte structure and functionality under conditions of ischemia-reperfusion. We further proposed that even "mild", undiagnosed peroxisomal defects can significantly impact cardiac performance following ischemia. This poses novel challenges for the risk assessment of cardiac pathologies.

Aswin Vinod Muthachikavil, Alexander von Appen, Thomas D Kühne
Computational Characterization of the Role of LEM2/LaminA Interactions on the Stability of BAF-Dimer Using Molecular Simulations.
Proteins, Art. No. doi: 10.1002/prot.70105 (2025)
Open Access DOI

The effect of the presence of the BAF-binding LEM-domain and LaminA Ig-fold on the stability of the BAF dimer was studied qualitatively using non-equilibrium pull simulations and quantitatively through the calculation of the potential of mean force profile along BAF-BAF separation distance. We find that hydrophobicity plays a significant role in stabilizing the BAF dimer when LEM-domain and LaminA are bound. The role of LEM-domain and LaminA in stabilizing the BAF dimer is explored by quantifying the strength of interaction between them, which are critical components of the nuclear lamina.

Anna Czarkwiani^*, Macrina Lobo^*, Lizbeth Airais Bolaños Castro^*, Andreas Petzold, Fabian Rost, René Maehr^#, Maximina H Yun^#
Molecular basis for de novo thymus regeneration in a vertebrate, the axolotl.
Sci Immunol, 10(114) Art. No. 9903 (2025)
DOI

The thymus is the primary site of T cell development, central to the establishment of self-tolerance and adaptive immune function. In mammals, the thymus undergoes age-related involution, resulting in a global decline in immune function. The thymus has some regenerative ability that relies on pre-existing thymic remnants but is insufficient to prevent involution. Here, we show that the juvenile axolotl (Ambystoma mexicanum) is able to regenerate its thymus de novo after complete removal, constituting an exception among vertebrates. Using single-cell transcriptomics and genetic and transplantation approaches, we demonstrate that de novo thymus regeneration results in the restoration of morphology, cell-type diversity, and function. FOXN1, although it has a conserved role in thymus organogenesis, is dispensable for the initiation of thymic regeneration. In contrast, we identify midkine signaling as a possible early driver of de novo thymus regeneration. This study demonstrates an instance of organ-level regeneration of the lymphoid system, which could guide future clinical strategies seeking to promote thymus regrowth.

Ruben I Calderon, Nirvay Sah, Molly Huang, Sampada Kallol, Ryan H Kittle, Walee B Shaik, Ahmed Abdelbaki, Jennifer N Chousal, Robert Morey, Tony Bui, Alejandra Mitre, Norah M E Fogarty, Claudia Gerri, Zoe Manalo, Claire Zheng, Peter De Hoff, Pratik Home, Kathy K Niakan, Heidi Cook-Andersen, Kathleen M Fisch, Soumen Paul, Francesca Soncin
VGLL1 contributes to both the transcriptome and epigenome of the developing trophoblast compartment.
Proc Natl Acad Sci U.S.A., 122(48) Art. No. e2508432122 (2025)
Open Access DOI

The trophectoderm (TE), the first lineage specified during mammalian development, initiates implantation and gives rise to placental trophoblasts. While animal models have elucidated key conserved signaling pathways involved in early TE specification, including bone morphogenetic protein (BMP), WNT, and HIPPO, species-specific differences during early development emphasize the need for human-specific models. We previously identified VGLL1, a coactivator of TEAD transcription factors, as a human-specific placental marker. In this study, we employed a pluripotent stem cell (PSC)-based model of TE induction by BMP4 to investigate chromatin remodeling and transcriptional dynamics during TE formation. BMP4-induced chromatin accessibility changes promoted a trophoblast gene expression program, while mesoderm lineage markers were only transiently expressed upon canonical WNT activation. We found that VGLL1 was expressed downstream of key TE transcription factors (GATA2/3, TFAP2A/C) but was essential for establishment of full trophoblast identity by up-regulating the epidermal growth factor receptor (EGFR) and reinforcing GATA3 expression through positive feedback. Notably, VGLL1 enhanced canonical WNT signaling via direct regulation of WNT receptors and effectors. We also identified KDM6B, a histone demethylase that removes H3K27me3 repressive marks, as a direct VGLL1 target. KDM6B facilitated activation of bivalent promoters associated with TE markers, linking epigenetic regulation to lineage identity. Our findings establish a mechanistic framework positioning VGLL1 as a central regulator that integrates HIPPO, BMP, and WNT signaling pathways to drive establishment of human TE.

Patrick M McCall^#, Kyoohyun Kim, Anna Shevchenko, Martine Ruer-Gruß, Jan Peychl, Jochen Guck, Andrej Shevchenko, Anthony Hyman, Jan Brugués^#
A label-free method for measuring the composition of multicomponent biomolecular condensates.
Nat Chem, 17(12) 1891-1902 (2025)
Open Access DOI

Many subcellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively because they either require fluorescent labels, which can perturb composition, or can distinguish only one or two components. Here we describe a label-free method based on quantitative phase imaging and analysis of tie-lines and refractive index to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex ageing dynamics for condensates composed of full-length proteins. We then use analysis of tie-lines and refractive index to simultaneously resolve the concentrations of five macromolecular solutes in multicomponent condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an unexpected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multicomponent condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

Kristin Böhlig, Juan M Iglesias-Artola, Antonino Asaro, Hjoerdis Mathilda Lennartz, Anna C Link, Björn Drobot, André Nadler
Bifunctional Probes Reveal the Rules of Intracellular Ether Lipid Transport.
Angew Chem Int Ed Engl, 64(46) Art. No. e202513360 (2025)
Open Access DOI

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Ether lipids play a significant role in physiology and human health. However, their cellular functions remain largely unknown due to a lack of tools for identifying their subcellular localization and interacting proteins. Here, we address this methodological gap by synthesizing minimally modified bifunctional ether lipid probes by introducing diazirine and alkyne groups. To interrogate the subcellular kinetics of intracellular ether lipid transport in mammalian cells, we used a combination of fluorescence imaging, machine learning-assisted image analysis, and mathematical modelling. We find that alkyl-linked ether lipids are transported up to twofold faster than vinyl-linked species (plasmalogens), pointing to yet undiscovered cellular lipid transport machinery able to distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non-vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl- and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in-depth characterization of intracellular ether lipid dynamics.

Leonie F Forth^#, Burkhard Malorny, Markus Bönn, Erik Brinks, Grégoire Denay, Carlus Deneke, Hosny El-Adawy, Jennie Fischer, Jannika Fuchs, Ekkehard Hiller, Nancy Bretschneider, Sylvia Kleta, Stefanie Lüth, Tilman Schultze, Henning Petersen, Michaela Projahn, Christian Schäfers, Kerstin Stingl, Andreas J Stroehlein, Laura Uelze, Kathrin Szabo, Anne Wöhlke, Jörg Linde^#
An inter-laboratory study characterizes the impact of bioinformatic approaches on genome-based cluster detection for foodborne bacterial pathogens.
Front Microbiol, 16 Art. No. 1629731 (2025)
Open Access DOI

Accurate assignment of whole-genome sequences to clusters in foodborne outbreak investigations remains challenging. Variability in bioinformatics tools and quality metrics significantly impacts clustering outcomes. This study assessed inter-laboratory variance in cluster identification by providing four datasets of 50 raw Illumina paired-end sequences covering Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Campylobacter jejuni. Following general rules of a specified guideline, participants applied in-house protocols for read quality assessment, 7-gene MLST, cgMLST, and SNP calling, then assigned samples to predefined focus clusters based on allele distance (AD) and mutations. Results revealed that differences in the interpretation of raw sequence and genome assembly quality influenced sample inclusion and finally cluster composition. Here, intra-species contamination was the most significant factor driving variability in decisions on whether to include or exclude samples. With one exception, 7-gene Multilocus-Sequence Typing (MLST) yielded consistent sequence types using different bioinformatics tools. The largest influence on cgMLST-defined clusters was the inclusion or exclusion of samples. Regarding bioinformatics, cgMLST was mainly reproducible. For S. enterica, discrepancies due to different software (Ridom SeqSphere+ vs. ChewieSnake) were larger than discrepancies due to different schemas. For other species, different schemas introduced larger discrepancies than different software. Most notably, C. jejuni cluster assignment was strongly affected by cgMLST schemas differing by a factor of two in the number of loci. SNP calling using Snippy produced concordant results across participants, except for C. jejuni when recombination filtering was used. This study highlights the impact caused by different interpretations of quality values when assessing clusters. Low-resolution cgMLST schemas were unsuitable for Campylobacter jejuni, and clustering near cut-off values was sensitive to bioinformatics tool selection. Standardized protocols are essential for reliable inter-laboratory comparison in foodborne pathogen surveillance.

Jessica Thiel, Duran Sürün, Desiree C Brändle, Madeleine Teichert, Stephan R Künzel, Ulrike Friedrich, Andreas Dahl, Kristin Schubert, Ignacy Rzagalinski, Andrej Shevchenko, Sofia Traikov, Peter Mirtschink, Lisa Wagenführ, Frank Buchholz, Kristina Hölig, Torsten Tonn, Romy Kronstein-Wiedemann
Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.
Stem Cell Rev Rep, 21(8) 2637-2653 (2025)
Open Access DOI

In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.

Meritxell Huch^#, Mansi Srivastava^#, Alex Eve
Beyond the beginning - development that lasts a lifetime.
Development, 152(20) Art. No. dev205313 (2025)
DOI

Basusree Ghosh^*, Patrick M McCall^*, Kristian Le Vay, Archishman Ghosh, Lars Hubatsch, David Thomas Gonzales, Jan Brugués, Hannes Mutschler^#, T-Y Dora Tang^#
RNA-peptide interactions tune the ribozyme activity within coacervate microdroplet dispersions.
Nat Commun, 16(1) Art. No. 8765 (2025)
Open Access DOI

Membrane-free complex coacervate microdroplets are compelling models for primitive compartmentalisation with the ability to form from biological molecules. However, understanding how molecular interactions can influence physicochemical properties and catalytic activity of membrane-free compartments is still in its infancy. This is important for defining the function of membrane-free compartments during the origin of life as well as in modern biology. Here, we use RNA-peptide coacervate microdroplets prepared with prebiotically relevant amino acids and a minimal hammerhead ribozyme. This is a model system to probe the relationship between coacervate composition, its properties and ribozyme activity. We show that ribozyme catalytic activity is inhibited within the coacervate compared to buffer solution, whilst variations in peptide sequence can modulate rates and yield of the ribozyme within the coacervate droplet by up to 15-fold. The apparent ribozyme rate constant is anti-correlated with its concentration and correlated to its diffusion coefficient within the coacervates. Our results provide a relationship between the physicochemical properties of the coacervate microenvironment and the catalytic activity of the ribozyme where membrane-free compartments could provide a selection pressure to drive molecular evolution on prebiotic earth.

Anna Hadarovich, David Kuster, Maria Luisa Romero Romero, Agnes Toth-Petroczy
On the Evolution of Biomolecular Condensates: From Prebiotic Origins to Subcellular Diversity.
Annu Rev Cell Dev Biol, 41(1) 403-432 (2025)
Open Access DOI

Biomolecular condensates provide a way to compartmentalize subcellular components with high temporal and spatial resolution, enabling rapid responses to signals and environmental changes. While the formation, components, and function of some condensates are well-characterized, their presence across organisms, their evolutionary history, and their origin are less well-understood. Here, we review the diversity of condensate components and highlight that not only disordered but also fully structured proteins are capable of driving condensate formation. We compare how proteomes of condensates overlap within and across species, and we present functionally analogous condensates across organisms. Additionally, we discuss the potential role of condensation in early life, suggesting that phase separation could have facilitated the selection and concentration of prebiotic molecules, promoting essential biochemical processes. We conclude that condensate-related organization principles are ubiquitously used across organisms from bacteria to mammals, and they potentially played a key role in prebiotic evolution, serving as primitive compartments for early biochemical processes.

Subham Biswas, Rahul Grover, Cordula Reuther, Chetan Poojari, Reza Shaebani, Shweta Nandakumar, Mona Gruenewald, Amir Zablotsky, Jochen S Hub, Stefan Diez, Karin John^#, Laura Schaedel^#
Tau accelerates tubulin exchange in the microtubule lattice.
Nat Phys, Art. No. doi: 10.1038/s41567-025-03003-7 (2025)
Open Access DOI

Microtubules are cytoskeletal filaments characterized by dynamic instability at their tips and a dynamic lattice that undergoes continuous tubulin loss and incorporation. Tau, a neuronal microtubule-associated protein, is well known for its role in stabilizing microtubule tips and promoting microtubule bundling. Here we demonstrate that tau also modulates microtubule lattice dynamics. Although tau lacks enzymatic activity, it significantly accelerates tubulin exchange within the lattice, particularly at topological defect sites. Our findings indicate that tau enhances lattice anisotropy by stabilizing longitudinal tubulin-tubulin interactions while destabilizing lateral ones, thereby enhancing the mobility and annihilation of lattice defects. These results challenge the traditional view of tau as merely a passive stabilizer, revealing its active role in dynamically remodelling the microtubule lattice structure.

Julia Vorhauser, Theodoros I Roumeliotis, David Coupe, Jacky K Leung, Lu Yu, Kristin Böhlig, Thomas Zerjatke, Ingmar Glauche, André Nadler, Jyoti S Choudhary, Jorg Mansfeld
A redox switch in p21-CDK feedback during G2 phase controls the proliferation-cell cycle exit decision.
Mol Cell, 85(17) 3241-3255 (2025)
Open Access DOI

Reactive oxygen species (ROS) influence cell proliferation and fate decisions by oxidizing cysteine residues (S-sulfenylation) of proteins, but specific targets and underlying regulatory mechanisms remain poorly defined. Here, we employ redox proteomics to identify cell-cycle-coordinated S-sulfenylation events and investigate their functional role in proliferation control. Although ROS levels rise during cell cycle progression, the overall oxidation of the proteome remains constant, with dynamic S-sulfenylation restricted to a subset of cysteines. Among these, we identify a critical redox-sensitive cysteine residue (C41) in the cyclin-dependent kinase (CDK) inhibitor p21. C41 oxidation regulates the interaction of p21 with CDK2 and CDK4, controlling a double-negative feedback loop that determines p21 stability. When C41 remains reduced, p21's half-life increases in the G2 phase, resulting in more p21 inheritance to daughter cells, suppressing proliferation and promoting senescence after irradiation. Notably, we identify dynamic S-sulfenylation on further cell cycle regulators, implying coordination of cell cycle and redox control.

Anaïs Bailles^#, Giulia Serafini, Heino Andreas, Christoph Zechner, Carl D. Modes, Pavel Tomancak^#
Anisotropic stretch biases the self-organization of actin fibers in multicellular Hydra aggregates.
Proc Natl Acad Sci U.S.A., 122(32) Art. No. e2423437122 (2025)
Open Access DOI

During development, groups of cells generate shape by coordinating their mechanical properties through an interplay of self-organization and prepatterning. Hydra displays a striking planar pattern of actin fibers at the organism scale, and mechanics influence the morphogenesis of biological structures during its prepatterned regeneration. However, how mechanics participate in the formation of an ordered pattern from a totally disordered state remains unknown. To study this, we used cellular aggregates formed from dissociated Hydra cells, which initially lose all actin polarity yet regenerate a long-range actin pattern. We showed quantitatively that the actin meshwork evolves from a disordered symmetric state to an ordered state in which rotational symmetry is broken, and translation symmetry is partially broken, with the nematic and smectic order parameters increasing over days. During the first hours, the actin meshwork displayed spatial heterogeneity in the nematic order parameter, and ordered domains separated by line defects progressively grew and fused. This suggests that local cell-cell interactions drive the transition from disorder to order. To understand the mechanism of ordering, we perturbed the tissue's physical constraints. We showed that while topology and geometry do not have a direct effect, anisotropic stretch biases the emerging orientation of the actin meshwork within hours. Surprisingly, although a Wnt head organizer is expected to play a role in the actin ordering, the stretch-associated alignment happened without the prior formation of a head organizer. This demonstrates the role of tissue mechanics in the alignment of the actin fibers during the disorder-to-order transition.

Xiangchun Ju^#, Shin-Yu Lee, Richard Ågren, Luiz Carlos Machado, Jiawei Xing, Chika Azama, Michael C Roy, Toshihiro Endo, Wieland Huttner, Adam Siepel, Izumi Fukunaga^#, Hugo Zeberg^#, Svante Pääbo^#
The activity and expression of adenylosuccinate lyase were reduced during modern human evolution, affecting brain and behavior.
Proc Natl Acad Sci U.S.A., 122(32) Art. No. e2508540122 (2025)
Open Access DOI

Adenylosuccinate lyase (ADSL), an enzyme that is crucial for purine biosynthesis, carries an amino acid substitution that is present in almost all humans today but absent in Neandertals and Denisovans. This substitution reduces the stability of the enzyme, but what functional consequences it has are unknown. Here, we show that when introduced into mice, this substitution causes substrates of the enzyme to accumulate in amounts that correlate negatively with ADSL expression levels. In the brain, where the expression of the enzyme is low, the substitution results in particularly high substrate levels. When the behavior of the mice is analyzed, female mice expressing the modern human-like version of ADSL access water more efficiently for drinking than their wild-type littermates. In addition to the amino acid substitution, a haplotype in the ADSL gene occurs at a carrier frequency of >97% in present-day humans and exhibits evidence of positive selection. It is associated with less ADSL expression as well as with increased concentrations of succinyladenosine, one of the substrates of the enzyme, in cerebrospinal fluid. Thus, two genetic changes have reduced ADSL activity in human tissues since modern and archaic humans separated, affecting purine biosynthesis, particularly in the brain.

Johannes Pahlke, Ivo F. Sbalzarini
On the Computational Power of Particle Methods
Fundamenta Informaticae, 194(1) Art. No. 3 (2025)
Open Access PDF DOI

We investigate the computational power of particle methods, a well-established class of algorit hms with applications in scientific computing and computer simulation. The computational power of a compute model determines the class of problems it can solve. Automata theory allows describing the computational power of abstract machines (automata) and the problems they can solve. At the top of the Chomsky hierarchy of formal languages and grammars are Turing machines, which resemble the concept on which most modern computers are built. Although particle methods can be interpreted as automata based on their formal definition, their computational power has so far not been studied. We address this by analyzing Turing completeness of particle methods. In particular, we prove two sets of restrictions under which a particle method is still Turing powerful, and we show when it loses Turing powerfulness. This contributes to understanding the theoretical foundations of particle methods and provides insight into the powerfulness of computer simulations.

David Grommisch^*, Evelien Eenjes^*, Maeve L Troost, Maria Genander
Epithelial architecture and signaling activity in the adult human esophagus.
Front Cell Dev Biol, 13 Art. No. 1632255 (2025)
Open Access DOI

Barrier epithelia function to shield the inside of our bodies from external stressors and pathogens. The esophageal epithelium is no exception, providing protection while at the same time transporting food to the stomach. Although many epithelial tissues are comparable between humans and mice, the human esophageal epithelium displays unique features in both progenitor cell organization and tissue architecture compared to the mouse. These differences have limited our understanding of the adult human esophagus, hindering the development of therapeutic strategies targeting human esophageal disease. Herein, we contrast the esophageal epithelial architecture and progenitor cell populations in mice and humans and discuss the role of a tentative human-specific progenitor cell population located in the submucosal gland ducts. Furthermore, we review current models available to study the human esophageal epithelium, focusing predominantly on adult primary organoids and epithelioids as well as the generation of human developmental esophageal epithelial cells from induced pluripotent stem cells. Finally, we discuss signaling activity implicated in maintaining normal human epithelial homeostasis, and how these pathways contribute to disease development. We aim to provide a comprehensive outlook on our current understanding of the human esophageal epithelium, while simultaneously highlighting unanswered questions in esophageal epithelial maintenance.

Kevin Nzumbi Mutemi, Oleg Simakov^#, Leslie Pan, Luca Santangeli, Ryan Wesley Null, Mette Handberg-Thorsager, Bruno C Vellutini, Kevin A. Peterson, Bastian Fromm, Tomas Larsson, Emily Savage, Mireia Osuna Lopez, Rajna Hercog, Jan Provaznik, Diana Ordoñez-Rueda, Nayara Azevedo, Eve Gazave, Michel Vervoort, Pavel Tomancak, Wenhua Tan, Sylke Winkler, Vladimir Benes, Jerome Hui, Conrad Helm, B Duygu Özpolat^#, Detlev Arendt^#
A genome resource for the marine annelid Platynereis spp.
BMC Genomics, 26(1) Art. No. 665 (2025)
Open Access DOI

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii (laboratory culture reference and a single individual assembly) and of the closely related P. massiliensis and P. megalops (single individual assembly) to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ~ 1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 105 miRNA loci. We further explore the high genetic variation (~ 3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

Angus Inman, Elisabeth Spiritosanto, Bridget L Evans, Judith E Lutton, Masazumi Tada, Till Bretschneider, Pierre A. Haas^#, Michael Smutny^#
A multi-tiered mechanical mechanism shapes the early neural plate.
Nat Commun, 16(1) Art. No. 6187 (2025)
Open Access DOI

The formation of complex tissues during embryonic development requires an intricate spatiotemporal coordination of local mechanical processes regulating global tissue morphogenesis. Here, we uncover a novel mechanism that mechanically regulates the shape of the anterior neural plate (ANP), a vital forebrain precursor, during zebrafish gastrulation. Combining in vivo and in silico approaches we reveal that the ANP is shaped by global tissue flows regulated by distinct force-generating processes. We show that mesendoderm migration and E-cadherin-dependent differential tissue interactions control distinct flow regimes in the neuroectoderm. Initial opposing flows lead to neuroectoderm cell internalisation and progressive multilayer tissue folding which in turn provide forces driving ANP tissue reshaping. We find that convergent extension is dispensable for internalisation but required for ANP tissue extension. Our results highlight how spatiotemporal regulation and coupling of different mechanical processes between tissues in the embryo control the first internalisation and folding events of the developing brain.

Justina Stark, Ivo F. Sbalzarini^#, Michael Brand^#
The people behind the papers - Justina Stark, Ivo Sbalzarini and Michael Brand.
Development, 152(13) Art. No. dev205004 (2025)
DOI

Morphogens are secreted from a local source and form long-range gradients. Existing computational models to study morphogen gradients typically simplify the tissue geometry. In a new study, Justina Stark and colleagues investigate the contribution of the porous 3D tissue geometry to long-range Fgf8a gradients in the early zebrafish embryo. To learn more about the people behind this work, we caught up with first author Justina Stark, and corresponding authors Ivo Sbalzarini (Professor at TU Dresden and Group Leader at the Max Planck Institute of Molecular Cell Biology and Genetics) and Michael Brand (Professor at the Center for Regenerative Therapies, TU Dresden).

Justina Stark^#, Rohit Krishnan Harish, Ivo F. Sbalzarini, Michael Brand^#
Morphogen gradients are regulated by porous media characteristics of the developing tissue.
Development, 152(13) Art. No. dev204312 (2025)
Open Access DOI

Long-range morphogen gradients have been proposed to form by morphogen diffusion from a localized source to distributed sinks in the target tissue. The role of the complex tissue geometry in this process is, however, less well understood and has not been explicitly resolved in existing models. Here, we numerically reconstruct pore-scale 3D geometries of zebrafish epiboly from light-sheet microscopy volumes. In these high-resolution 3D geometries, we simulate Fgf8a gradient formation in the tortuous extracellular space. Our simulations show that when realistic embryo geometries are considered, a source-diffusion-degradation mechanism with additional binding to extracellular matrix polymers is sufficient to explain emergence and robust maintenance of Fgf8a gradients. The predicted normalized gradient is robust against changes in source and sink rates but sensitive to changes in the pore connectivity of the extracellular space, with lower connectivity leading to steeper and shorter gradients. This demonstrates the importance of considering realistic geometries when studying morphogen gradients.

Gonen Golani^#, Manas Seal, Mrityunjoy Kar, Anthony Hyman, Daniella Goldfarb, Samuel Safran^#
Mesoscale properties of protein clusters determine the size and nature of liquid-liquid phase separation (LLPS).
Communications Physics, 8(1) Art. No. 226 (2025)
Open Access DOI

The observation of Liquid-Liquid Phase Separation (LLPS) in biological cells has dramatically shifted the paradigm that soluble proteins are uniformly dispersed in the cytoplasm or nucleoplasm. The LLPS region is preceded by a one-phase solution, where recent experiments have identified clusters in an aqueous solution with 102-103 proteins. Here, we theoretically consider a core-shell model with mesoscale core, surface, and bending properties of the clusters' shell and contrast two experimental paradigms for the measured cluster size distributions of the Cytoplasmic Polyadenylation Element Binding-4 (CPEB4) and Fused in Sarcoma (FUS) proteins. The fits to the theoretical model and earlier electron paramagnetic resonance (EPR) experiments suggest that the same protein may exhibit hydrophilic, hydrophobic, and amphiphilic conformations, which act to stabilize the clusters. We find that CPEB4 clusters are much more stable compared to FUS clusters, which are less energetically favorable. This suggests that in CPEB4, LLPS consists of large-scale aggregates of clusters, while for FUS, clusters coalesce to form micron-scale LLPS domains.

Eugene Christo V R, Esther Charlotte Sophia Kloth, Filippo Nisini, Cordula Reuther, Stefan Diez
Lowering Ionic Strength Improves the Sensitivity of Microtubule Gliding Assay Based Molecular Detection.
Nano Lett, 25(20) 8194-8202 (2025)
Open Access DOI

Microtubule gliding assays provide a unique mechanism for molecular detection in which binding of analytes to the microtubule lattice reduces the microtubule gliding speed. The reduction in the gliding speed correlates with the density of the bound analytes, enabling its quantification. Although promising, this technique is still in the proof-of-concept stage. Improving the sensitivity and limit of detection of the assay could make the technique comparable to that of advanced molecular detection methods. This study demonstrates that reducing the ionic strength of the buffer increases the sensitivity of the assay by enhancing the interactions between kinesin and microtubules. When using a low ionic strength buffer (BRB10) compared with a standard buffer (BRB80), we observed a more pronounced reduction in microtubule gliding speed in the presence of analytes, improving the detection limit. Therefore, this approach offers a simple and scalable way to improve the sensitivity of motor-based detection assays.

Lucrezia Camilla Ferme^#, Allyson Q Ryan, Robert Haase, Carl D. Modes, Caren Norden^#
Timely neurogenesis drives the transition from nematic to crystalline nuclear packing during retinal morphogenesis.
Sci Adv, 11(19) Art. No. 6843 (2025)
Open Access DOI

Correct organogenesis depends on the timely coordination of developmental processes, such as cell proliferation, differentiation, and migration. This coordination is particularly critical in crowded tissues, such as pseudostratified epithelia (PSE) that are often found as organ precursors. They are composed of elongated epithelial cells with densely packed nuclei aligned along the apicobasal axis. While cell cycle-dependent nuclear movements in PSE are well studied, less is known about how nuclear packing influences tissue morphogenesis. To investigate this, we analyzed nuclear shapes, sizes, and neighborhood statistics in zebrafish neuroepithelia, focusing on the retinal PSE. We found that nuclei exhibit elongated shapes and biaxial nematic-like orientational order but remain positionally disordered. During retinal development, nuclear packing density increases, approaching theoretical limits. This occurs when the tissue transitions to a laminated structure and nuclear shapes are remodeled. Timely neurogenesis is critical as failure to initiate neurogenesis leads to tissue deformations. These findings highlight the influence of nuclear shape and positioning for organ morphogenesis.

Sharif Iqbal, Simon Andersson, Ernesta Nesta, Nalle Pentinmikko, Ashish Kumar, Sawan Kumar Jha, Daniel Borshagovski, Anna T Webb, Nadja Gebert, Emma W Viitala, Alexandra Ritchie, Sandra Scharaw, Emilia Kuuluvainen, Hjalte List Larsen, Tuure Saarinen, Anne Juuti, Ari Ristimäki, Michael Jeltsch, Alessandro Ori, Markku Varjosalo, Kirsi H Pietiläinen, Saara Ollila, Kim B Jensen, Menno J Oudhoff, Pekka Katajisto
Fetal-like reversion in the regenerating intestine is regulated by mesenchymal asporin.
Cell Stem Cell, 32(4) 613-626 (2025)
DOI

Mesenchymal cells and the extracellular matrix (ECM) support epithelium during homeostasis and regeneration. However, the role of the mesenchyme in epithelial conversion into a fetal-like regenerative state after damage is not known. We modeled epithelial regeneration by culturing intestinal epithelium on decellularized small intestinal scaffolds (iECM) and identify asporin (Aspn), an ECM-bound proteoglycan, as a critical mediator of epithelial fetal-like reprogramming. After damage, transient increase in Aspn expression by the pericryptal fibroblasts induces epithelial transforming growth factor β (TGF-β)-signaling via CD44 and promotes timely epithelial reprogramming. Temporal control of Aspn is lost in old mice, and after damage, the persistently high level of Aspn stagnates epithelium in the regenerative state. Increase in Wnt signaling can resolve the stagnated regenerative program of the old epithelium, promoting restoration of tissue function. In summary, we establish a platform for modeling epithelial injury responses ex vivo and show that the mesenchymal Aspn-producing niche modulates tissue repair by regulating epithelial fetal-like reprogramming.

Adrian Pascal Nievergelt
Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.
Plant J, 122(1) Art. No. e70140 (2025)
Open Access DOI

The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.

James Cleland, Hanh Thi-Kim Vu, Johanna Dickmann, Andrei Rozanski, Steffen Werner, Andrea Schuhmann, Anna Shevchenko, Jochen Rink
A comparative analysis of planarian regeneration specificity reveals tissue polarity contributions of the axial cWnt signalling gradient.
Elife, Art. No. doi: 10.7554/eLife.105707.1 (2025)
Open Access DOI

Lara M Hoepfner, Adrian Pascal Nievergelt^#, Fabrizio Matrino, Martin Scholz, Helen E Foster, Jonathan Rodenfels, Alexander von Appen, Michael Hippler^#, Gaia Pigino^#
Unwrapping the Ciliary Coat: High-Resolution Structure and Function of the Ciliary Glycocalyx.
Adv Sci (Weinh), Art. No. e2413355 (2025)
Open Access DOI

The glycocalyx, a highly heterogeneous glycoprotein layer of cilia regulates adhesion and force transduction and is involved in signaling. The high-resolution molecular architecture of this layer is currently not understood. The structure of the ciliary coat is described in the green alga Chlamydomonas reinhardtii by cryo-electron tomography and proteomic approaches and the high-resolution cryoEM structure of the main component, FMG1B is solved. FMG1B is described as a mucin orthologue which lacks the major O-glycosylation of mammalian mucins but is N-glycosylated. FMG1A, a previously undescribed isoform of FMG1B is expressed in C. reinhardtii. By microflow-based adhesion assays, increased surface adhesion in the glycocalyx deficient double-mutant fmg1b-fmg1a is observed. It is found this mutant is capable of surface-gliding, with neither isoform required for extracellular force transduction by intraflagellar transport. The results find FMG1 to form a protective layer with adhesion-regulative instead of adhesion-conferring properties and an example of an undescribed class of mucins.

Michela Milani, Francesco Starinieri, Anna Fabiano, Stefano Beretta, Tiziana Plati, Cesare Canepari, Mauro Biffi, Fabio Russo, Valeria Berno, Rossana Norata, Francesca Sanvito, Ivan Merelli, Luigi Aloia, Meritxell Huch, Luigi Naldini, Alessio Cantore
Identification of hepatocyte-primed cholangiocytes in the homeostatic liver by in vivo lentiviral gene transfer to mice and non-human primates.
Cell Rep, 44(3) Art. No. 115341 (2025)
Open Access DOI

Liver regeneration is supported by hepatocytes and, in certain conditions, biliary epithelial cells (BECs). BECs are facultative liver stem cells that form organoids in culture and engraft in damaged livers. However, BEC heterogeneity in the homeostatic liver remains to be fully elucidated. Here, we exploit systemic lentiviral vector (LV) administration to achieve efficient and lifelong gene transfer to BECs in mice. We find that LV-marked BECs retain organoid formation potential and predominantly respond to liver damage; however, they are less clonogenic and display a hepatocyte-primed transcriptome compared to untransduced BECs. We thus identify a BEC subset committed to hepatocyte lineage in the absence of liver damage, characterized by a transcriptional network orchestrated by hepatocyte nuclear factor 4α. We also report in vivo targeting of such BECs in non-human primates. This work highlights intrinsic BEC heterogeneity and that in vivo LV gene transfer to the liver may persist following BEC-mediated repair of hepatic damage.

Georgina A Stooke-Vaughan^*, Sangwoo Kim^*, Shuo-Ting Yen, Kevin Son, Samhita P Banavar, James Giammona, David Kimelman, Otger Campàs
The physical roles of different posterior tissues in zebrafish axis elongation.
Nat Commun, 16(1) Art. No. 1839 (2025)
Open Access DOI

Shaping embryonic tissues requires spatiotemporal changes in genetic and signaling activity as well as in tissue mechanics. Studies linking specific molecular perturbations to changes in the tissue physical state remain sparse. Here we study how specific genetic perturbations affecting different posterior tissues during zebrafish body axis elongation change their physical state, the resulting large-scale tissue flows, and posterior elongation. Using a custom analysis software to reveal spatiotemporal variations in tissue fluidity, we show that dorsal tissues are most fluid at the posterior end, rigidify anterior of this region, and become more fluid again yet further anteriorly. In the absence of notochord (noto mutants) or when the presomitic mesoderm is substantially reduced (tbx16 mutants), dorsal tissues elongate normally. Perturbations of posterior-directed morphogenetic flows in dorsal tissues (vangl2 mutants) strongly affect the speed of elongation, highlighting the essential role of dorsal cell flows in delivering the necessary material to elongate the axis.

Joel Jonsson, Bevan Cheeseman, Ivo F. Sbalzarini
APR-CNN: Convolutional Neural Networks for the Adaptive Particle Representation of Large Microscopy Images.
Transactions on Machine Learning Research, Art. No. 5qKI2dkrjL (2025)
Open Access PDF

We present APR-CNN, a novel class of convolutional neural networks designed for efficient and scalable three-dimensional microscopy image analysis. APR-CNNs operate natively on a sparse, multi-resolution image representation known as the Adaptive Particle Representation (APR). This significantly reduces memory and compute requirements compared to traditional pixel-based CNNs. We introduce APR-native layers for convolution, pooling, and upsampling, along with hybrid architectures that combine APR and pixel layers to balance accuracy and computational efficiency. We show in benchmarks that APR-CNNs achieve comparable segmentation accuracy to pixel-based CNNs while drastically reducing memory usage and inference time. We further showcase the potential of APR-CNNs in large-scale volumetric image analysis, reducing inference times from weeks to days. This opens up new avenues for applying deep learning to large, high-resolution, three-dimensional biomedical datasets with constrained computational resources.

Yitong Xu, Anna Chao, Melissa Rinaldin, Alison Kickuth, Jan Brugués, Stefano Di Talia
The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.
Curr Biol, 35(3) 655-664 (2025)
DOI

Anaphase is tightly controlled spatiotemporally to ensure proper separation of chromosomes.1,2,3 The mitotic spindle, the self-organized microtubule structure driving chromosome segregation, scales in size with the available cytoplasm.4,5,6,7 Yet, the relationship between spindle size and chromosome movement remains poorly understood. Here, we address this relationship during the cleavage divisions of the Drosophila blastoderm. We show that the speed of chromosome separation gradually decreases during the four nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in spindle length, ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, we find that two processes contribute to controlling the speed at which chromosomes move in anaphase: the activity of molecular motors important for microtubule depolymerization and sliding and the cell cycle oscillator. Specifically, we found that the levels of multiple kinesin-like proteins important for microtubule depolymerization, as well as kinesin-5, contribute to setting the speed of chromosome separation. This observation is further supported by the scaling of poleward flux rate with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Thus, our work suggests a link between the biochemical rate of mitotic exit and the forces exerted by the spindle. Collectively, we propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase.

Aditya Chhatre, Ludek Stepanek, Adrian Pascal Nievergelt, Gonzalo Alvarez Viar, Stefan Diez^#, Gaia Pigino^#
Tubulin tyrosination/detyrosination regulate the affinity and sorting of intraflagellar transport trains on axonemal microtubule doublets.
Nat Commun, 16(1) Art. No. 1055 (2025)
Open Access DOI

Cilia assembly and function rely on the bidirectional transport of components between the cell body and ciliary tip via Intraflagellar Transport (IFT) trains. Anterograde and retrograde IFT trains travel along the B- and A-tubules of microtubule doublets, respectively, ensuring smooth traffic flow. However, the mechanism underlying this segregation remains unclear. Here, we test whether tubulin detyrosination (enriched on B-tubules) and tyrosination (enriched on A-tubules) have a role in IFT logistics. We report that knockout of tubulin detyrosinase VashL in Chlamydomonas reinhardtii causes frequent IFT train stoppages and impaired ciliary growth. By reconstituting IFT train motility on de-membranated axonemes and synthetic microtubules, we show that anterograde and retrograde trains preferentially associate with detyrosinated and tyrosinated microtubules, respectively. We propose that tubulin tyrosination/detyrosination is crucial for spatial segregation and collision-free IFT train motion, highlighting the significance of the tubulin code in ciliary transport.

A Sophie Brumm, Afshan McCarthy, Claudia Gerri, Todd Fallesen, Laura Woods, Riley McMahon, Athanasios Papathanasiou, Kay Elder, Phil Snell, Leila Christie, Patricia Garcia, Valerie Shaikly, Mohamed Taranissi, Paul Serhal, Rabi A Odia, Mina Vasilic, Anna Osnato, Peter J Rugg-Gunn, Ludovic Vallier, Caroline S Hill, Kathy K Niakan
Initiation and maintenance of the pluripotent epiblast in pre-implantation human development is independent of NODAL signaling.
Dev Cell, 60(2) 174-185 (2025)
Open Access DOI

The human blastocyst contains the pluripotent epiblast from which human embryonic stem cells (hESCs) can be derived. ACTIVIN/NODAL signaling maintains expression of the transcription factor NANOG and in vitro propagation of hESCs. It is unknown whether this reflects a functional requirement for epiblast development in human embryos. Here, we characterized NODAL signaling activity during pre-implantation human development. We showed that NANOG is an early molecular marker restricted to the nascent human pluripotent epiblast and was initiated prior to the onset of NODAL signaling. We further demonstrated that expression of pluripotency-associated transcription factors NANOG, SOX2, OCT4, and KLF17 were maintained in the epiblast in the absence of NODAL signaling activity. Genome-wide transcriptional analysis showed that NODAL signaling inhibition did not decrease NANOG transcription or impact the wider pluripotency-associated gene regulatory network. These data suggest differences in the signaling requirements regulating pluripotency in the pre-implantation human epiblast compared with existing hESC culture.

Oliver Gafvert, Peter Grindrod, Heather A Harrington, Catherine F Higham, Desmond J Higham, Ka Man Yim
On the hidden layer-to-layer topology of the representations of reality realised within neural networks.
Eng Comput, Art. No. doi: 10.1108/EC-06-2024-0508 (2025)
DOI

PurposeConsider an information processing algorithm that is designed to process an input data object onto an output data object via a number of successive internal {\it layers} and mappings between them. The possible activation state within each layer can be represented as a cube within Euclidean space of a high dimension (e.g. equal to the number of artificial neurons at that level). Multiple instances of such input objects produce a point cloud within each layer's cube: this is the "representation of the reality" at that layer, as sampled by the set of input objects.Design/methodology/approachMost neural networks reduce the dimension of each layer's cube from layer to successive layer. This gives the false impression of refining the inner representations of reality, distilling it down to fewer dimensions from which to discriminate or to infer outcomes (whatever is the aim). However, the representation of reality realised within each layer's cube is a manifold, a curved subset embedded within it and of much lower dimension. Investigations show that such manifolds may not always be reducing in their local dimension. Instead, the manifold may become folded over and over, filling up further dimensions and creating non-realistic (unforeseeable) proximities.FindingsWe discuss some of the likely consequences of these relatively unforeseen characteristics and, in particular, the possible vulnerability of such algorithms to non-realistic perturbations. We consider a possible response to this issue.Practical implicationsNew forms of calibration are necessary, using geometric/topological loss functions, as opposed to simple (variation-limiting) regularisation terms.Originality/valueWe apply persistent homology methods to understand how the images of the point cloud (representing the sampled reality) change as they pass from layer to layer.

Jan Fischer, Mariëlle Alders, Marcel M A M Mannens, David Genevieve, Karl Hackmann, Evelin Schröck, Bekim Sadikovic, Joseph Porrmann
Validation of a hypomorphic variant in CDK13 as the cause of CHDFIDD with autosomal recessive inheritance through determination of an episignature.
Clin Epigenetics, 17(1) Art. No. 5 (2025)
Open Access DOI

Autosomal dominant CDK13-related disease is characterized by congenital heart defects, dysmorphic facial features, and intellectual developmental disorder (CHDFIDD). Heterozygous pathogenic variants, particularly missense variants in the kinase domain, have previously been described as disease causing. Using the determination of a methylation pattern and comparison with an established episignature, we reveal the first hypomorphic variant in the kinase domain of CDK13, leading to a never before described autosomal recessive form of CHDFIDD in a boy with characteristic features. This highlights the utility of episignatures in variant interpretation, as well as a potential novel diagnostic approach in unsolved cases or for disease prognosis.

Temitope Opeyemi Oriowo, Ioannis Chrysostomakis, Sebastian Martin, Sandra Kukowka, Thomas Brown, Sylke Winkler, Eugene W Myers, Astrid Böhne, Madlen Stange
A chromosome-level, haplotype-resolved genome assembly and annotation for the Eurasian minnow (Leuciscidae: Phoxinus phoxinus) provide evidence of haplotype diversity.
GigaScience, 14 Art. No. giae116 (2025)
Open Access DOI

In this study, we present an in-depth analysis of the Eurasian minnow (Phoxinus phoxinus) genome, highlighting its genetic diversity, structural variations, and evolutionary adaptations. We generated an annotated haplotype-phased, chromosome-level genome assembly (2n = 50) by integrating high-fidelity (HiFi) long reads and chromosome conformation capture data (Hi-C).

Mehrez Gammoudi, Isabel L. Dittmann, Johannes Girstmair, Pavel Tomançak, Bernhard Egger, Veronica N. Bulnes
A new Phrikoceros species (Polycladida: Cotylea) from Ghar El Melh lagoon (Tunisia) with some remarks on the morphology of the genera Phrikoceros and Thytthosoceros.
Biologia, 80(1) 101-115 (2025)
DOI

We describe Phrikoceros jannetae sp. n., a new cotylean polyclad species from Ghar El Melh lagoon, a natural seawater lagoon situated in the north of Tunisia. The new species is characterized by the dorsal translucent white to brownish dorsal surface, with black round dots, red microdots, white blotches arranged as discrete lateral streaks, a mid-dorsal darker band and an interrupted slender black rim. We provide some insights into the biology of this species including the plastic tentacle configuration and the variability of body form and outline within the same specimen. Phrikoceros jannetae sp. n. was found among tunicates of the species Ciona intestinalis.

Joan Antoni Soler^*, Anupam Singh^*, Marino Zerial, Shashi Thutupalli
Motor Function of the Two-Component EEA1-Rab5 Revealed by dcFCCS.
Methods Mol Biol, 2881 87-115 (2025)
DOI

Fluorescence correlation spectroscopy (FCS) enables the measurement of fluctuations at fast timescales (typically few nanoseconds) and with high spatial resolution (tens of nanometers). This single-molecule measurement has been used to characterize single-molecule transport and flexibility of polymers and biomolecules such as DNA and RNA. Here, we apply this technique as dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to identify the motor function of the tethering protein EEA1 and the small GTPase Rab5 by probing the flexibility changes through end-monomer fluctuations.

Tina Subic, Ivo F. Sbalzarini
Loss of bimolecular reactions in reaction-diffusion master equations is consistent with diffusion limited reaction kinetics in the mean field limit.
J Chem Phys, 161(23) Art. No. 234107 (2024)
Open Access PDF DOI

We show that the resolution-dependent loss of bimolecular reactions in spatiotemporal Reaction-Diffusion Master Equations (RDMEs) is in agreement with the mean-field Collins-Kimball (C-K) theory of diffusion-limited reaction kinetics. The RDME is a spatial generalization of the chemical master equation, which enables studying stochastic reaction dynamics in spatially heterogeneous systems. It uses a regular Cartesian grid to partition space into locally well-mixed reaction compartments and treats diffusion as a jump reaction between neighboring grid cells. As the chance for reactants to be in the same grid cell decreases for smaller cell widths, the RDME loses bimolecular reactions in finer grids. We show that for a single homo-bimolecular reaction, the mesh spacing can be interpreted as the reaction radius of a well-mixed C-K rate. Then, the bimolecular reaction loss is consistent with diffusion-limited kinetics in the mean-field steady state. In this interpretation, the constant in a bimolecular reaction propensity is no longer the macroscopic reaction rate but the rate of the ballistic C-K step. For the same grid resolution, different diffusion models in RDME, such as those based on finite differences and Gaussian jumps, represent different reaction radii.

Wieland Huttner
Human-specific gene ARHGAP11B-potentially an additional tool in the treatment of neurodegenerative diseases?
Front Mol Med, 4 Art. No. 1465647 (2024)
Open Access DOI

Maarten P Bebelman, Lenka Belicova, Marino Zerial
The people behind the papers - Maarten Bebelman, Lenka Belicova and Marino Zerial.
Dev Genes Evol, 151(22) Art. No. dev204486 (2024)
DOI

Hepatoblasts are the bipotential progenitors of liver cells that differentiate into either hepatocytes or cholangiocytes. In their study, Marino Zerial and colleagues reveal how hepatoblast polarisation and lumen morphogenesis are linked to hepatocyte cell fate determination. To know more about their work, we spoke to the first authors, Maarten Bebelman and Lenka Belicova, and the corresponding author, Marino Zerial, Director and Scientific Member at the Max Planck Institute of Molecular Cell Biology and Genetics.

Tiffany A Kosch^*^#, María Torres-Sánchez^*^#, H Christoph Liedtke, Kyle Summers, Maximina H Yun, Andrew J Crawford, Simon T Maddock, Md Sabbir Ahammed, Victor L N Araújo, Lorenzo V Bertola, Gary M Bucciarelli, Albert Carné, Céline M Carneiro, Kin O Chan, Ying Chen, Angelica Crottini, Jessica M da Silva, Robert D Denton, Carolin Dittrich, Gonçalo Espregueira Themudo, Katherine A Farquharson, Natalie J Forsdick, Edward Gilbert, Jing Che, Barbara A Katzenback, Ramachandran Kotharambath, Nicholas A Levis, Roberto Márquez, Glib Mazepa, Kevin P Mulder, Hendrik Müller, Mary J O'Connell, Pablo Orozco-terWengel, Gemma Palomar, Alice Petzold, David W Pfennig, Karin S Pfennig, Michael S Reichert, Jacques Robert, Mark D Scherz, Karen Siu-Ting, Anthony A Snead, Matthias Stöck, Adam M M Stuckert, Jennifer L Stynoski, Rebecca D Tarvin, Katharina C Wollenberg Valero
The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation.
BMC Genomics, 25(1) Art. No. 1025 (2024)
Open Access DOI

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomic resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomic resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, anti-predator strategies, and resilience and adaptive responses. They also serve as essential models for studying broad genomic traits, such as evolutionary genome expansions and contractions, as they exhibit the widest range of genome sizes among all animal taxa and possess multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The emergence of long-read sequencing technologies, combined with advanced molecular and computational techniques that improve scaffolding and reduce computational workloads, is now making it possible to address some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC, https://mvs.unimelb.edu.au/amphibian-genomics-consortium ) in early 2023. This burgeoning community already has more than 282 members from 41 countries. The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and call on the research and conservation communities to unite as part of the AGC to enable amphibian genomics research to "leap" to the next level.

Kathryn Turnbull^#, Helge Paternoga, Esther von der Weth, Artyom A Egorov, Agnieszka A Pochopien, Yujie Zhang, Lilit Nersisyan, Tõnu Margus, Marcus J O Johansson, Vicent Pelechano, Daniel N Wilson^#, Vasili Hauryliuk^#
The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.
Nucleic Acids Res, 52(19) 12005-12020 (2024)
Open Access DOI

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

Máire Ní Leathlobhair^#, Anna Frangou, Ben Kinnersley, Alex J Cornish, Daniel Chubb, Eszter Lakatos, Prabhu Arumugam, Andreas J Gruber, Philip Law, Avraam Tapinos, G Maria Jakobsdottir, Iliana Peneva, Atef Sahli, Evie M Smyth, Richard Y Ball, Rushan Sylva, Ksenija Benes, Dan Stark, Robin J Young, Alexander T J Lee, Vincent Wolverson, Richard S Houlston, Alona Sosinsky, Andrew Protheroe, Matthew J Murray^#, David C Wedge^#, Clare Verrill^#, Testicular Cancer Genomics England Clinical Interpretation Partnership Consortium; Genomics England Research Consortium
Genomic landscape of adult testicular germ cell tumours in the 100,000 Genomes Project.
Nat Commun, 15(1) Art. No. 9247 (2024)
Open Access DOI

Testicular germ cell tumours (TGCT), which comprise seminoma and non-seminoma subtypes, are the most common cancers in young men. In this study, we present a comprehensive whole genome sequencing analysis of adult TGCTs. Leveraging samples from participants recruited via the UK National Health Service and data from the Genomics England 100,000 Genomes Project, our results provide an extended description of genomic elements underlying TGCT pathogenesis. This catalogue offers a comprehensive, high-resolution map of copy number alterations, structural variation, and key global genome features, including mutational signatures and analysis of extrachromosomal DNA amplification. This study establishes correlations between genomic alterations and histological diversification, revealing divergent evolutionary trajectories among TGCT subtypes. By reconstructing the chronological order of driver events, we identify a subgroup of adult TGCTs undergoing relatively late whole genome duplication. Additionally, we present evidence that human leukocyte antigen loss is a more prevalent mechanism of immune disruption in seminomas. Collectively, our findings provide valuable insights into the developmental and immune modulatory processes implicated in TGCT pathogenesis and progression.

Dominica Cao, Sumit Garai, James DiFrisco^#, Jesse V Veenvliet^#
The logic of monsters: development and morphological diversity in stem-cell-based embryo models.
Interface Focus, 14(5) Art. No. 20240023 (2024)
Open Access DOI

Organoids and stem-cell-based embryo models (SEMs) are imperfect organ or embryo representations that explore a much larger space of possible forms, or morphospace, compared to their in vivo counterparts. Here, we discuss SEM biology in light of seminal work by Pere Alberch, a leading figure in early evo-devo, interpreting SEMs as developmental 'monstrosities' in the Alberchian sense. Alberch suggested that ordered patterns in aberrant development-i.e. 'the logic of monsters'-reveal developmental constraints on possible morphologies. In the same vein, we detail how SEMs have begun to shed light on structural features of normal development, such as developmental variability, the relative importance of internal versus external constraints, boundary conditions and design principles governing robustness and canalization. We argue that SEMs represent a powerful experimental tool to explore and expand developmental morphospace and propose that the 'monstrosity' of SEMs can be leveraged to uncover the 'hidden' rules and developmental constraints that robustly shape and pattern the embryo.

Shanshan Xu, Maria E Gierisch, Enrica Barchi, Ina Poser, Simon Alberti, Florian A Salomons, Nico P Dantuma
Chemical inhibition of the integrated stress response impairs the ubiquitin-proteasome system.
Commun Biol, 7(1) Art. No. 1282 (2024)
Open Access DOI

Inhibitors of the integrated stress response (ISR) have been used to explore the potential beneficial effects of reducing the activation of this pathway in diseases. As the ISR is in essence a protective response, there is, however, a risk that inhibition may compromise the cell's ability to restore protein homeostasis. Here, we show that the experimental compound ISRIB impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Accumulation of a UPS reporter substrate that is intercepted by ribosome quality control was comparable to the level observed after blocking the UPS with a proteasome inhibitor. Consistent with impairment of the cytosolic UPS, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. Our data suggest that the persistent protein translation during proteotoxic stress in the absence of a functional ISR increases the pool of DRiPs, thereby hindering the efficient clearance of cytosolic substrates by the UPS.

Gonzalo Alvarez Viar, Nikolai Klena, Fabrizio Martino, Adrian Pascal Nievergelt, Davide Bolognini, Paola Capasso, Gaia Pigino
Protofilament-specific nanopatterns of tubulin post-translational modifications regulate the mechanics of ciliary beating.
Curr Biol, 34(19) 4464-4475 (2024)
Open Access DOI

Controlling ciliary beating is essential for motility and signaling in eukaryotes. This process relies on the regulation of various axonemal proteins that assemble in stereotyped patterns onto individual microtubules of the ciliary structure. Additionally, each axonemal protein interacts exclusively with determined tubulin protofilaments of the neighboring microtubule to carry out its function. While it is known that tubulin post-translational modifications (PTMs) are important for proper ciliary motility, the mode and extent to which they contribute to these interactions remain poorly understood. Currently, the prevailing understanding is that PTMs can confer functional specialization at the level of individual microtubules. However, this paradigm falls short of explaining how the tubulin code can manage the complexity of the axonemal structure where functional interactions happen in defined patterns at the sub-microtubular scale. Here, we combine immuno-cryo-electron tomography (cryo-ET), expansion microscopy, and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nanopatterns at a sub-microtubular scale. These nanopatterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are indispensable for their regulation during ciliary beating. Our findings offer a new paradigm for understanding how different tubulin PTMs, such as glycylation, glutamylation, acetylation, tyrosination, and detyrosination, can coexist within the ciliary structure and specialize individual protofilaments for the regulation of diverse protein complexes. The identification of a ciliary tubulin nanocode by cryo-ET suggests the need for high-resolution studies to better understand the molecular role of PTMs in other cellular compartments beyond the cilium.

Takashi Namba, Wieland Huttner
What Makes Us Human: Insights from the Evolution and Development of the Human Neocortex.
Annu Rev Cell Dev Biol, 40(1) 427-452 (2024)
Open Access DOI

"What makes us human?" is a central question of many research fields, notably anthropology. In this review, we focus on the development of the human neocortex, the part of the brain with a key role in cognition, to gain neurobiological insight toward answering this question. We first discuss cortical stem and progenitor cells and human-specific genes that affect their behavior. We thus aim to understand the molecular foundation of the expansion of the neocortex that occurred in the course of human evolution, as this expansion is generally thought to provide a basis for our unique cognitive abilities. We then review the emerging evidence pointing to differences in the development of the neocortex between present-day humans and Neanderthals, our closest relatives. Finally, we discuss human-specific genes that have been implicated in neuronal circuitry and offer a perspective for future studies addressing the question of what makes us human.

Alfonso Martinez Arias, Nicolas Rivron, Naomi Moris, Patrick P L Tam, Cantas Alev, Jianping Fu, Anna-Katerina Hadjantonakis, Jacob H Hanna, Gabriella Minchiotti, Olivier Pourquie, Guojun Sheng, Liliana Solnica Krezel, Jesse V Veenvliet, Aryeh Warmflash
Criteria for the standardization of stem-cell-based embryo models.
Nat Cell Biol, 26(10) 1625-1628 (2024)
DOI

Manfred Schartl^#, Joost M Woltering, Iker Irisarri, Kang Du, Susanne Kneitz, Martin Pippel, Thomas Brown, Paolo Franchini, Jing Li, Ming Li, Mateus Adolfi, Sylke Winkler, Josane de Freitas Sousa, Zhuoxin Chen, Sandra Jacinto, Evgeny Z Kvon, Luis Rogério Correa de Oliveira, Erika Monteiro, Danielson Baia Amaral, Thorsten Burmester, Domitille Chalopin, Alexander Suh, Eugene W Myers, Oleg Simakov, Igor Schneider, Axel Meyer^#
The genomes of all lungfish inform on genome expansion and tetrapod evolution.
Nature, 634(8032) 96-103 (2024)
DOI

The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

Metin G Davutoglu, Veikko Geyer, Lukas Niese, Johannes R Soltwedel, Marcelo L Zoccoler, Valeria Sabatino, Robert Haase, Nils Kröger, Stefan Diez^#, Nicole Poulsen^#
Gliding motility of the diatom Craspedostauros australis coincides with the intracellular movement of raphid-specific myosins.
Commun Biol, 7(1) Art. No. 1187 (2024)
Open Access DOI

Raphid diatoms are one of the few eukaryotes capable of gliding motility, which is remarkably fast and allows for quasi-instantaneous directional reversals. Besides other mechanistic models, it has been suggested that an actomyosin system provides the force for diatom gliding. However, in vivo data on the dynamics of actin and myosin in diatoms are lacking. In this study, we demonstrate that the raphe-associated actin bundles required for diatom movement do not exhibit a directional turnover of subunits and thus their dynamics do not contribute directly to force generation. By phylogenomic analysis, we identified four raphid diatom-specific myosins in Craspedostauros australis (CaMyo51A-D) and investigated their in vivo localization and dynamics through GFP-tagging. Only CaMyo51B-D but not CaMyo51A exhibited coordinated movement during gliding, consistent with a role in force generation. The characterization of raphid diatom-specific myosins lays the foundation for unraveling the molecular mechanisms that underlie the gliding motility of diatoms.

Mario Ivanković^*, Jeremias N Brand^*, Luca Pandolfini, Thomas Brown, Martin Pippel, Andrei Rozanski, Til Schubert, Markus Grohme, Sylke Winkler, Laura Robledillo, Meng Zhang, Azzurra Codino, Stefano Gustincich, Miquel Vila-Farré, Shu Zhang, Argyris Papantonis, André Marques, Jochen Rink
A comparative analysis of planarian genomes reveals regulatory conservation in the face of rapid structural divergence.
Nat Commun, 15(1) Art. No. 8215 (2024)
Open Access DOI

The planarian Schmidtea mediterranea is being studied as a model species for regeneration, but the assembly of planarian genomes remains challenging. Here, we report a high-quality haplotype-phased, chromosome-scale genome assembly of the sexual S2 strain of S. mediterranea and high-quality chromosome-scale assemblies of its three close relatives, S. polychroa, S. nova, and S. lugubris. Using hybrid gene annotations and optimized ATAC-seq and ChIP-seq protocols for regulatory element annotation, we provide valuable genome resources for the planarian research community and a first comparative perspective on planarian genome evolution. Our analyses reveal substantial divergence in protein-coding sequences and regulatory regions but considerable conservation within promoter and enhancer annotations. We also find frequent retrotransposon-associated chromosomal inversions and interchromosomal translocations within the genus Schmidtea and, remarkably, independent and nearly complete losses of ancestral metazoan synteny in Schmidtea and two other flatworm groups. Overall, our results suggest that platyhelminth genomes can evolve without syntenic constraints.

Bernadette J Stolz^*, Jagdeep Dhesi^*, Joshua A Bull, Heather A Harrington, Helen M Byrne, Iris H R Yoon
Relational Persistent Homology for Multispecies Data with Application to the Tumor Microenvironment.
Bull Math Biol, 86(11) Art. No. 128 (2024)
Open Access DOI

Topological data analysis (TDA) is an active field of mathematics for quantifying shape in complex data. Standard methods in TDA such as persistent homology (PH) are typically focused on the analysis of data consisting of a single entity (e.g., cells or molecular species). However, state-of-the-art data collection techniques now generate exquisitely detailed multispecies data, prompting a need for methods that can examine and quantify the relations among them. Such heterogeneous data types arise in many contexts, ranging from biomedical imaging, geospatial analysis, to species ecology. Here, we propose two methods for encoding spatial relations among different data types that are based on Dowker complexes and Witness complexes. We apply the methods to synthetic multispecies data of a tumor microenvironment and analyze topological features that capture relations between different cell types, e.g., blood vessels, macrophages, tumor cells, and necrotic cells. We demonstrate that relational topological features can extract biological insight, including the dominant immune cell phenotype (an important predictor of patient prognosis) and the parameter regimes of a data-generating model. The methods provide a quantitative perspective on the relational analysis of multispecies spatial data, overcome the limits of traditional PH, and are readily computable.

Ann M Mc Cartney^*, Giulio Formenti^*, Alice Mouton^*, Diego De Panis, Luísa S Marins, Henrique G Leitão, Genevieve Diedericks, Joseph Kirangwa, Marco Morselli, Judit Salces-Ortiz, Nuria Escudero, Alessio Iannucci, Chiara Natali, Hannes Svardal, Rosa Fernández, Tim De Pooter, Geert Joris, Mojca Strazisar, Jonathan Wood, Katie E Herron, Ole Seehausen, Phillip C Watts, Felix Shaw, Robert P Davey, Alice Minotto, José M Fernández, Astrid Böhne, Carla Alegria, Tyler Alioto, Paulo C Alves, Isabel R Amorim, Jean-Marc Aury, Niclas Backstrom, Petr Baldrian, Laima Baltrunaite, Endre Barta, Bertrand BedHom, Caroline Belser, Johannes Bergsten, Laurie Bertrand, Helena Bilandija, Mahesh Binzer-Panchal, Iliana Bista, Mark L Blaxter, Paulo A V Borges, Guilherme Borges Dias, Mirte Bosse, Tom Brown, Rémy Bruggmann, Elena Buena-Atienza, Josephine Burgin, Elena Buzan, Alessia Cariani, Nicolas Casadei, Matteo Chiara, Sergio Chozas, Fedor Čiampor, Angelica Crottini, Corinne Cruaud, Fernando Cruz, Love Dalen, Alessio De Biase, Javier Del Campo, Teo Delic, Alice B Dennis, Martijn F L Derks, Maria Angela Diroma, Mihajla Djan, Simone Duprat, Klara Eleftheriadi, Philine G D Feulner, Jean-François Flot, Giobbe Forni, Bruno Fosso, Pascal Fournier, Christine Fournier-Chambrillon, Toni Gabaldon, Shilpa Garg, Carmela Gissi, Luca Giupponi, Jessica Gomez-Garrido, Josefa González, Miguel Grilo, Björn Grüning, Thomas Guerin, Nadege Guiglielmoni, Marta Gut, Marcel P Haesler, Christoph Hahn, Balint Halpern, Peter W Harrison, Julia Heintz, Maris Hindrikson, Jacob Höglund, Kerstin Howe, Graham M Hughes, Benjamin Istace, Mark J Cock, Franc Janžekovič, Zophonias O Jonsson, Sagane Joye-Dind, Janne J Koskimäki, Boris Krystufek, Justyna Kubacka, Heiner Kuhl, Szilvia Kusza, Karine Labadie, Meri Lähteenaro, Henrik Lantz, Anton Lavrinienko, Lucas Leclère, Ricardo J Lopes, Ole D Madsen, Ghislaine Magdelenat, Giulia Magoga, Tereza Manousaki, Tapio Mappes, Joao Pedro Marques, Gemma I Martinez Redondo, Florian Maumus, Shane A McCarthy, Hendrik-Jan Megens, José Melo-Ferreira, Sofia L Mendes, Matteo Montagna, Joao Moreno, Mai-Britt Mosbech, Monica Moura, Zuzana Musilova, Eugene W Myers, Will J Nash, Alexander Nater, Pamela Nicholson, Manuel Niell, Reindert Nijland, Benjamin Noel, Karin Noren, Pedro H Oliveira, Remi-Andre Olsen, Lino Ometto, Rebekah A Oomen, Stephan Ossowski, Vaidas Palinauskas, Snaebjorn Palsson, Jerome P Panibe, Joana Pauperio, Martina Pavlek, Emilie Payen, Julia Pawlowska, Jaume Pellicer, Graziano Pesole, João Pimenta, Martin Pippel, Anna Maria Pirttilä, Nikos Poulakakis, Jeena Rajan, Rúben M C Rego, Roberto Resendes, Philipp Resl, Ana Riesgo, Patrik Rodin-Morch, Ana Rosa Soares, Carlos Rodríguez Fernandes, Maria M Romeiras, Guilherme Roxo, Lukas Rüber, Maria Jose Ruiz-Lopez, Urmas Saarma, Luis Fernando Delgadillo Silva, Manuela Sim-Sim, Lucile Soler, Vitor C Sousa, Carla Sousa Santos, Alberto Spada, Milomir Stefanovic, Viktor Steger, Josefin Stiller, Matthias Stöck, Torsten H Struck, Hiranya Sudasinghe, Riikka Tapanainen, Christian Tellgren-Roth, Helena Trindade, Yevhen Tukalenko, Ilenia Urso, Benoit Vacherie, Steven M Van Belleghem, Kees Van Oers, Carlos Vargas-Chavez, Nevena Velickovic, Noel Vella, Adriana Vella, Cristiano Vernesi, Sara Vicente, Sara Villa, Olga Vinnere Pettersson, Filip A M Volckaert, Judit Voros, Patrick Wincker, Sylke Winkler, Claúdio Ciofi, Robert M Waterhouse, Camila J Mazzoni
The European Reference Genome Atlas: piloting a decentralised approach to equitable biodiversity genomics.
NPJ Biodivers, 3(1) Art. No. 28 (2024)
Open Access DOI

A genomic database of all Earth's eukaryotic species could contribute to many scientific discoveries; however, only a tiny fraction of species have genomic information available. In 2018, scientists across the world united under the Earth BioGenome Project (EBP), aiming to produce a database of high-quality reference genomes containing all ~1.5 million recognized eukaryotic species. As the European node of the EBP, the European Reference Genome Atlas (ERGA) sought to implement a new decentralised, equitable and inclusive model for producing reference genomes. For this, ERGA launched a Pilot Project establishing the first distributed reference genome production infrastructure and testing it on 98 eukaryotic species from 33 European countries. Here we outline the infrastructure and explore its effectiveness for scaling high-quality reference genome production, whilst considering equity and inclusion. The outcomes and lessons learned provide a solid foundation for ERGA while offering key learnings to other transnational, national genomic resource projects and the EBP.

Jochen Krattenmacher, Manuel Lera-Ramirez, Alexandre Beber, Stepan Herynek, Lenka Grycova, Xiaocheng Liu, Pavel Neuzil, Francois Nedelec, Stefan Diez^#, Marcus Braun^#, Zdenek Lansky^#
Ase1 selectively increases the lifetime of antiparallel microtubule overlaps.
Curr Biol, 34(17) 4071-4080 (2024)
Open Access DOI

Microtubules (MTs) are dynamically unstable polar biopolymers switching between periods of polymerization and depolymerization, with the switch from the polymerization to the depolymerization phase termed catastrophe and the reverse transition termed rescue.1 In presence of MT-crosslinking proteins, MTs form parallel or anti-parallel overlaps and self-assemble reversibly into complex networks, such as the mitotic spindle. Differential regulation of MT dynamics in parallel and anti-parallel overlaps is critical for the self-assembly of these networks.2,3 Diffusible MT crosslinkers of the Ase1/MAP65/PRC1 family associate with different affinities to parallel and antiparallel MT overlaps, providing a basis for this differential regulation.4,5,6,7,8,9,10,11 Ase1/MAP65/PRC1 family proteins directly affect MT dynamics12 and recruit other proteins that locally alter MT dynamics, such as CLASP or kinesin-4.7,13,14,15,16 However, how Ase1 differentially regulates MT stability in parallel and antiparallel bundles is unknown. Here, we show that Ase1 selectively promotes antiparallel MT overlap longevity by slowing down the depolymerization velocity and by increasing the rescue frequency, specifically in antiparallelly crosslinked MTs. At the retracting ends of depolymerizing MTs, concomitant with slower depolymerization, we observe retention and accumulation of Ase1 between crosslinked MTs and on isolated MTs. We hypothesize that the ability of Ase1 to reduce the dissociation of tubulin subunits is sufficient to promote its enrichment at MT ends. A mathematical model built on this idea shows good agreement with the experiments. We propose that differential regulation of MT dynamics by Ase1 contributes to mitotic spindle assembly by specifically stabilizing antiparallel overlaps, compared to parallel overlaps or isolated MTs.

Michael F Staddon
How the zebra got its stripes: Curvature-dependent diffusion orients Turing patterns on three-dimensional surfaces.
Phys Rev E, 110(3) Art. No. 034402 (2024)
Open Access DOI

Many animals have patterned fur, feathers, or scales, such as the stripes of a zebra. Turing models, or reaction- diffusion systems, are a class of mathematical models of interacting species that have been successfully used to generate animal-like patterns for many species. When diffusion of the inhibitor is high enough relative to the activator, a diffusion-driven instability can spontaneously form patterns. However, it is not just the type of pattern but also the orientation that matters, and it remains unclear how patterns are oriented in practice. Here, we propose a mechanism by which the curvature of the surface influences the rate of diffusion, and can recapture the correct orientation of stripes on models of a zebra and of a cat in numerical simulations. Previous work has shown how anisotropic diffusion can give stripe forming reaction-diffusion systems a bias in orientation. From the observation that zebra stripes run around the direction of highest curvature, that is around the torso and legs, we apply this result by modifying the anisotropic diffusion rates based on the local curvature. These results show how local geometry can influence the reaction dynamics to give robust, global-scale patterns. Overall, this model proposes a coupling between the system geometry and reaction-diffusion dynamics that can give global control over the patterning by using only local curvature information. Such a model can give shape and positioning information in animal development without the need for spatially dependent morphogen gradients.

Alex J Cornish, Andreas J Gruber, Ben Kinnersley, Daniel Chubb, Anna Frangou, Giulio Caravagna, Boris Noyvert, Eszter Lakatos, Henry M Wood, Steve Thorn, Richard Culliford, Claudia Arnedo-Pac, Jacob Househam, William Cross, Amit Sud, Philip Law, Máire Ní Leathlobhair, Aliah Hawari, Connor Woolley, Kitty Sherwood, Nathalie Feeley, Güler Gül, Juan Fernandez-Tajes, Luis Zapata, Ludmil B Alexandrov, Nirupa Murugaesu, Alona Sosinsky, Jonathan Mitchell, Nuria Lopez-Bigas, Philip Quirke, David N Church, Ian P M Tomlinson, Andrea Sottoriva, Trevor A Graham, David C Wedge, Richard S Houlston
The genomic landscape of 2,023 colorectal cancers.
Nature, 633(8028) 127-136 (2024)
Open Access PDF DOI

Colorectal carcinoma (CRC) is a common cause of mortality1, but a comprehensive description of its genomic landscape is lacking2-9. Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichia colipks+ colibactin in rectal cancers10 and the importance of the SBS93 signature11-13, which suggests that diet or smoking is a risk factor. Immune-escape driver mutations14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1), highlighting the role of whole-genome sequencing in optimizing patient care.

Dominik Kopczynski^*, Christer S. Ejsing^*, Jeffrey G McDonald, Takeshi Bamba, Erin S Baker, Justine Bertrand-Michel, Britta Brügger, Cristina Coman, Shane R Ellis, Timothy J Garrett, William J Griffiths, Xue Li Guan, Xianlin Han, Marcus Höring, Michal Holčapek, Nils Hoffmann, Kevin Huynh, Rainer Lehmann, Jace W Jones, Rima Kaddurah-Daouk, Harald C Köfeler, Peter J Meikle, Thomas O Metz, Valerie B O'Donnell, Daisuke Saigusa, Dominik Schwudke, Andrej Shevchenko, Federico Torta, Juan Antonio Vizcaíno, Ruth Welti, Markus R Wenk, Denise Wolrab, Yu Xia, Kim Ekroos^#, R Ahrends^#, Gerhard Liebisch^#
The lipidomics reporting checklist a framework for transparency of lipidomic experiments and repurposing resource data.
J Lipid Res, 65(9) Art. No. 100621 (2024)
Open Access DOI

The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.

Lei Xing^#, Wieland Huttner^#, Takashi Namba^#
Role of cell metabolism in the pathophysiology of brain size-associated neurodevelopmental disorders.
Neurobiol Dis, 199 Art. No. 106607 (2024)
Open Access DOI

Cell metabolism is a key regulator of human neocortex development and evolution. Several lines of evidence indicate that alterations in neural stem/progenitor cell (NPC) metabolism lead to abnormal brain development, particularly brain size-associated neurodevelopmental disorders, such as microcephaly. Abnormal NPC metabolism causes impaired cell proliferation and thus insufficient expansion of NPCs for neurogenesis. Therefore, the production of neurons, which is a major determinant of brain size, is decreased and the size of the brain, especially the size of the neocortex, is significantly reduced. This review discusses recent progress understanding NPC metabolism, focusing in particular on glucose metabolism, fatty acid metabolism and amino acid metabolism (e.g., glutaminolysis and serine metabolism). We provide an overview of the contributions of these metabolic pathways to brain development and evolution, as well as to the etiology of neurodevelopmental disorders. Furthermore, we discuss the advantages and disadvantages of various experimental models to study cell metabolism in the developing brain.

Benedikt Kuhn, Jonathan Zöller, Iwan Zimmermann, Tim Gemeinhardt, Dogukan H Özkul, Julian D Langer, Markus A Seeger, Eric R Geertsma
Interdomain-linkers control conformational transitions in the SLC23 elevator transporter UraA.
Nat Commun, 15(1) Art. No. 7518 (2024)
Open Access DOI

Uptake of nucleobases and ascorbate is an essential process in all living organisms mediated by SLC23 transport proteins. These transmembrane carriers operate via the elevator alternating-access mechanism, and are composed of two rigid domains whose relative motion drives transport. The lack of large conformational changes within these domains suggests that the interdomain-linkers act as flexible tethers. Here, we show that interdomain-linkers are not mere tethers, but have a key regulatory role in dictating the conformational space of the transporter and defining the rotation axis of the mobile transport domain. By resolving a wide inward-open conformation of the SLC23 elevator transporter UraA and combining biochemical studies using a synthetic nanobody as conformational probe with hydrogen-deuterium exchange mass spectrometry, we demonstrate that interdomain-linkers control the function of transport proteins by influencing substrate affinity and transport rate. These findings open the possibility to allosterically modulate the activity of elevator proteins by targeting their linkers.

Benjamin M Stormo, Grace A McLaughlin, Ameya P Jalihal, Logan K Frederick, Sierra J Cole, Ian Seim, Fred S Dietrich, Ashutosh Chilkoti, Amy Gladfelter
Intrinsically disordered sequences can tune fungal growth and the cell cycle for specific temperatures.
Curr Biol, 34(16) 3722-3734 (2024)
DOI

Temperature can impact every reaction essential to a cell. For organisms that cannot regulate their own temperature, adapting to temperatures that fluctuate unpredictably and on variable timescales is a major challenge. Extremes in the magnitude and frequency of temperature changes are increasing across the planet, raising questions as to how the biosphere will respond. To examine mechanisms of adaptation to temperature, we collected wild isolates from different climates of the fungus Ashbya gossypii, which has a compact genome of only ∼4,600 genes. We found control of the nuclear division cycle and polarized morphogenesis, both critical processes for fungal growth, were temperature sensitive and varied among the isolates. The phenotypes were associated with naturally varying sequences within the glutamine-rich region (QRR) IDR of an RNA-binding protein called Whi3. This protein regulates both nuclear division and polarized growth via its ability to form biomolecular condensates. In cells and in cell-free reconstitution assays, we found that temperature tunes the properties of Whi3-based condensates. Exchanging Whi3 sequences between isolates was sufficient to rescue temperature-sensitive phenotypes, and specifically, a heptad repeat sequence within the QRR confers temperature-sensitive behavior. Together, these data demonstrate that sequence variation in the size and composition of an IDR can promote cell adaptation to growth at specific temperature ranges. These data demonstrate the power of IDRs as tuning knobs for rapid adaptation to environmental fluctuations.

Robert G. Parton^#, Kai Simons^#
The Biology of Lipids.
Cold Spring Harb Perspect Biol, 16(8) Art. No. a041713 (2024)
DOI

Lipids are the defining features of cellular membranes. They act collectively to form a variety of different structures, and understanding their complex behavior represents an early example of systems biology. A multidisciplinary approach is needed to analyse the functions of lipids in biological systems, and new work is providing fascinating insights into their roles in membrane biology, metabolism, signaling, subcellular dynamics and various disease processes.

Leila Haj Abdullah Alieh^*, Beatriz C de Toledo^*, Anna Hadarovich, Agnes Toth-Petroczy, Federico Calegari
Characterization of alternative splicing during mammalian brain development reveals the extent of isoform diversity and potential effects on protein structural changes.
Biol Open, 13(10) Art. No. bio061721 (2024)
Open Access DOI

Regulation of gene expression is critical for fate commitment of stem and progenitor cells during tissue formation. In the context of mammalian brain development, a plethora of studies have described how changes in the expression of individual genes characterize cell types across ontogeny and phylogeny. However, little attention has been paid to the fact that different transcripts can arise from any given gene through alternative splicing (AS). Considered a key mechanism expanding transcriptome diversity during evolution, assessing the full potential of AS on isoform diversity and protein function has been notoriously difficult. Here, we capitalize on the use of a validated reporter mouse line to isolate neural stem cells, neurogenic progenitors and neurons during corticogenesis and combine the use of short- and long-read sequencing to reconstruct the full transcriptome diversity characterizing neurogenic commitment. Extending available transcriptional profiles of the mammalian brain by nearly 50,000 new isoforms, we found that neurogenic commitment is characterized by a progressive increase in exon inclusion resulting in the profound remodeling of the transcriptional profile of specific cortical cell types. Most importantly, we computationally infer the biological significance of AS on protein structure by using AlphaFold2, revealing how radical protein conformational changes can arise from subtle changes in isoforms sequence. Together, our study reveals that AS has a greater potential to impact protein diversity and function than previously thought, independently from changes in gene expression.

Julia Flock^*, Yexin Xie^*, Regis P. Lemaitre, Karine Lapouge, Kim Remans
The Use of Baculovirus-Mediated Gene Expression in Mammalian Cells for Recombinant Protein Production.
Methods Mol Biol, 2810 29-53 (2024)
DOI

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.

Rasmus K Norrild, Thomas O Mason, Lars Boyens-Thiele, Soumik Ray, Joachim B Mortensen, Anatol Fritsch, Juan M Iglesias-Artola, Louise K Klausen, Emil G P Stender, Henrik Jensen, Alexander K Buell
Taylor Dispersion-Induced Phase Separation for the Efficient Characterisation of Protein Condensate Formation.
Angew Chem Int Ed Engl, 63(25) Art. No. e202404018 (2024)
Open Access DOI

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid-liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion-induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL-3 and Ddx4. Uniquely accessible to this method, we find an unexpected re-entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α-synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS-modifying compounds can be screened highly efficiently.

Alexandra M Garfinkel^*, Efe Ilker^*, Hidenobu Miyazawa^*, Kathrin Schmeisser^*, Jason M Tennessen^*
Historic obstacles and emerging opportunities in the field of developmental metabolism - lessons from Heidelberg.
Development, 151(12) Art. No. dev202937 (2024)
DOI

The field of developmental metabolism is experiencing a technological revolution that is opening entirely new fields of inquiry. Advances in metabolomics, small-molecule sensors, single-cell RNA sequencing and computational modeling present new opportunities for exploring cell-specific and tissue-specific metabolic networks, interorgan metabolic communication, and gene-by-metabolite interactions in time and space. Together, these advances not only present a means by which developmental biologists can tackle questions that have challenged the field for centuries, but also present young scientists with opportunities to define new areas of inquiry. These emerging frontiers of developmental metabolism were at the center of a highly interactive 2023 EMBO workshop 'Developmental metabolism: flows of energy, matter, and information'. Here, we summarize key discussions from this forum, emphasizing modern developmental biology's challenges and opportunities.

Andy Göbel, Sophie Pählig, Anja Motz, Dorit Breining, Sofia Traikov, Lorenz C Hofbauer, Tilman D Rachner
Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase.
Biochem Biophys Res Commun, 710 Art. No. 149841 (2024)
DOI

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4-2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to - 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4-2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4-2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4-2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

Naresh Yandrapalli
Complex Emulsions as an Innovative Pharmaceutical Dosage form in Addressing the Issues of Multi-Drug Therapy and Polypharmacy Challenges.
Pharmaceutics, 16(6) Art. No. 707 (2024)
Open Access DOI

This review explores the intersection of microfluidic technology and complex emulsion development as a promising solution to the challenges of formulations in multi-drug therapy (MDT) and polypharmacy. The convergence of microfluidic technology and complex emulsion fabrication could herald a transformative era in multi-drug delivery systems, directly confronting the prevalent challenges of polypharmacy. Microfluidics, with its unparalleled precision in droplet formation, empowers the encapsulation of multiple drugs within singular emulsion particles. The ability to engineer emulsions with tailored properties-such as size, composition, and release kinetics-enables the creation of highly efficient drug delivery vehicles. Thus, this innovative approach not only simplifies medication regimens by significantly reducing the number of necessary doses but also minimizes the pill burden and associated treatment termination-issues associated with polypharmacy. It is important to bring forth the opportunities and challenges of this synergy between microfluidic-driven complex emulsions and multi-drug therapy poses. Together, they not only offer a sophisticated method for addressing the intricacies of delivering multiple drugs but also align with broader healthcare objectives of enhancing treatment outcomes, patient safety, and quality of life, underscoring the importance of dosage form innovations in tackling the multifaceted challenges of modern pharmacotherapy.

Jan Tiemann^#, Matthew McGinity, Ivo F. Sbalzarini, Ulrik Günther^#
Live and Interactive 3D Photomanipulation under the Microscope using Virtual Reality.
In: CHI'24 : extended abstracts of the 2024 CHI Conference on Human Factors in Computing Systems (2024) Ch. 228(Eds.) Florian Mueller, New York, ACM (2024)
Open Access PDF DOI

State-of-the-art microscopes, as used in cell biology, are not only capable of capturing 3D images, but also permit manipulation of (sub-)cellular structures using techniques such as optical traps, optogenetics or laser ablation. However, such microscopes are still controlled using 2D interfaces, prohibiting actual 3-dimensional manipulation. We present microscenery, a virtual reality (VR) microscope control software, designed to facilitate 3D laser ablation experiments. We combine microscopy automation with VR rendering and intuitive controller-based input to empower biologists with the precision of laser-based techniques while providing the full 3D spatial context of their sample. We describe the design goals and architecture of the software and illustrate the potential of the system by conducting a brief expert review study for 3D ablation experiments. Our results suggest VR is not only an effective interface for microscopic manipulations, but can enable novel experiments which are either impossible with traditional 2D interfaces, or prohibitively time-consuming.

Buqing Yi, Eva Patrasová, Lenka Šimůnková, Fabian Rost, Sylke Winkler, Alexa Laubner, Susanne Reinhardt, Andreas Dahl, Alexander Dalpke
Investigating the cause of a 2021 winter wave of COVID-19 in a border region in eastern Germany: a mixed-methods study, August to November 2021.
Epidemiol Infect, 152 Art. No. e87 (2024)
Open Access DOI

It is so far unclear how the COVID-19 winter waves started and what should be done to prevent possible future waves. In this study, we deciphered the dynamic course of a winter wave in 2021 in Saxony, a state in Eastern Germany neighbouring the Czech Republic and Poland. The study was carried out through the integration of multiple virus genomic epidemiology approaches to track transmission chains, identify emerging variants and investigate dynamic changes in transmission clusters. For identified local variants of interest, functional evaluations were performed. Multiple long-lasting community transmission clusters have been identified acting as driving force for the winter wave 2021. Analysis of the dynamic courses of two representative clusters indicated a similar transmission pattern. However, the transmission cluster caused by a locally occurring new Delta variant AY.36.1 showed a distinct transmission pattern, and functional analyses revealed a replication advantage of it. This study indicated that long-lasting community transmission clusters starting since early autumn caused by imported or locally occurring variants all contributed to the development of the 2021 winter wave. The information we achieved might help future pandemic prevention.

Christine Desroches Altamirano, Moo-Koo Kang, Mareike A Jordan, Tom Borianne, Irem Dilmen, Maren Gnädig, Alexander von Appen, Alf Honigmann, Titus Franzmann, Simon Alberti
eIF4F is a thermo-sensing regulatory node in the translational heat shock response.
Mol Cell, 84(9) 1727-1741 (2024)
Open Access DOI

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Bethan Clark, Aaron Hickey, Aleksandra Marconi, Bettina Fischer, Joel Elkin, Rita Mateus, M Emília Santos
Developmental plasticity and variability in the formation of egg-spots, a pigmentation ornament in the cichlid Astatotilapia calliptera.
Evol Dev, 26(3) Art. No. e12475 (2024)
Open Access DOI

Vertebrate pigmentation patterns are highly diverse, yet we have a limited understanding of how evolutionary changes to genetic, cellular, and developmental mechanisms generate variation. To address this, we examine the formation of a sexually-selected male ornament exhibiting inter- and intraspecific variation, the egg-spot pattern, consisting of circular yellow-orange markings on the male anal fins of haplochromine cichlid fishes. We focus on Astatotilapia calliptera, the ancestor-type species of the Malawi cichlid adaptive radiation of over 850 species. We identify a key role for iridophores in initializing egg-spot aggregations composed of iridophore-xanthophore associations. Despite adult sexual dimorphism, aggregations initially form in both males and females, with development only diverging between the sexes at later stages. Unexpectedly, we found that the timing of egg-spot initialization is plastic. The earlier individuals are socially isolated, the earlier the aggregations form, with iridophores being the cell type that responds to changes to the social environment. Furthermore, we observe apparent competitive interactions between adjacent egg-spot aggregations, which strongly suggests that egg-spot patterning results mostly from cell-autonomous cellular interactions. Together, these results demonstrate that A. calliptera egg-spot development is an exciting model for investigating pigment pattern formation at the cellular level in a system with developmental plasticity, sexual dimorphism, and intraspecific variation. As A. calliptera represents the ancestral bauplan for egg-spots, these findings provide a baseline for informed comparisons across the incredibly diverse Malawi cichlid radiation.

Cara F Webster, Michael Smotherman, Martin Pippel, Thomas Brown, Sylke Winkler, Myrtani Pieri, Meike Mai, Eugene W Myers, Emma Teeling, Sonja Vernes, Bat1K Consortium
The genome sequence of Tadarida brasiliensis I. Geoffroy Saint-Hilaire, 1824 [Molossidae; Tadarida].
Wellcome Open Res, 9 Art. No. 98 (2024)
Open Access DOI

We present a genome assembly from an individual male Tadarida brasiliensis (The Brazilian free-tailed bat; Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.28 Gb in span. The majority of the assembly is scaffolded into 25 chromosomal pseudomolecules, with the X and Y sex chromosomes assembled.

Lewis Marsh, Felix Y Zhou, Xiau Qin, Xin Lu, Helen M Byrne, Heather A Harrington
Detecting Temporal shape changes with the Euler Characteristic Transform.
Transactions of Mathematics and its Applications , 8 Art. No. tnae002 (2024)
Open Access DOI

Organoids are multi-cellular structures that are cultured in vitro from stem cells to resemble specific organs (e.g., brain, liver) in their three-dimensional composition. Dynamic changes in the shape and composition of these model systems can be used to understand the effect of mutations and treatments in health and disease. In this paper, we propose a new technique in the field of topological data analysis for DEtecting Temporal shape changes with the Euler Characteristic Transform (DETECT). DETECT is a rotationally invariant signature of dynamically changing shapes. We demonstrate our method on a data set of segmented videos of mouse small intestine organoid experiments and show that it outperforms classical shape descriptors. We verify our method on a synthetic organoid data set and illustrate how it generalizes to 3D. We conclude that DETECT offers rigorous quantification of organoids and opens up computationally scalable methods for distinguishing different growth regimes and assessing treatment effects.

Mukesh Kumar, Canan Has, Khanh Lam-Kamath, Sophie Ayciriex, Deepshe Dewett, Mina Bashir, Clara Poupault, Kai Schuhmann, Henrik Thomas, Oskar Knittelfelder, Bharath Kumar Raghuraman, R Ahrends, Jens Rister^#, Andrej Shevchenko^#
Lipidome Unsaturation Affects the Morphology and Proteome of the Drosophila Eye.
J Proteome Res, 23(4) 1188-1199 (2024)
Open Access DOI

Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster, which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food. We developed a series of semisynthetic foods to manipulate the length and unsaturation of fatty acid moieties in GPLs and singled out proteins whose abundance is specifically affected by membrane lipid unsaturation in the Drosophila eye. Unexpectedly, we identified a group of proteins that have muscle-related functions and increased their abundances under unsaturated eye lipidome conditions. In contrast, the abundance of two stress response proteins, Turandot A and Smg5, is decreased by lipid unsaturation. Our findings could guide the genetic dissection of homeostatic mechanisms that maintain visual function when the eye is exposed to environmental and dietary challenges.

Laura Meißner, Lukas Niese, Irene Schüring, Aniruddha Mitra, Stefan Diez
Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.
EMBO J, 43(7) 1244-1256 (2024)
Open Access DOI

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

Ferenc Molnár, Szabolcs Horvát, Ana R. Ribeiro Gomes, Jorge Martinez Armas, Botond Molnár, Robert F Hevner, Kenneth Knoblauch, Henry Kennedy, Zoltan Toroczkai
Predictability of cortico-cortical connections in the mammalian brain.
Network Neuroscience, 8(1) 138-157 (2024)
Open Access DOI

Despite a five order of magnitude range in size, the brains of mammals share many anatomical and functional characteristics that translate into cortical network commonalities. Here we develop a machine learning framework to quantify the degree of predictability of the weighted interareal cortical matrix. Partial network connectivity data were obtained with retrograde tract-tracing experiments generated with a consistent methodology, supplemented by projection length measurements in a nonhuman primate (macaque) and a rodent (mouse). We show that there is a significant level of predictability embedded in the interareal cortical networks of both species. At the binary level, links are predictable with an area under the ROC curve of at least 0.8 for the macaque. Weighted medium and strong links are predictable with an 85%-90% accuracy (mouse) and 70%-80% (macaque), whereas weak links are not predictable in either species. These observations reinforce earlier observations that the formation and evolution of the cortical network at the mesoscale is, to a large extent, rule based. Using the methodology presented here, we performed imputations on all area pairs, generating samples for the complete interareal network in both species. These are necessary for comparative studies of the connectome with minimal bias, both within and across species. Revealed by tract-tracing datasets, communication between the functional areas of the cortex operates via a complex, dense, and weighted network of physical connections with little apparent regularity. Although there are studies showing the existence of nonrandom topological features, their extent has not been clear. Employing a machine learning-based approach, which efficiently extracts structural models from such datasets, here we show that there is a significant amount of regularity embedded in the mammalian connectome. This regularity allows predicting interareal connections and their weights with good accuracy and can be used to infer properties of experimentally untested connections. The structural models are well learned even with small training sets, without overfitting, suggesting the existence of a low-dimensional, universal mechanism for mesoscale cortical network formation and evolution.

Paula Cubillos, Nora Ditzer, Annika Kolodziejczyk, Gustav Schwenk, Janine Hoffmann, Theresa M Schütze, Razvan Derihaci, Cahit Birdir, Johannes Em Köllner, Andreas Petzold, Mihail Sarov, Ulrich Martin, Katherine S. Long, Pauline Wimberger, Mareike Albert
The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex.
EMBO J, 43(8) 1388-1419 (2024)
Open Access DOI

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Landi Sun^*^#, Jana Meissner^*, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang^#
Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.
Bio Protoc, 14(4) Art. No. e4940 (2024)
Open Access DOI

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Colette Dehay^#, Wieland Huttner^#
Development and evolution of the primate neocortex from a progenitor cell perspective.
Development, 151(4) Art. No. dev199797 (2024)
DOI

The generation of neurons in the developing neocortex is a major determinant of neocortex size. Crucially, the increase in cortical neuron numbers in the primate lineage, notably in the upper-layer neurons, contributes to increased cognitive abilities. Here, we review major evolutionary changes affecting the apical progenitors in the ventricular zone and focus on the key germinal zone constituting the foundation of neocortical neurogenesis in primates, the outer subventricular zone (OSVZ). We summarize characteristic features of the OSVZ and its key stem cell type, the basal (or outer) radial glia. Next, we concentrate on primate-specific and human-specific genes, expressed in OSVZ-progenitors, the ability of which to amplify these progenitors by targeting the regulation of the cell cycle ultimately underlies the evolutionary increase in upper-layer neurons. Finally, we address likely differences in neocortical development between present-day humans and Neanderthals that are based on human-specific amino acid substitutions in proteins operating in cortical progenitors.

Nikolai P Jaschke, Dorit Breining, Maura Hofmann, Sophie Pählig, Ulrike Baschant, Reinhard Oertel, Sofia Traikov, Tatyana Grinenko, Francesco Saettini, Andrea Biondi, Myrto Stylianou, Henrik Bringmann, Cuiling Zhang, Tomomi M Yoshida, Heike Weidner, Wolfram C Poller, Filip K Swirski, Andy Göbel, Lorenz C Hofbauer, Martina Rauner, Christoph Scheiermann, Andrew Wang, Tilman D Rachner
Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.
Immunity, 57(2) 364-378 (2024)
Open Access DOI

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Maresca Marcello, Davide Accardi, Sebastian T. Bundschuh, Jeffrey Oegema, Andrey Andreev, Emmanuel G. Reynaud, Jan Peychl
Light-Sheet Microscopy Technology in the Multiuser Environment of a Core Imaging Facility – Practical Considerations.
In: Light Sheet Fluorescence Microscopy. (Eds.) Emmanuel G. Reynaud, Weinheim, Wiley-VCH (2024), 365-381 Ch. 12
DOI

New imaging technologies are always a challenge when they enter core imaging facilities or become a shared system that accommodates several users, projects, and demands. Not only they need to be set up and run smoothly in this multiuser environment (that requires staff training, maintenance, and scheduling), but they are needed either for short time imaging sessions on fixed samples, or sometimes blocked for several days for a long-term life imaging of developmental events, while delivering large amount of data. To help other imaging centers to establish efficient imaging process and smooth user experience, here we present the experience from two different imaging facilities that have used light sheet microscopes even before the first commercial systems became available. The first imaging platform has implemented three commercial light-sheet systems (2 × Lightsheet Z.1, Zeiss and 1 × LS1, Viventis) in the multiuser environment of the core light microscopy facility of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden (Germany), which serves about 220 users per year, and it has an average volume of annual booked instrument time of 40 000 hours. The second is the Centre for Cell Imaging at Liverpool University (United Kingdom), part of the Liverpool Shared Research Facilities network, which serves about 130 users per year and has an average volume of annual booked instrument time of about 24 000 hours. The CCI was the first open access core facility in United Kingdom to set up a Zeiss Lightsheet Z.1 microscope. Here we share some practical tips for those who plan to implement this technology in their institutes.

Kathrin Schmeisser^#, Damla Kaptan, Bharath Kumar Raghuraman, Andrej Shevchenko, Jonathan Rodenfels, Sider Penkov, Teymuras V. Kurzchalia^#
Mobilization of cholesterol induces the transition from quiescence to growth in Caenorhabditis elegans through steroid hormone and mTOR signaling.
Commun Biol, 7(1) Art. No. 121 (2024)
Open Access DOI

Recovery from the quiescent developmental stage called dauer is an essential process in C. elegans and provides an excellent model to understand how metabolic transitions contribute to developmental plasticity. Here we show that cholesterol bound to the small secreted proteins SCL-12 or SCL-13 is sequestered in the gut lumen during the dauer state. Upon recovery from dauer, bound cholesterol undergoes endocytosis into lysosomes of intestinal cells, where SCL-12 and SCL-13 are degraded and cholesterol is released. Free cholesterol activates mTORC1 and is used for the production of dafachronic acids. This leads to promotion of protein synthesis and growth, and a metabolic switch at the transcriptional level. Thus, mobilization of sequestered cholesterol stores is the key event for transition from quiescence to growth, and cholesterol is the major signaling molecule in this process.

Priyanka Bhatia, Marc Bickle, Amay A Agrawal, Buster Truss, Aikaterina Nikolaidi, Kathrin Brockmann, Lydia Reinhardt, Stefanie Vogel, Eva M Szegoe, Arun Pal, Andreas Hermann, Ivan Mikicic, Maximina H Yun, Björn H Falkenburger, Jared Sterneckert
Axonal Lysosomal Assays for Characterizing the Effects of LRRK2 G2019S.
Biology (Basel), 13(1) Art. No. 58 (2024)
Open Access DOI

The degeneration of axon terminals before the soma, referred to as "dying back", is a feature of Parkinson's disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to "dying back" pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics.

Benjamin Seelbinder, Susan Wagner, Manavi Jain, Elena Erben, Sergei Klykov, Iliya D. Stoev, Venkat Raghavan Krishnaswamy, Moritz Kreysing
Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.
Elife, 13 Art. No. e76421 (2024)
Open Access DOI

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Andrea I Luppi^*, Manesh Girn^*, Fernando E Rosas, Christopher Timmermann, Leor Roseman, David Erritzoe, David J Nutt, Emmanuel A Stamatakis, R Nathan Spreng, Lei Xing, Wieland Huttner, Robin L Carhart-Harris
A role for the serotonin 2A receptor in the expansion and functioning of human transmodal cortex.
Brain, 147(1) 56-80 (2024)
DOI

Integrating independent but converging lines of research on brain function and neurodevelopment across scales, this article proposes that serotonin 2A receptor (5-HT2AR) signalling is an evolutionary and developmental driver and potent modulator of the macroscale functional organization of the human cerebral cortex. A wealth of evidence indicates that the anatomical and functional organization of the cortex follows a unimodal-to-transmodal gradient. Situated at the apex of this processing hierarchy-where it plays a central role in the integrative processes underpinning complex, human-defining cognition-the transmodal cortex has disproportionately expanded across human development and evolution. Notably, the adult human transmodal cortex is especially rich in 5-HT2AR expression and recent evidence suggests that, during early brain development, 5-HT2AR signalling on neural progenitor cells stimulates their proliferation-a critical process for evolutionarily-relevant cortical expansion. Drawing on multimodal neuroimaging and cross-species investigations, we argue that, by contributing to the expansion of the human cortex and being prevalent at the apex of its hierarchy in the adult brain, 5-HT2AR signalling plays a major role in both human cortical expansion and functioning. Owing to its unique excitatory and downstream cellular effects, neuronal 5-HT2AR agonism promotes neuroplasticity, learning and cognitive and psychological flexibility in a context-(hyper)sensitive manner with therapeutic potential. Overall, we delineate a dual role of 5-HT2ARs in enabling both the expansion and modulation of the human transmodal cortex.

Marek Uvizl, Sébastien J Puechmaille, Sarahjane Power, Martin Pippel, Samuel Carthy, Wilfried Haerty, Eugene W Myers, Emma Teeling, Zixia Huang
Comparative Genome Microsynteny Illuminates the Fast Evolution of Nuclear Mitochondrial Segments (NUMTs) in Mammals.
Mol Biol Evol, 41(1) Art. No. msad278 (2024)
Open Access DOI

The escape of DNA from mitochondria into the nuclear genome (nuclear mitochondrial DNA, NUMT) is an ongoing process. Although pervasively observed in eukaryotic genomes, their evolutionary trajectories in a mammal-wide context are poorly understood. The main challenge lies in the orthology assignment of NUMTs across species due to their fast evolution and chromosomal rearrangements over the past 200 million years. To address this issue, we systematically investigated the characteristics of NUMT insertions in 45 mammalian genomes and established a novel, synteny-based method to accurately predict orthologous NUMTs and ascertain their evolution across mammals. With a series of comparative analyses across taxa, we revealed that NUMTs may originate from nonrandom regions in mtDNA, are likely found in transposon-rich and intergenic regions, and unlikely code for functional proteins. Using our synteny-based approach, we leveraged 630 pairwise comparisons of genome-wide microsynteny and predicted the NUMT orthology relationships across 36 mammals. With the phylogenetic patterns of NUMT presence-and-absence across taxa, we constructed the ancestral state of NUMTs given the mammal tree using a coalescent method. We found support on the ancestral node of Fereuungulata within Laurasiatheria, whose subordinal relationships are still controversial. This study broadens our knowledge on NUMT insertion and evolution in mammalian genomes and highlights the merit of NUMTs as alternative genetic markers in phylogenetic inference.

Romina Piscitello-Gómez^*, Franz Gruber^*, Abhijeet Krishna, Charlie Duclut, Carl D. Modes, Marko Popović, Frank Jülicher, Natalie Dye, Suzanne Eaton
Core PCP mutations affect short-time mechanical properties but not tissue morphogenesis in the Drosophila pupal wing.
Elife, 12 Art. No. e85581 (2023)
Open Access DOI

How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing of Drosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. Previously, we showed that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned (Etournay et al., 2015). Here, we show that these active cellular behaviors are not guided by the core planar cell polarity (PCP) pathway, a conserved signaling system that guides tissue development in many other contexts. We find no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation they do not significantly affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes during Drosophila pupal wing morphogenesis do not require core PCP as an orientational guiding cue.

Abhinav Singh, Ivo F. Sbalzarini, Anas Obeidat
Entropically damped artificial compressibility for the discretization corrected particle strength exchange method in incompressible fluid mechanics.
Computers & Fluids, 267 Art. No. 106074 (2023)
PDF DOI

We present a consistent mesh-free numerical scheme for solving the incompressible Navier–Stokes equations. Our method is based on entropically damped artificial compressibility for imposing the incompressibility constraint explicitly, and the Discretization-Corrected Particle Strength Exchange (DC-PSE) method to consistently discretize the differential operators on mesh-free particles. We further couple our scheme with Brinkman penalization to solve the Navier–Stokes equations in complex geometries. The method is validated using the 3D Taylor–Green vortex flow and the lid-driven cavity flow problem in 2D and 3D, where we also compare our method with hr-SPH and report better accuracy for DC-PSE. In order to validate DC-PSE Brinkman penalization, we study flow past obstacles, such as a cylinder, and report excellent agreement with previous studies.

Andrew C Marshall, Jerry Cummins, Simon Kobelke, Tianyi Zhu, Jocelyn Widagdo, Victor Anggono, Anthony Hyman, Archa H Fox^#, Charles S Bond^#, Mihwa Lee^#
Different Low-complexity Regions of SFPQ Play Distinct Roles in the Formation of Biomolecular Condensates.
J Mol Biol, 435(24) Art. No. 168364 (2023)
Open Access DOI

Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, we find that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Our data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.

Dennis Schifferl, Manuela Scholze-Wittler, Alba Villaronga Luque, Milena Pustet, Lars Wittler, Jesse V Veenvliet, Frederic Koch^#, Bernhard G Herrmann^#
Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.
Development, 150(22) Art. No. dev202111 (2023)
Open Access DOI

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Zheng Shen, Daxiao Sun, Adriana Savastano, Sára Joana Varga, Maria-Sol Cima-Omori, Stefan Becker, Alf Honigmann, Markus Zweckstetter
Multivalent Tau/PSD-95 interactions arrest in vitro condensates and clusters mimicking the postsynaptic density.
Nat Commun, 14(1) Art. No. 6839 (2023)
Open Access DOI

Alzheimer's disease begins with mild memory loss and slowly destroys memory and thinking. Cognitive impairment in Alzheimer's disease has been associated with the localization of the microtubule-associated protein Tau at the postsynapse. However, the correlation between Tau at the postsynapse and synaptic dysfunction remains unclear. Here, we show that Tau arrests liquid-like droplets formed by the four postsynaptic density proteins PSD-95, GKAP, Shank, Homer in solution, as well as NMDA (N-methyl-D-aspartate)-receptor-associated protein clusters on synthetic membranes. Tau-mediated condensate/cluster arrest critically depends on the binding of multiple interaction motifs of Tau to a canonical GMP-binding pocket in the guanylate kinase domain of PSD-95. We further reveal that competitive binding of a high-affinity phosphorylated peptide to PSD-95 rescues the diffusional dynamics of an NMDA truncated construct, which contains the last five amino acids of the NMDA receptor subunit NR2B fused to the C-terminus of the tetrameric GCN4 coiled-coil domain, in postsynaptic density-like condensates/clusters. Taken together, our findings propose a molecular mechanism where Tau modulates the dynamic properties of the postsynaptic density.

Arthur Hernandez, Michael F Staddon, Michael Moshe, M Cristina Marchetti
Finite elasticity of the vertex model and its role in rigidity of curved cellular tissues.
Soft Matter, 19(40) 7744-7752 (2023)
DOI

Using a mean field approach and simulations, we study the non-linear mechanical response of the vertex model (VM) of biological tissue to compression and dilation. The VM is known to exhibit a transition between solid and fluid-like, or floppy, states driven by geometric incompatibility. Target perimeter and area set a target shape which may not be geometrically achievable, thereby engendering frustration. Previously, an asymmetry in the linear elastic response was identified at the rigidity transition between compression and dilation. Here we show that the asymmetry extends away from the transition point for finite strains. Under finite compression, an initially solid VM can completely relax perimeter tension, resulting in a drop discontinuity in the mechanical response. Conversely, an initially floppy VM under dilation can rigidify and have a higher response. These observations imply that re-scaling of cell area shifts the transition between rigid and floppy states. Based on this insight, we calculate the re-scaling of cell area engendered by intrinsic curvature and write a prediction for the rigidity transition in the presence of curvature. The shift of the rigidity transition in the presence of curvature for the VM provides a new metric for predicting tissue rigidity from image data of curved tissues in a manner analogous to the flat case.

Fang-Chu Lin, Heidi L van de Wouw, Otger Campàs, Ellen M Sletten, Jeffrey I Zink
Synthesis of Fluorous Ferrofluids and Effects of the Nanoparticle Coatings on Field- and Temperature-Dependent Magnetizations.
Chem Mater, 35(19) 7957-7966 (2023)
Open Access DOI

Ferrofluids have been extensively employed in industrial, environmental, and biomedical areas. Among them, fluorous ferrofluids are of particular interest because of the biorthogonal nature of perfluorocarbons (PFCs). However, the noninteracting nature of PFCs as well as challenges in functionalization of nanoparticle surfaces with fluorous ligands has limited their applications, especially in biomedicine. In particular, commercially available fluorous ferrofluids are stabilized using ionic surfactants with charged groups that physically interact with a wide range of charged biological molecules. In this paper, we developed a unique two-phase ligand attachment strategy to render stable fluorous ferrofluids using nonionic surfactants. The superparamagnetic Fe3O4 or MnFe2O4 core of the magnetic nanoparticles, the magnetic component of the ferrofluid, was coated with a silica shell containing abundant surface hydroxyl groups, thereby enabling the installation of fluorous ligands through stable covalent, neutral, siloxane bonds. We explored chemistry-material relationships between different ligands and PFC solvents and found that low-molecular-weight ligands can assist with the installation of high-molecular-weight ligands (4000-8000 g/mol), allowing us to systematically control the size and thickness of ligand functionalization on the nanoparticle surface. By zero-field-cooled magnetization measurements, we studied how the ligands affect magnetic dipole orientation forces and observed a curve flattening that is only associated with the ferrofluids. This work provided insight into ferrofluids' dependence on interparticle interactions and contributed a methodology to synthesize fluorous ferrofluids with nonionic surfactants that exhibit both magnetic and chemical stability. We believe that the doped MnFe2O4 fluorous ferrofluid has the highest combination of stability and magnetization reported to date.

Tenneille E Ludwig^*, Peter W Andrews^*, Ivana Barbaric, Nissim Benvenisty, Anita Bhattacharyya, Jeremy M Crook, Laurence M Daheron, Jonathan S Draper, Lyn Healy, Meritxell Huch, Mandar M Inamdar, Kim B Jensen, Andreas Kurtz, Madeline Lancaster, Prisca Liberali, Matthias Lutolf, Christine L Mummery, Martin F Pera, Yoji Sato, Noriko Shimasaki, Austin Smith, Jihwan Song, Claudia Spits, Glyn Stacey, Christine A Wells, Tongbiao Zhao, Jack T Mosher
ISSCR standards for the use of human stem cells in basic research.
Stem Cell Rep, 18(9) 1744-1752 (2023)
Open Access DOI

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Yi-Heng Tai, Daniel Engels, Giuseppe Locatelli, Ioanna Emmanouilidis, Caroline Fecher, Delphine Theodorou, Stephan A Müller, Simon Licht-Mayer, Mario Kreutzfeldt, Ingrid Wagner, Natalia Prudente de Mello, Sofia-Natsouko Gkotzamani, Laura Trovò, Arek Kendirli, Almir Aljović, Michael O Breckwoldt, Ronald Naumann, Florence M Bareyre, Fabiana Perocchi, Don Mahad, Doron Merkler, Stefan F Lichtenthaler, Martin Kerschensteiner^#, Thomas Misgeld^#
Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions.
Nat Metab, 5(8) 1364-1381 (2023)
Open Access DOI

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.

Anastasia Shatilovich^*, Vamshidhar Gade^*, Martin Pippel, Tarja T Hoffmeyer, Alexei V Tchesunov, Lewis Stevens, Sylke Winkler, Graham M Hughes, Sofia Traikov, Michael Hiller, Elisaveta Rivkina, Philipp H Schiffer^#, Eugene W Myers, Teymuras V. Kurzchalia^#
A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva.
PLoS Genet, 19(7) Art. No. e1010798 (2023)
Open Access DOI

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

Dhawal Choudhary, Laura Mediani, Mario Avellaneda, Sveinn Bjarnason, Simon Alberti, Edgar Boczek, Pétur O Heidarsson, Alessandro Mossa^#, Serena Carra^#, Sander J Tans^#, Ciro Cecconi^#
Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.
J Am Chem Soc, 145(28) 15188-15196 (2023)
Open Access DOI

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

Veikko Geyer^#, Stefan Diez^#
Horizontal Magnetic Tweezers to Directly Measure the Force-Velocity Relationship for Multiple Kinesin Motors.
Small, 19(30) Art. No. e2300558 (2023)
Open Access DOI

Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8 µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities which we attribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.

Kristian Le Vay^*^#, Elia Salibi^*, Basusree Ghosh, T Y Dora Tang^#, Hannes Mutschler^#
Ribozyme activity modulates the physical properties of RNA-peptide coacervates.
Elife, 12 Art. No. e83543 (2023)
Open Access DOI

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

Aparajita Lahree, João Mello-Vieira, Maria M Mota
The nutrient games - Plasmodium metabolism during hepatic development.
Trends Parasitol, 39(6) 445-460 (2023)
Open Access DOI

Malaria is a febrile illness caused by species of the protozoan parasite Plasmodium and is characterized by recursive infections of erythrocytes, leading to clinical symptoms and pathology. In mammals, Plasmodium parasites undergo a compulsory intrahepatic development stage before infecting erythrocytes. Liver-stage parasites have a metabolic configuration to facilitate the replication of several thousand daughter parasites. Their metabolism is of interest to identify cellular pathways essential for liver infection, to kill the parasite before onset of the disease. In this review, we summarize the current knowledge on nutrient acquisition and biosynthesis by liver-stage parasites mostly generated in murine malaria models, gaps in knowledge, and challenges to create a holistic view of the development and deficiencies in this field.

Jan Simon Schuhmacher, Susanne Tom Dieck, Savvas Christoforidis, Cedric Landerer, Jimena Davila Gallesio, Lena Hersemann, Sarah Seifert, Ramona Schäfer, Angelika Giner, Agnes Toth-Petroczy, Yannis Kalaidzidis, Katherine E Bohnsack, Markus T Bohnsack, Erin M Schuman, Marino Zerial
The Rab5 effector FERRY links early endosomes with mRNA localization.
Mol Cell, 83(11) 1839-1855 (2023)
Open Access DOI

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

Meritxell Huch
Stem cell-derived organoid models: defying the Hayflick limit.
Nat Rev Genet, 24(6) Art. No. 348 (2023)
DOI

Dennis Quentin^*, Jan Simon Schuhmacher^*, Barbara Klink, Janelle Lauer, Tanvir R Shaikh, Pim J Huis In 't Veld, Luisa M Welp, Henning Urlaub, Marino Zerial^#, Stefan Raunser^#
Structural basis of mRNA binding by the human FERRY Rab5 effector complex.
Mol Cell, 83(11) 1856-1871 (2023)
Open Access DOI

The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.

Tzer Han Tan, Jifeng Liu, Anne Grapin-Botton
Mapping and exploring the organoid state space using synthetic biology.
Semin Cell Dev Biol, 141 23-32 (2023)
DOI

The functional relevance of an organoid is dependent on the differentiation, morphology, cell arrangement and biophysical properties, which collectively define the state of an organoid. For an organoid culture, an individual organoid or the cells that compose it, these state variables can be characterised, most easily by transcriptomics and by high-content image analysis. Their states can be compared to their in vivo counterparts. Current evidence suggests that organoids explore a wider state space than organs in vivo due to the lack of niche signalling and the variability of boundary conditions in vitro. Using data-driven state inference and in silico modelling, phase diagrams can be constructed to systematically sort organoids along biochemical or biophysical axes. These phase diagrams allow us to identify control strategies to modulate organoid state. To do so, the biochemical and biophysical environment, as well as the cells that seed organoids, can be manipulated.

Jane C Stinchcombe, Yukako Asano, Christopher J G Kaufman, Kristin Böhlig, Christopher J Peddie, Lucy M Collinson, André Nadler, Gillian M Griffiths
Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse.
Science, 380(6647) 818-823 (2023)
DOI

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

Haruka Oda, Yuko Sato, Shigehiro A Kawashima, Yusuke Fujiwara, Máté Pálfy, Edlyn Wu, Nadine Vastenhouw, Motomu Kanai, Hiroshi Kimura
Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo.
Biol Open, 12(5) Art. No. bio059783 (2023)
Open Access DOI

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

Manuela Völkner, Felix Wagner, Thomas Kurth, Alex Sykes, Claudia Del Toro Runzer, Lynn J A Ebner, Cagri Kavak, Vasileia Ismini Alexaki, Peter Cimalla, Mirko Mehner, Edmund Koch, Mike Karl
Modeling inducible neuropathologies of the retina with differential phenotypes in organoids.
Front Cell Neurosci, 17 Art. No. 1106287 (2023)
Open Access DOI

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

Johannes Pahlke
A unifying mathematical definition enables the theoretical study of the algorithmic class of particle methods.
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2023)
Open Access

Mathematical definitions provide a precise, unambiguous way to formulate concepts. They also provide a common language between disciplines. Thus, they are the basis for a well-founded scientific discussion. In addition, mathematical definitions allow for deeper insights into the defined subject based on mathematical theorems that are incontrovertible under the given definition. Besides their value in mathematics, mathematical definitions are indispensable in other sciences like physics, chemistry, and computer science. In computer science, they help to derive the expected behavior of a computer program and provide guidance for the design and testing of software. Therefore, mathematical definitions can be used to design and implement advanced algorithms. One class of widely used algorithms in computer science is the class of particle-based algorithms, also known as particle methods. Particle methods can solve complex problems in various fields, such as fluid dynamics, plasma physics, or granular flows, using diverse simulation methods, including Discrete Element Methods (DEM), Molecular Dynamics (MD), Reproducing Kernel Particle Methods (RKPM), Particle Strength Exchange (PSE), and Smoothed Particle Hydrodynamics (SPH). Despite the increasing use of particle methods driven by improved computing performance, the relation between these algorithms remains formally unclear. In particular, particle methods lack a unifying mathematical definition and precisely defined terminology. This prevents the determination of whether an algorithm belongs to the class and what distinguishes the class. Here we present a rigorous mathematical definition for determining particle methods and demonstrate its importance by applying it to several canonical algorithms and those not previously recognized as particle methods. Furthermore, we base proofs of theorems about parallelizability and computational power on it and use it to develop scientific computing software. Our definition unified, for the first time, the so far loosely connected notion of particle methods. Thus, it marks the necessary starting point for a broad range of joint formal investigations and applications across fields.

Michael F Staddon, Arthur Hernandez^#, Mark J Bowick^#, Michael Moshe^#, M Cristina Marchetti^#
The role of non-affine deformations in the elastic behavior of the cellular vertex model.
Soft Matter, 19(17) 3080-3091 (2023)
Open Access DOI

The vertex model of epithelia describes the apical surface of a tissue as a tiling of polygonal cells, with a mechanical energy governed by deviations in cell shape from preferred, or target, area, A0, and perimeter, P0. The model exhibits a rigidity transition driven by geometric incompatibility as tuned by the target shape index, . For with p*(6) the perimeter of a regular hexagon of unit area, a cell can simultaneously attain both the preferred area and preferred perimeter. As a result, the tissue is in a mechanically soft compatible state, with zero shear and Young's moduli. For p0 < p*(6), it is geometrically impossible for any cell to realize the preferred area and perimeter simultaneously, and the tissue is in an incompatible rigid solid state. Using a mean-field approach, we present a complete analytical calculation of the linear elastic moduli of an ordered vertex model. We analyze a relaxation step that includes non-affine deformations, leading to a softer response than previously reported. The origin of the vanishing shear and Young's moduli in the compatible state is the presence of zero-energy deformations of cell shape. The bulk modulus exhibits a jump discontinuity at the transition and can be lower in the rigid state than in the fluid-like state. The Poisson's ratio can become negative which lowers the bulk and Young's moduli. Our work provides a unified treatment of linear elasticity for the vertex model and demonstrates that this linear response is protocol-dependent.

Mateusz Susik, Ivo F. Sbalzarini
Analysis of the Hamiltonian Monte Carlo genotyping algorithm on PROVEDIt mixtures including a novel precision benchmark.
Forensic Sci Int Genet, 64 Art. No. 102840 (2023)
Open Access PDF DOI

We provide an internal validation study of a recently published precise DNA mixture algorithm based on Hamiltonian Monte Carlo sampling (Susik et al., 2022). We provide results for all 428 mixtures analysed by Riman et al. (2021) and compare the results with two state-of-the-art software products: STRmix™ v2.6 and Euroformix v3.4.0. The comparison shows that the Hamiltonian Monte Carlo method provides reliable values of likelihood ratios (LRs) close to the other methods. We further propose a novel large-scale precision benchmark and quantify the precision of the Hamiltonian Monte Carlo method, indicating its improvements over existing solutions. Finally, we analyse the influence of the factors discussed by Buckleton et al. (2022).

Stanislav Vinopal^*, Sebastian Dupraz^*, Eissa Alfadil, Thorben Pietralla, Shweta Bendre, Michael Stiess, Sven Falk, Germán Camargo Ortega, Nicola Maghelli, Iva M Tolić, Jiří Smejkal, Magdalena Götz, Frank Bradke
Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain.
Neuron, 111(8) 1241-1263 (2023)
Open Access DOI

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.

Federica Luppino, Ivan Adzhubei, Christopher Cassa^#, Agnes Toth-Petroczy^#
DeMAG predicts the effects of variants in clinically actionable genes by integrating structural and evolutionary epistatic features.
Nat Commun, 14(1) Art. No. 2230 (2023)
Open Access DOI

Despite the increasing use of genomic sequencing in clinical practice, the interpretation of rare genetic variants remains challenging even in well-studied disease genes, resulting in many patients with Variants of Uncertain Significance (VUSs). Computational Variant Effect Predictors (VEPs) provide valuable evidence in variant assessment, but they are prone to misclassifying benign variants, contributing to false positives. Here, we develop Deciphering Mutations in Actionable Genes (DeMAG), a supervised classifier for missense variants trained using extensive diagnostic data available in 59 actionable disease genes (American College of Medical Genetics and Genomics Secondary Findings v2.0, ACMG SF v2.0). DeMAG improves performance over existing VEPs by reaching balanced specificity (82%) and sensitivity (94%) on clinical data, and includes a novel epistatic feature, the 'partners score', which leverages evolutionary and structural partnerships of residues. The 'partners score' provides a general framework for modeling epistatic interactions, integrating both clinical and functional information. We provide our tool and predictions for all missense variants in 316 clinically actionable disease genes (demag.org) to facilitate the interpretation of variants and improve clinical decision-making.

Claudia Gerri^#, Afshan McCarthy, Gwen Mei Scott, Marius Regin, Panagiotis Stamatiadis, Sophie Brumm, Claire S Simon, Janet Lee, Cristina Montesinos, Caroline Hassitt, Sarah Hockenhull, Daniel Hampshire, Kay Elder, Phil Snell, Leila Christie, Ali A Fouladi-Nashta, Hilde Van de Velde, Kathy K Niakan^#
A conserved role of the Hippo signalling pathway in initiation of the first lineage specification event across mammals.
Development, 150(8) Art. No. dev201112 (2023)
Open Access DOI

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.

Juan M Iglesias-Artola, André Nadler
The Road to Quantitative Lipid Biochemistry in Living Cells.
Acc Chem Res, 56(7) 810-820 (2023)
Open Access DOI

ConspectusTraditional cell biological techniques are not readily suitable for studying lipid signaling events because genetic perturbations are much slower than the interconversion of lipids in complex metabolic networks. For this reason, novel chemical biological approaches have been developed. One approach is to chemically modify a lipid with a so-called "caging group" that renders it inactive, but this cage can be removed photochemically inside cells to release the bioactive molecule. These caged compounds offer unique advantages for studying the kinetics of cellular biochemistry and have been extensively used in the past. However, a limitation of conventional caged compounds is their ability to diffuse freely inside the cell, which does not permit localized activation below optical precision. This poses a challenge for studying lipid signaling as lipid function inside cells is tightly linked to their parent membrane. An ideal lipid probe should, therefore, be restricted to a single organelle membrane or preferentially to a single leaflet. We first demonstrated the plasma-membrane-specific photorelease of fatty acids by employing sulfonated caging groups. Using these caged fatty acid probes we demonstrated that lipid localization determines signaling outcome. Generalizing this approach, we designed a so-called "click cage" that can be coupled to lipids and offers the possibility to attach organelle targeting groups via click chemistry. Using this strategy, we have synthesized plasma membrane, lysosomal, mitochondria, and endoplasmic-reticulum-targeted lipids that can be used to dissect organelle-specific signaling events. To reduce the synthetic effort associated with generating caged compounds, we designed a coumarin triflate reagent that allows the direct functionalization of phosphate- or carboxylate-containing compounds. With this novel reagent, we synthesized a small library of photocaged G-protein-coupled receptor (GPCR) ligands to study the underlying lipid signaling dynamics. Most recently, we have focused on quantifying the kinetics of lipid signaling for different diacylglycerol (DAG) species using plasma-membrane-targeted caged DAGs. Using this approach, we quantitatively measured lipid-protein affinities and lipid transbilayer dynamics in living cells. After analyzing DAGs with different acyl chain length and saturation degree, we discovered that affinities can vary by up to an order of magnitude. This finding clearly shows that cells are able to distinguish between individual DAG species, thereby demonstrating that lipid diversity matters in cellular signal processing. Although the recent advances have yielded valuable tools to study lipid signaling, challenges remain on specifically targeting the different leaflets of organelle membranes. Furthermore, it is necessary to simplify the experimental approaches required for parametrizing and corroborating quantitative kinetic models of lipid signaling. In the future, we envision that the application of leaflet-specific caged lipids to model membrane systems will be of crucial importance for understanding lipid asymmetry.

Sarita Hebbar^#, Sofia Traikov, Catrin Hälsig, Elisabeth Knust^#
Modulating the Kynurenine pathway or sequestering toxic 3-hydroxykynurenine protects the retina from light-induced damage in Drosophila.
PLoS Genet, 19(3) Art. No. e1010644 (2023)
Open Access DOI

Tissue health is regulated by a myriad of exogenous or endogenous factors. Here we investigated the role of the conserved Kynurenine pathway (KP) in maintaining retinal homeostasis in the context of light stress in Drosophila melanogaster. cinnabar, cardinal and scarlet are fly genes that encode different steps in the KP. Along with white, these genes are known regulators of brown pigment (ommochrome) biosynthesis. Using white as a sensitized genetic background, we show that mutations in cinnabar, cardinal and scarlet differentially modulate light-induced retinal damage. Mass Spectrometric measurements of KP metabolites in flies with different genetic combinations support the notion that increased levels of 3-hydroxykynurenine (3OH-K) and Xanthurenic acid (XA) enhance retinal damage, whereas Kynurenic Acid (KYNA) and Kynurenine (K) are neuro-protective. This conclusion was corroborated by showing that feeding 3OH-K results in enhanced retinal damage, whereas feeding KYNA protects the retina in sensitized genetic backgrounds. Interestingly, the harmful effects of free 3OH-K are diminished by its sub-cellular compartmentalization. Sequestering of 3OH-K enables the quenching of its toxicity through conversion to brown pigment or conjugation to proteins. This work enabled us to decouple the role of these KP genes in ommochrome formation from their role in retinal homeostasis. Additionally, it puts forward new hypotheses on the importance of the balance of KP metabolites and their compartmentalization in disease alleviation.

Shanshan Xu, Maria E Gierisch, Anna Katharina Schellhaus, Ina Poser, Simon Alberti, Florian A Salomons, Nico P Dantuma
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment.
EMBO J, 42(3) Art. No. e111802 (2023)
Open Access DOI

The role of cytosolic stress granules in the integrated stress response has remained largely enigmatic. Here, we studied the functionality of the ubiquitin-proteasome system (UPS) in cells that were unable to form stress granules. Surprisingly, the inability of cells to form cytosolic stress granules had primarily a negative impact on the functionality of the nuclear UPS. While defective ribosome products (DRiPs) accumulated at stress granules in thermally stressed control cells, they localized to nucleoli in stress granule-deficient cells. The nuclear localization of DRiPs was accompanied by redistribution and enhanced degradation of SUMOylated proteins. Depletion of the SUMO-targeted ubiquitin ligase RNF4, which targets SUMOylated misfolded proteins for proteasomal degradation, largely restored the functionality of the UPS in the nuclear compartment in stress granule-deficient cells. Stress granule-deficient cells showed an increase in the formation of mutant ataxin-1 nuclear inclusions when exposed to thermal stress. Our data reveal that stress granules play an important role in the sequestration of cytosolic misfolded proteins, thereby preventing these proteins from accumulating in the nucleus, where they would otherwise infringe nuclear proteostasis.

Nicole Poulsen^#, Helene Hennig, Veikko Geyer, Stefan Diez, Richard Wetherbee, Sorel Fitz-Gibbon, Matteo Pellegrini, Nils Kröger^#
On the role of cell surface associated, mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae).
J Phycol, 59(1) 54-69 (2023)
Open Access DOI

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (Frustule Associated Components), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC-MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.

Zuyao Ni, Michele Wölk, Geoff Jukes, Karla Mendivelso Espinosa, R Ahrends, Lucila Aimo, Jorge Alvarez-Jarreta, Simon Andrews, Robert Andrews, Alan Bridge, Geremy C Clair, Matthew J Conroy, Eoin Fahy, Caroline Gaud, Laura Goracci, Jürgen Hartler, Nils Hoffmann, Dominik Kopczyinki, Ansgar Korf, Andrea F Lopez-Clavijo, Adnan Malik, Jacobo Miranda Ackerman, Martijn R Molenaar, Claire O'Donovan, Tomás Pluskal, Andrej Shevchenko, Denise Slenter, Gary Siuzdak, Martina Kutmon, Hiroshi Tsugawa, Egon L Willighagen, Jianguo Xia, Valerie B O'Donnell^#, Maria Fedorova^#
Guiding the choice of informatics software and tools for lipidomics research applications.
Nat Methods, 20(2) 193-204 (2023)
DOI

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

Hiroaki Ishikawa, Jeremy Moore, Dennis R Diener, Markus Delling, Wallace F. Marshall
Testing the ion-current model for flagellar length sensing and IFT regulation.
Elife, 12 Art. No. e82901 (2023)
Open Access DOI

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

Patrick Cahan^#, Barbara Treutlein^#
A conversation with ChatGPT on the role of computational systems biology in stem cell research.
Stem Cell Rep, 18(1) 1-2 (2023)
Open Access DOI

Jingyuan Zhu, Aseem Salhotra, Christoph Robert Meinecke, Pradheebha Surendiran, Roman Lyttleton, Danny Reuter, Hillel Kugler, Stefan Diez, Alf Mansson^#, Heiner Linke^#, Till Korten^#
Solving the 3-Satisfiability Problem Using Network-Based Biocomputation.
Adv Intell Syst, 4(12) Art. No. 2200202 (2022)
Open Access DOI

The 3-satisfiability Problem (3-SAT) is a demanding combinatorial problem that is of central importance among the nondeterministic polynomial (NP) complete problems, with applications in circuit design, artificial intelligence, and logistics. Even with optimized algorithms, the solution space that needs to be explored grows exponentially with the increasing size of 3-SAT instances. Thus, large 3-SAT instances require excessive amounts of energy to solve with serial electronic computers. Network-based biocomputation (NBC) is a parallel computation approach with drastically reduced energy consumption. NBC uses biomolecular motors to propel cytoskeletal filaments through nanofabricated networks that encode mathematical problems. By stochastically exploring possible paths through the networks, the cytoskeletal filaments find possible solutions. However, to date, no NBC algorithm for 3-SAT has been available. Herein, an algorithm that converts 3-SAT into an NBC-compatible network format is reported and four small 3-SAT instances (with up to three variables and five clauses) using the actin-myosin biomolecular motor system are experimentally solved. Because practical polynomial conversions to 3-SAT exist for many important NP complete problems, the result opens the door to enable NBC to solve small instances of a wide range of problems.

Tina Subic, Ivo F. Sbalzarini
A Gaussian jump process formulation of the reaction-diffusion master equation enables faster exact stochastic simulations.
J Chem Phys, 157(19) Art. No. 194110 (2022)
Open Access PDF DOI

We propose a Gaussian jump process model on a regular Cartesian lattice for the diffusion part of the Reaction-Diffusion Master Equation (RDME). We derive the resulting Gaussian RDME (GRDME) formulation from analogy with a kernel-based discretization scheme for continuous diffusion processes and quantify the limits of its validity relative to the classic RDME. We then present an exact stochastic simulation algorithm for the GRDME, showing that the accuracies of GRDME and RDME are comparable, but exact simulations of the GRDME require only a fraction of the computational cost of exact RDME simulations. We analyze the origin of this speedup and its scaling with problem dimension. The benchmarks suggest that the GRDME is a particularly beneficial model for diffusion-dominated systems in three dimensional spaces, often occurring in systems biology and cell biology.

Caitlyn L McCafferty^#, Ophelia Papoulas, Mareike A Jordan, Gabriel Hoogerbrugge, Candice Nichols, Gaia Pigino, David W Taylor, John Wallingford, Edward M Marcotte^#
Integrative modeling reveals the molecular architecture of the intraflagellar transport A (IFT-A) complex.
Elife, 11 Art. No. e81977 (2022)
Open Access DOI

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.

Ioannis Nellas, K Venkatesan Iyer, Juan M Iglesias-Artola, Martin Pippel, André Nadler, Suzanne Eaton, Natalie Dye
Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc.
EMBO Rep, 23(11) Art. No. e54025 (2022)
Open Access DOI

Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.

Sean P A Ritter, Logan A Brand, Shelby L Vincent, Albert Remus R Rosana, Allison Lewis, Denise S Whitford, George W Owttrim
Multiple Light-Dark Signals Regulate Expression of the DEAD-Box RNA Helicase CrhR in Synechocystis PCC 6803.
Cells, 11(21) Art. No. 3397 (2022)
Open Access DOI

Since oxygenic photosynthesis evolved in the common ancestor of cyanobacteria during the Archean, a range of sensing and response strategies evolved to allow efficient acclimation to the fluctuating light conditions experienced in the diverse environments they inhabit. However, how these regulatory mechanisms are assimilated at the molecular level to coordinate individual gene expression is still being elucidated. Here, we demonstrate that integration of a series of three distinct light signals generate an unexpectedly complex network regulating expression of the sole DEAD-box RNA helicase, CrhR, encoded in Synechocystis sp. PCC 6803. The mechanisms function at the transcriptional, translational and post-translation levels, fine-tuning CrhR abundance to permit rapid acclimation to fluctuating light and temperature regimes. CrhR abundance is enhanced 15-fold by low temperature stress. We initially confirmed that the primary mechanism controlling crhR transcript accumulation at 20 °C requires a light quantity-driven reduction of the redox poise in the vicinity of the plastoquinone pool. Once transcribed, a specific light quality cue, a red light signal, was required for crhR translation, far-red reversal of which indicates a phytochrome-mediated mechanism. Examination of CrhR repression at 30 °C revealed that a redox- and light quality-independent light signal was required to initiate CrhR degradation. The crucial role of light was further revealed by the observation that dark conditions superseded the light signals required to initiate each of these regulatory processes. The findings reveal an unexpected complexity of light-dark sensing and signaling that regulate expression of an individual gene in cyanobacteria, an integrated mechanism of environmental perception not previously reported.

Meritxell Huch^#, Mina Gouti^#
Once upon a dish: the next frontier in engineering multicellular systems.
Development, 149(20) Art. No. dev200744 (2022)
DOI

In June 2022, the second meeting on 'Engineering Multicellular Systems', organized by the European Molecular Biology Laboratory and the Institute of Bioengineering of Catalonia, took place in Barcelona. Stem cell and systems biologists, physicists and engineers from all over the world gathered to discuss how recent breakthroughs in organoid technologies, engineering and mechanobiology are boosting our understanding of early morphogenesis, organogenesis and organ function with applications in tissue engineering, disease modeling and drug screening. The meeting was organized with sustainability in mind, and included an ethics session and an outreach public activity.

Camilo Riquelme-Guzmán, Stephanie L Tsai, Karen Carreon Paz, Congtin Nguyen, David Oriola, Maritta Schuez, Jan Brugués, Joshua D. Currie, Tatiana Sandoval-Guzman
Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration.
Elife, 11 Art. No. e79966 (2022)
Open Access DOI

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

Clifford Brangwynne^#, Anthony A. Hyman^#, Michael K Rosen^#
The path to condensates—19th Wiley Prize in Biomedical Sciences laureates share their discovery stories.
Nat Sci, 2(4) Art. No. e20210091 (2022)
Open Access DOI

Preface by Prof. Titia de Lange, Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA The 19th Annual Wiley Prize in Biomedical Sciences celebrated a breakthrough in cell biology: how membrane‐less cellular compartments are formed. The existence of membrane‐less organelles, often called bodies or puncta, has been known for a long time, but what exactly they represented and how they were formed was not known. This problem was solved by a physicist, Clifford Brangwynne, a cell biologist, Anthony Hyman, and a chemist, Michael Rosen. Each, synergistically, made groundbreaking contributions to the discovery that membrane‐less organelles are liquid–liquid phase‐separated entities. The two independent discoveries leading to the principle that multivalent low‐affinity interactions between selected sets of macromolecules, some containing intrinsically disordered regions, formed a molecular condensate with unique dynamic properties, gave birth to the large, blossoming field of biomolecular condensates. The implications of those findings have influenced almost all further research of intracellular processes, including RAS signaling, immune synapses, DNA repair, transcriptional activation, and the functions of nuclear pores, the nucleolus and centrosomes. In this perspective article, the laureates of the award take us on their personal and professional trip that led to their scientific discoveries. Their stories are a celebration of the interdisciplinary essence of Natural Sciences and the potential unlocked when scientists from different fields work together to solve mysteries.

Jeffrey G McDonald^*, Christer S. Ejsing^*, Dominik Kopczynski^*, Michal Holčapek^*, Junken Aoki, Makoto Arita, Masanori Arita, Erin S Baker, Justine Bertrand-Michel, John A Bowden, Britta Brügger, Shane R Ellis, Maria Fedorova, William J Griffiths, Xianlin Han, Jürgen Hartler, Nils Hoffmann, Jeremy P Koelmel, Harald C Köfeler, Todd W Mitchell, Valerie B O'Donnell, Daisuke Saigusa, Dominik Schwudke, Andrej Shevchenko, Candice Z Ulmer, Markus R Wenk, Michael Witting, Denise Wolrab, Yu Xia^#, R Ahrends^#, Gerhard Liebisch^#, Kim Ekroos
Introducing the Lipidomics Minimal Reporting Checklist.
Nat Metab, 4(9) 1086-1088 (2022)
DOI

Mukesh Kumar, Canan Has, Khanh Lam-Kamath, Sophie Ayciriex, Deepshe Dewett, Mina Bashir, Clara Poupault, Kai Schuhmann, Oskar Knittelfelder, Bharath Kumar Raghuraman, R Ahrends, Jens Rister^#, Andrej Shevchenko^#
Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Drosophila Eye Proteome.
Biomolecules, 12(8) Art. No. 1083 (2022)
Open Access DOI

The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.

Mark Leaver^#, Eduardo Moreno, Merve Kayhan, Angela McGaughran, Christian Rödelsperger, Ralf J Sommer, Anthony Hyman^#
Adaptation to environmental temperature in divergent clades of the nematode Pristionchus pacificus.
Evolution, 76(8) 1660-1673 (2022)
Open Access DOI

Because of ongoing climate change, populations of organisms are being subjected to stressful temperatures more often. This is especially problematic for ectothermic organisms, which are likely to be more sensitive to changes in temperature. Therefore, we need to know if ectotherms have adapted to environmental temperature and, if so, what are the evolutionary mechanisms behind such adaptation. Here, we use the nematode Pristionchus pacificus as a case study to investigate thermal adaptation on the Indian Ocean island of La Réunion, which experiences a range of temperatures from coast to summit. We study the evolution of high-temperature tolerance by constructing a phylogenetic tree of strains collected from many different thermal niches. We show that populations of P. pacificus at low altitudes have higher fertility at warmer temperatures. Most likely, this phenotype has arisen recently and at least twice independently, consistent with parallel evolution. We also studied low-temperature tolerance and showed that populations from high altitudes have increased their fertility at cooler temperatures. Together, these data indicate that P. pacificus strains on La Réunion are subject to divergent selection, adapting to hot and cold niches at the coast and summit of the volcano. Precisely defining these thermal niches provides essential information for models that predict the impact of future climate change on these populations.

Hugo van den Hoek^*, Nikolai Klena^*, Mareike A Jordan, Gonzalo Alvarez Viar, Ricardo D Righetto, Miroslava Schaffer, Philipp S Erdmann, William Wan, Stefan Geimer, Jürgen M Plitzko, Wolfgang Baumeister, Gaia Pigino^#, Virginie Hamel^#, Paul Guichard^#, Benjamin D Engel^#
In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains.
Science, 377(6605) 543-548 (2022)
DOI

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

Fereshteh R Najafabadi^*, Mark Leaver^*, Stephan W. Grill
Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis.
Mol Biol Cell, 33(8) Art. No. ar74 (2022)
DOI

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.

Henrike Indrischek, John A Hammer, Anja Machate, Nikolai Hecker, Bogdan Kirilenko, Juliana G. Roscito, Stefan Hans, Caren Norden, Michael Brand^#, Michael Hiller^#
Vision-related convergent gene losses reveal SERPINE3's unknown role in the eye.
Elife, 11 Art. No. e77999 (2022)
Open Access DOI

Despite decades of research, knowledge about the genes that are important for development and function of the mammalian eye and are involved in human eye disorders remains incomplete. During mammalian evolution, mammals that naturally exhibit poor vision or regressive eye phenotypes have independently lost many eye-related genes. This provides an opportunity to predict novel eye-related genes based on specific evolutionary gene loss signatures. Building on these observations, we performed a genome-wide screen across 49 mammals for functionally uncharacterized genes that are preferentially lost in species exhibiting lower visual acuity values. The screen uncovered several genes, including SERPINE3, a putative serine proteinase inhibitor. A detailed investigation of 381 additional mammals revealed that SERPINE3 is independently lost in 18 lineages that typically do not primarily rely on vision, predicting a vision-related function for this gene. To test this, we show that SERPINE3 has the highest expression in eyes of zebrafish and mouse. In the zebrafish retina, serpine3 is expressed in Müller glia cells, a cell type essential for survival and maintenance of the retina. A CRISPR-mediated knockout of serpine3 in zebrafish resulted in alterations in eye shape and defects in retinal layering. Furthermore, two human polymorphisms that are in linkage with SERPINE3 are associated with eye-related traits. Together, these results suggest that SERPINE3 has a role in vertebrate eyes. More generally, by integrating comparative genomics with experiments in model organisms, we show that screens for specific phenotype-associated gene signatures can predict functions of uncharacterized genes.

Eleonora Patsenker, Veera Raghavan Thangapandi, Oskar Knittelfelder, Alessandra Palladini, Michaela Hefti, Jane Beil-Wagner, Gerhard Rogler, Thorsten Buch, Andrej Shevchenko, Jochen Hampe, Felix Stickel
The PNPLA3 variant I148M reveals protective effects toward hepatocellular carcinoma in mice via restoration of omega-3 polyunsaturated fats.
J Nutr Biochem, 108 Art. No. 109081 (2022)
DOI

Alcohol consumption and high caloric diet are leading causes of progressive fatty liver disease. Genetic variant rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3 rs738409 C>G) has been repeatedly described as one of the major risk loci for alcoholic liver cirrhosis (ALC) and hepatocellular carcinoma (HCC) in humans, however, the mechanism behind this association is incompletely understood. We generated mice carrying the rs738409 variant (PNPLA3 I148M) in order to detect genotype-phenotype relationships in mice upon chow and alcohol-high fat/high sugar diet (EtOH/WD). We could clearly demonstrate that the presence of rs738409 per se is sufficient to induce spontaneous development of steatosis after 1 year in mice on a chow diet, whereas in the setting of unhealthy diet feeding, PNPLA3 I148M did not affect hepatic inflammation or fibrosis, but induced a striking lipid remodeling, microvesicular steatosis and protected from HCC formation. Using shot gun lipidomics, we detected a striking restoration of reduced long chain-polyunsaturated fatty acids (LC-PUFA)-containing TGs, docosapentaenoic acid (C22:5 n3) and omega-3-derived eicosanoids (5-HEPE, 20-HEPE, 19,20-EDP, 21-HDHA) in PNPLA3 I148M mice upon EtOH/WD. At the molecular level, PNPLA3 I148M modulated enzymes for fatty acid and TG transport and metabolism. These findings suggest (dietary) lipids as an important and independent driver of hepatic tumorigenesis. Genetic variant in PNPLA3 exerted protective effects in mice, conflicting with findings in humans. Species-related differences in physiology and metabolism should be taken into account when modeling unhealthy human lifestyle, as genetic mouse models may not always allow for translation of insight gained in humans.

Madeline Lancaster, Samantha A Morris, Takanori Takebe, Li Qian, Shaorong Gao, Meritxell Huch
Anniversary reflections: Inspiring discoveries and the future of the field.
Cell Stem Cell, 29(6) 879-881 (2022)
DOI

Cell Stem Cell was launched in 2007, and this year marks its 15th anniversary. To recognize this occasion, we asked six advisory board members to reflect on inspiring discoveries reported in Cell Stem Cell and how these breakthroughs connect to their vision for the future of the field.

Maximina H Yun^#, Toshinori Hayashi^#, Andras Simon
Standardized gene and genetic nomenclature for the newt Pleurodeles waltl.
Dev Dyn, 251(6) 911-912 (2022)
Open Access DOI

Alastair W Skeffington^#, Marc Gentzel, Andre Ohara, Alexander Milentyev, Christoph Heintze, Lorenz Böttcher, Stefan Görlich, Andrej Shevchenko, Nicole Poulsen, Nils Kröger^#
Shedding light on silica biomineralization by comparative analysis of the silica-associated proteomes from three diatom species.
Plant J, 110(6) 1700-1716 (2022)
DOI

Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

Rana Amini, Archit Bhatnagar, Raimund Schlüßler, Stephanie Möllmert, Jochen Guck, Caren Norden
Amoeboid-like migration ensures correct horizontal cell layer formation in the developing vertebrate retina.
Elife, 11 Art. No. e76408 (2022)
Open Access DOI

Migration of cells in the developing brain is integral for the establishment of neural circuits and function of the central nervous system. While migration modes during which neurons employ predetermined directional guidance of either preexisting neuronal processes or underlying cells have been well explored, less is known about how cells featuring multipolar morphology migrate in the dense environment of the developing brain. To address this, we here investigated multipolar migration of horizontal cells in the zebrafish retina. We found that these cells feature several hallmarks of amoeboid-like migration that enable them to tailor their movements to the spatial constraints of the crowded retina. These hallmarks include cell and nuclear shape changes, as well as persistent rearward polarization of stable F-actin. Interference with the organization of the developing retina by changing nuclear properties or overall tissue architecture, hampers efficient horizontal cell migration and layer formation showing that cell-tissue interplay is crucial for this process. In view of the high proportion of multipolar migration phenomena observed in brain development, the here uncovered ameboid-like migration mode might be conserved in other areas of the developing nervous system.

Fritzi Ott^*, Christiane Körner^*, Kim Werner, Martin Gericke, Ines Liebscher, Donald Lobsien, Silvia Radrezza, Andrej Shevchenko, Ute Hofmann, Jürgen Kratzsch, Rolf Gebhardt, Thomas Berg, Madlen Matz-Soja
Hepatic Hedgehog Signaling Participates in the Crosstalk between Liver and Adipose Tissue in Mice by Regulating FGF21.
Cells, 11(10) Art. No. 1680 (2022)
Open Access DOI

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.

Iliya D. Stoev, Florian Huhn, Nicola Maghelli, Elena Erben, Sebastian T. Bundschuh, Benjamin Seelbinder, Catarina Nabais, Britta Schroth-Diez, Ines Höfer, Jan Peychl, Moritz Kreysing, Gert Rapp
FLUCS - Focused Light-Induced Cytoplasmic Streaming : Optically controlled microflows for biology and the life sciences.
Imaging & Microscopy, 24(2) 22-24 (2022)
Open Access

FLUCS is a new photomanipulation technique that allows scientists to optically generate and precisely control microscopic flows within cells, embryos and other viscous fluids. The development of the module is the result of a collaboration between engineers and scientists from Rapp OptoElectronic, the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, and Olympus Europe. The integration of the Rapp FLUCS module into the Olympus IXplore SpinSR spinning disk confocal microscope system allows to generate microflows in the specimen and to image the resulting dynamic biological processes at high spatial and temporal resolution. The system is now available as a finalized and tested product to a broad scientific community worldwide. https://analyticalscience.wiley.com/do/10.1002/was.0004000255; https://analyticalscience.wiley.com/do/10.1002/was.00070072/full/blaetterkatalog_sim0222.pdf

Foram M Joshi, Gonzalo Alvarez Viar, Gaia Pigino, Hauke Drechsler^#, Stefan Diez^#
Fabrication of High Aspect Ratio Gold Nanowires within the Microtubule Lumen.
Nano Lett, 22(9) 3659-3667 (2022)
DOI

Gold nanowires have great potential use as interconnects in electronic, photonic, and optoelectronic devices. To date, there are various fabrication strategies for gold nanowires, each one associated with particular drawbacks as they utilize high temperatures, toxic chemicals, or expensive compounds to produce nanowires of suboptimal quality. Inspired by nanowire fabrication strategies that used higher-order biopolymer structures as molds for electroless deposition of gold, we here report a strategy for the growth of gold nanowires from seed nanoparticles within the lumen of microtubules. Luminal targeting of seed particles occurs through covalently linked Fab fragments of an antibody recognizing the acetylated lysine 40 on the luminal side of α-tubulin. Gold nanowires grown by electroless deposition within the microtubule lumen exhibit a homogeneous morphology and high aspect ratios with a mean diameter of 20 nm. Our approach is fast, simple, and inexpensive and does not require toxic chemicals or other harsh conditions.

Wei Dong^*, Xubo Zhang^*, Yue Kong, Zhenwen Zhao, Ali Mahmoud, Lixian Wu, Bernard Moussian^#, Jianzhen Zhang^#
CYP311A1 in the anterior midgut is involved in lipid distribution and microvillus integrity in Drosophila melanogaster.
Cell Mol Life Sci, 79(5) Art. No. 261 (2022)
DOI

Lipids are either taken up from food sources or produced internally in specialized tissues such as the liver. Among others, both routes of lipid metabolism involve cytochrome P450 monooxygenases (CYPs). We sought to analyze the function of Cyp311a1 that has been shown to be expressed in the midgut of the fruit fly Drosophila melanogaster. Using a GFP-tagged version of CYP311A1 that is expressed under the control of its endogenous promoter, we show that Cyp311a1 localizes to the endoplasmic reticulum in epithelial cells of the anterior midgut. In larvae with reduced Cyp311a1 expression in the anterior midgut, compared to control larvae, the apical plasma membrane of the respective epithelial cells contains less and shorter microvilli. In addition, we observed reduction of neutral lipids in the fat body, the insect liver, and decreased phosphatidylethanolamine (PE) and triacylglycerols (TAG) amounts in the whole body of these larvae. Probably as a consequence, they cease to grow and eventually die. The microvillus defects in larvae with reduced Cyp311a1 expression are restored by supplying PE, a major phospholipid of plasma membranes, to the food. Moreover, the growth arrest phenotype of these larvae is partially rescued. Together, these results suggest that the anterior midgut is an import hub in lipid distribution and that the midgut-specific CYP311A1 contributes to this function by participating in shaping microvilli in a PE-dependent manner.

Liliya Doronina^*^#, Graham M Hughes^*^#, Diana Moreno-Santillan, Colleen Lawless, Tadhg Lonergan, Louise Ryan, David Jebb, Bogdan Kirilenko, Jennifer M Korstian, Liliana M Dávalos, Sonja Vernes, Eugene W Myers, Emma Teeling, Michael Hiller, Lars S Jermiin, Jürgen Schmitz, Mark S Springer, David A Ray^#
Contradictory Phylogenetic Signals in the Laurasiatheria Anomaly Zone.
Genes (Basel), 13(5) Art. No. 766 (2022)
Open Access DOI

Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.

Sergey Nurk^*, Sergey Koren^*, Arang Rhie^*, Mikko Rautiainen^*, Andrey V Bzikadze, Alla Mikheenko, Mitchell R Vollger, Nicolas Altemose, Lev Uralsky, Ariel Gershman, Sergey Aganezov, Savannah J Hoyt, Mark Diekhans, Glennis A Logsdon, Michael Alonge, Stylianos E Antonarakis, Matthew Borchers, Gerard G Bouffard, Shelise Y Brooks, Gina V Caldas, Nae-Chyun Chen, Haoyu Cheng, Chen-Shan Chin, William Chow, Leonardo G de Lima, Philip C Dishuck, Richard Durbin, Tatiana Dvorkina, Ian T Fiddes, Giulio Formenti, Robert S Fulton, Arkarachai Fungtammasan, Erik Garrison, Patrick G S Grady, Tina A Graves-Lindsay, Ira M Hall, Nancy F Hansen, Gabrielle A Hartley, Marina Haukness, Kerstin Howe, Michael W Hunkapiller, Chirag Jain, Miten Jain, Erich D Jarvis, Peter Kerpedjiev, Melanie Kirsche, Mikhail Kolmogorov, Jonas Korlach, Milinn Kremitzki, Heng Li, Valerie V Maduro, Tobias Marschall, Ann M McCartney, J McDaniel, Danny E Miller, James C Mullikin, Eugene W Myers, Nathan D Olson, Benedict Paten, Paul Peluso, Pavel Pevzner, David Porubsky, Tamara Potapova, Evgeny I Rogaev, Jeffrey A Rosenfeld, Steven L Salzberg, Valerie A Schneider, Fritz J Sedlazeck, Kishwar Shafin, Colin J Shew, Alaina Shumate, Ying Sims, Arian F A Smit, Daniela C Soto, Ivan Sović, Jessica M Storer, Aaron Streets, Beth A Sullivan, Francoise Thibaud-Nissen, James Torrance, Justin Wagner, Brian Walenz, Aaron M Wenger, Jonathan Wood, Chunlin Xiao, Stephanie M Yan, Alice C Young, Samantha Zarate, Urvashi Surti, Rajiv C McCoy, Megan Y Dennis, Ivan A Alexandrov, Jennifer L Gerton, Rachel J O'Neill, Winston Timp, Justin M Zook, Michael C Schatz, Evan E Eichler^#, Karen H Miga^#, Adam M Phillippy^#
The complete sequence of a human genome.
Science, 376(6588) 44-53 (2022)
DOI

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

Pallavi Subramanian^#, Sofia Gargani, Alessandra Palladini, Margarita Chatzimike, Michal Grzybek, M Peitzsch, Anastasios D Papanastasiou, Iryna Pyrina, Vasileios Ntafis, Bettina Gercken, Mathias Lesche, Andreas Petzold, Anupam Sinha, Marina Nati, Veera Raghavan Thangapandi, Ioannis Kourtzelis, Margarita Andreadou, Anke Witt, Andreas Dahl, Ralph Burkhardt, Robert Haase, Antonio Domingues, Ian Henry, Nicola Zamboni, Peter Mirtschink, Kyoung-Jin Chung, Jochen Hampe, Ünal Coskun, Dimitris L Kontoyiannis^#, Trian Chavakis^#
The RNA binding protein human antigen R is a gatekeeper of liver homeostasis.
Hepatology, 75(4) 881-897 (2022)
Open Access DOI

NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC.

Diana A Robledo-Ruiz^#, Han Ming Gan^#, Parwinder Kaur, Olga Dudchenko, David Weisz, Ruqayya Khan, Erez Lieberman Aiden, Ekaterina Osipova, Michael Hiller, Hernán E Morales, Michael J L Magrath, Rohan H Clarke, Paul Sunnucks, Alexandra Pavlova
Chromosome-length genome assembly and linkage map of a critically endangered Australian bird: the helmeted honeyeater.
GigaScience, 11 Art. No. giac025 (2022)
Open Access DOI

The helmeted honeyeater (Lichenostomus melanops cassidix) is a Critically Endangered bird endemic to Victoria, Australia. To aid its conservation, the population is the subject of genetic rescue. To understand, monitor, and modulate the effects of genetic rescue on the helmeted honeyeater genome, a chromosome-length genome and a high-density linkage map are required.

Moritz Blumer, Tom Brown, Mariella Bontempo Freitas, Ana Luiza Destro, Juraci A Oliveira, Ariadna E Morales, Tilman Schell, Carola Greve, Martin Pippel, David Jebb, Nikolai Hecker, Alexis-Walid Ahmed, Bogdan Kirilenko, Maddy Foote, Axel Janke, Burton K Lim, Michael Hiller
Gene losses in the common vampire bat illuminate molecular adaptations to blood feeding.
Sci Adv, 8(12) Art. No. eabm6494 (2022)
Open Access DOI

Vampire bats are the only mammals that feed exclusively on blood. To uncover genomic changes associated with this dietary adaptation, we generated a haplotype-resolved genome of the common vampire bat and screened 27 bat species for genes that were specifically lost in the vampire bat lineage. We found previously unknown gene losses that relate to reduced insulin secretion (FFAR1 and SLC30A8), limited glycogen stores (PPP1R3E), and a unique gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2 and CTRL) and distinct pathogen diversity of blood (RNASE7) and predict the complete lack of cone-based vision in these strictly nocturnal bats (PDE6H and PDE6C). Notably, REP15 loss likely helped vampire bats adapt to high dietary iron levels by enhancing iron excretion, and the loss of CYP39A1 could have contributed to their exceptional cognitive abilities. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to blood feeding.

Felipe Mora-Bermúdez^#, Elena Taverna, Wieland Huttner^#
From stem and progenitor cells to neurons in the developing neocortex: key differences among hominids.
FEBS J, 289(6) 1524-1535 (2022)
Open Access DOI

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, e.g. brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.

Kang Du, Martin Pippel, Susanne Kneitz, Romain Feron, Irene da Cruz, Sylke Winkler, Brigitta Wilde, Edgar G Avila Luna, Gene Myers, Yann Guiguen, Constantino Macias Garcia, Manfred Schartl
Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.
Genome Res, 32(3) 583-594 (2022)
DOI

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Benjamin Dalton^*, David Oriola^*, Franziska Decker^*, Frank Jülicher^#, Jan Brugués^#
A gelation transition enables the self-organization of bipolar metaphase spindles.
Nat Phys, 18(3) 323-331 (2022)
Open Access DOI

The mitotic spindle is a highly dynamic bipolar structure that emerges from the self-organization of microtubules, molecular motors and other proteins. Sustained motor-driven poleward flows of dynamic microtubules play a key role in the bipolar organization of spindles. However, it is not understood how the local activity of motor proteins generates these large-scale coherent poleward flows. Here we show that a gelation transition enables long-range microtubule transport causing the spindles to self-organize into two oppositely polarized microtubule gels. Laser ablation experiments reveal that local active stresses generated at the spindle midplane propagate through the structure, thereby driving global coherent microtubule flows. Simulations show that microtubule gels undergoing rapid turnover can exhibit long stress relaxation times, in agreement with the long-range flows observed in experiments. Finally, our model predicts that in the presence of branching microtubule nucleation, either disrupting such flows or decreasing the network connectivity can lead to a microtubule polarity reversal in spindles. We experimentally confirm this inversion of polarity by abolishing the microtubule transport in spindles. Overall, we uncover a connection between spindle rheology and architecture in spindle self-organization. The activity of molecular motors drives the self-organization of cytoskeleton structures, leading to large-scale active flows. Now, experiments and simulations show how a gelation process enables such long-range transport in spindles.

Prasath Paramasivam^*, Christian Franke^*, Martin Stöter, Andreas Höijer, Stefano Bartesaghi, Alan Sabirsh, Lennart Lindfors, Marianna Yanez Arteta, Anders Dahlén, Annette Bak, Shalini Andersson, Yannis Kalaidzidis, Marc Bickle, Marino Zerial
Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.
J Cell Biol, 221(2) Art. No. e202110137 (2022)
Open Access PDF DOI

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.

Mengfei Gao
Expansion microscopy opens the door to exploring more challenges.
Nat Methods, 19(2) 147-148 (2022)
DOI

Franziska Wagner^*, Irina Ruf^*, Thomas Lehmann^*, Rebecca Hofmann, Sylvia Ortmann, Christian Schiffmann, Michael Hiller, Clara Stefen, Heiko Stuckas
Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.
J Evol Biol, 35(2) 225-239 (2022)
Open Access DOI

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

Colin Jackson^#, Agnes Toth-Petroczy, Rachel Kolodny, Florian Hollfelder, Monika Fuxreiter, Shina Caroline Lynn Kamerlin^#, Nobuhiko Tokuriki^#
Adventures on the Routes of Protein Evolution-In Memoriam Dan Salah Tawfik (1955-2021).
J Mol Biol, 434(7) Art. No. 167462 (2022)
DOI

Understanding how proteins evolved not only resolves mysteries of the past, but also helps address challenges of the future, particularly those relating to the design and engineering of new protein functions. Here we review the work of Dan S. Tawfik, one of the pioneers of this area, highlighting his seminal contributions in diverse fields such as protein design, high throughput screening, protein stability, fundamental enzyme-catalyzed reactions and promiscuity, that underpin biology and the origins of life. We discuss the influence of his work on how our models of enzyme and protein function have developed and how the main driving forces of molecular evolution were elucidated. The discovery of the rugged routes of evolution has enabled many practical applications, some which are now widely used.

Timothy J Welsh^*, Georg Krainer^*, Jorge R Espinosa^*, Jerelle A Joseph, Akshay Sridhar, Marcus Jahnel, William E Arter, Kathrin Saar, Simon Alberti^#, Rosana Collepardo-Guevara^#, Tuomas P J Knowles^#
Surface Electrostatics Govern the Emulsion Stability of Biomolecular Condensates.
Nano Lett, 22(2) 612-621 (2022)
DOI

Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions that in general have a propensity to fuse and coalesce; however, many condensates persist as independent droplets in the test tube and inside cells. This stability is crucial for their function, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modeling, we investigate how the nanoscale forces that sustain condensates impact their stability against fusion. By comparing peptide-RNA (PR25:PolyU) and proteinaceous (FUS) condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity. Moreover, measurements of single condensate zeta potentials reveal that such systems can constitute classically stable emulsions. Taken together, these results highlight the role of passive stabilization mechanisms in protecting biomolecular condensates against coalescence.

Harris A Lewin, Stephen Richards, Erez Lieberman Aiden, Miguel L Allende, John M Archibald, Miklós Bálint, Katharine B Barker, Benedikt Baumgartner, Katherine Belov, Giorgio Bertorelle, Mark L Blaxter, Jing Cai, Nicolette D Caperello, Keith Carlson, Juan Carlos Castilla-Rubio, Shu-Miaw Chaw, Lei Chen, Anna K Childers, Jonathan A Coddington, Dalia A Conde, Montserrat Corominas, Keith A Crandall, Andrew J Crawford, Federica DiPalma, Richard Durbin, ThankGod E Ebenezer, Scott V Edwards, Olivier Fedrigo, Paul Flicek, Giulio Formenti, Richard A Gibbs, M Thomas P Gilbert, Melissa M Goldstein, Jennifer Marshall Graves, Henry T Greely, Ilya Grigoriev, Kevin J Hackett, Neil Hall, David Haussler, Kristofer M Helgen, Carolyn J Hogg, Sachiko Isobe, Kjetill Sigurd Jakobsen, Axel Janke, Erich D Jarvis, Warren E Johnson, Steven J. M. Jones, Elinor K Karlsson, Paul J Kersey, Jin-Hyoung Kim, W John Kress, Shigehiro Kuraku, Mara K N Lawniczak, James H Leebens-Mack, Xueyan Li, Kerstin Lindblad-Toh, Xin Liu, Jose Victor Lopez, Tomas Marques-Bonet, Sophie Mazard, Jonna A K Mazet, Camila J Mazzoni, Eugene W Myers, Rachel J O'Neill, Sadye Paez, Hyun Park, Gene E Robinson, Cristina Roquet, Oliver A Ryder, Jamal S M Sabir, H Bradley Shaffer, Timothy M Shank, Jacob S Sherkow, Pamela S Soltis, Boping Tang, Leho Tedersoo, Marcela Uliano-Silva, Kun Wang, Xiaofeng Wei, Regina Wetzer, Julia L Wilson, Xun Xu, Huanming Yang, Anne D Yoder, Guojie Zhang
The Earth BioGenome Project 2020: Starting the clock.
Proc Natl Acad Sci U.S.A., 119(4) Art. No. e2115635118 (2022)
Open Access DOI

Mara K N Lawniczak, Richard Durbin, Paul Flicek, Kerstin Lindblad-Toh, Xiaofeng Wei, John M Archibald, William J Baker, Katherine Belov, Mark L Blaxter, Tomas Marques Bonet, Anna K Childers, Jonathan A Coddington, Keith A Crandall, Andrew J Crawford, Robert P Davey, Federica Di Palma, Qi Fang, Wilfried Haerty, Neil Hall, Katharina J Hoff, Kerstin Howe, Erich D Jarvis, Warren E Johnson, Rebecca N Johnson, Paul J Kersey, Xin Liu, Jose Victor Lopez, Eugene W Myers, Olga Vinnere Pettersson, Adam M Phillippy, Monica F Poelchau, Kim D Pruitt, Arang Rhie, Juan Carlos Castilla-Rubio, Sanjeeb Kumar Sahu, Nicholas A Salmon, Pamela S Soltis, David Swarbreck, Francoise Thibaud-Nissen, Sibo Wang, Jill L Wegrzyn, Guojie Zhang, He Zhang, Harris A Lewin, Stephen Richards
Standards recommendations for the Earth BioGenome Project.
Proc Natl Acad Sci U.S.A., 119(4) Art. No. e2115639118 (2022)
Open Access DOI

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Katherine S. Long^#, Wieland Huttner^#
The Role of the Extracellular Matrix in Neural Progenitor Cell Proliferation and Cortical Folding During Human Neocortex Development.
Front Cell Neurosci, 15 Art. No. 804649 (2022)
Open Access DOI

Extracellular matrix (ECM) has long been known to regulate many aspects of neural development in many different species. However, the role of the ECM in the development of the human neocortex is not yet fully understood. In this review we discuss the role of the ECM in human neocortex development and the different model systems that can be used to investigate this. In particular, we will focus on how the ECM regulates human neural stem and progenitor cell proliferation and differentiation, how the ECM regulates the architecture of the developing human neocortex and the effect of mutations in ECM and ECM-associated genes in neurodevelopmental disorders.

German Belenguer^*, Gianmarco Mastrogiovanni^*, Clare Pacini^*, Zoe Hall, Anna Dowbaj, Robert Arnes-Benito, Aleksandra Sljukic, Nicole Prior, Sofia Kakava, Charles R. Bradshaw, Susan E Davies, Michele Vacca, Kourosh Saeb-Parsy, Bon-Kyoung Koo, Meritxell Huch
RNF43/ZNRF3 loss predisposes to hepatocellular-carcinoma by impairing liver regeneration and altering the liver lipid metabolic ground-state.
Nat Commun, 13(1) Art. No. 334 (2022)
Open Access DOI

RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer.

Felipe Mora-Bermúdez^#, Wieland Huttner^#
What Are the Human-Specific Aspects of Neocortex Development?
Front Neurosci, 16 Art. No. 878950 (2022)
Open Access DOI

When considering what makes us human, the development of the neocortex, the seat of our higher cognitive abilities, is of central importance. Throughout this complex developmental process, neocortical stem and progenitor cells (NSPCs) exert a priming role in determining neocortical tissue fate, through a series of cellular and molecular events. In this Perspective article, we address five questions of relevance for potentially human-specific aspects of NSPCs, (i) Are there human-specific NSPC subtypes? (ii) What is the functional significance of the known temporal differences in NSPC dynamics between human and other great apes? (iii) Are there functional interactions between the human-specific genes preferentially expressed in NSPCs? (iv) Do humans amplify certain metabolic pathways for NSPC proliferation? and finally (v) Have differences evolved during human evolution, notably between modern humans and Neandertals, that affect the performance of key genes operating in NSPCs? We discuss potential implications inherent to these questions, and suggest experimental approaches on how to answer them, hoping to provide incentives to further understand key issues of human cortical development.

Marlena Stadtmüller, Alexa Laubner, Fabian Rost, Sylke Winkler, Eva Patrasová, Lenka Šimůnková, Susanne Reinhardt, Johanna Beil, Alexander Dalpke, Buqing Yi
Emergence and spread of a sub-lineage of SARS-CoV-2 Alpha variant B.1.1.7 in Europe, and with further evolution of spike mutation accumulations shared with the Beta and Gamma variants.
Virus Evol, 8(1) Art. No. veac010 (2022)
Open Access DOI

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution plays a significant role in shaping the dynamics of the coronavirus disease 2019 pandemic. To monitor the evolution of SARS-CoV-2 variants, through international collaborations, we performed genomic epidemiology analyses on a weekly basis with SARS-CoV-2 samples collected from a border region between Germany, Poland, and the Czech Republic in a global background. For identified virus mutant variants, active viruses were isolated and functional evaluations were performed to test their replication fitness and neutralization sensitivity against vaccine-elicited serum neutralizing antibodies. Thereby we identified a new B.1.1.7 sub-lineage carrying additional mutations of nucleoprotein G204P and open-reading-frame-8 K68stop. Of note, this B.1.1.7 sub-lineage is the predominant B.1.1.7 variant in several European countries such as Czech Republic, Austria, and Slovakia. The earliest samples belonging to this sub-lineage were detected in November 2020 in a few countries in the European continent, but not in the UK. We have also detected its further evolution with extra spike mutations D138Y and A701V, which are signature mutations shared with the Gamma and Beta variants, respectively. Antibody neutralization assay of virus variant isolations has revealed that the variant with extra spike mutations is 3.2-fold less sensitive to vaccine-elicited antibodies as compared to the other B.1.1.7 variants tested, indicating potential for immune evasion, but it also exhibited reduced replication fitness, suggesting lower transmissibility. The wide spread of this B.1.1.7 sub-lineage was related to the pandemic waves in early 2021 in various European countries. These findings about the emergence, spread, evolution, infection, and transmission abilities of this B.1.1.7 sub-lineage add to our understanding about the pandemic development in Europe and highlight the importance of international collaboration on virus mutant surveillance.

Benjamin Seelbinder, Manavi Jain, Elena Erben, Sergei Klykov, Iliya D. Stoev, Moritz Kreysing
Non-invasive Chromatin Deformation and Measurement of Differential Mechanical Properties in the Nucleus.
bioRxiv, Art. No. https://doi.org/10.1101/2021.12.15.472786 (2021)
Open Access PDF DOI

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C /μm) can lead to substantial intra-nuclear chromatin displacements (>1 μm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high rigidity and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Friederike I Tam, Mathias J. Gerl, Christian Klose, Michal Surma, Joseph A King, Maria Seidel, Kerstin Weidner, Veit Roessner, Kai Simons, Stefan Ehrlich
Adverse Effects of Refeeding on the Plasma Lipidome in Young Individuals With Anorexia Nervosa?
J Am Acad Child Adolesc Psychiatry, 60(12) 1479-1490 (2021)
DOI

Refeeding is the cornerstone of anorexia nervosa (AN) treatment, but little is known regarding the optimal pace and dietary composition or possible adverse effects of current clinical practices. Plasma lipids may be a moderating factor underlying unfavorable refeeding effects in AN, such as an abnormal central body fat distribution. The objective of this study was to analyze the plasma lipidome in the acutely underweight state of AN before and after refeeding.

Clara Stefen^#, Franziska Wagner, Marika Asztalos, Peter Giere, Peter Grobe, Michael Hiller, Rebecca Hofmann, Maria Jähde, Ulla Lächele, Thomas Lehmann, Sylvia Ortmann, Benjamin Peters, Irina Ruf, Christian Schiffmann, Nadja Thier, Gabriele Unterhitzenberger, Lars Vogt, Matthias Rudolf, Peggy Wehner, Heiko Stuckas^#
Phenotyping in the era of genomics: MaTrics-a digital character matrix to document mammalian phenotypic traits.
Mamm Biol, 102 235-249 (2021)
Open Access DOI

A new and uniquely structured matrix of mammalian phenotypes, MaTrics (Mammalian Tains for Comparative Genomics) in a digital form is presented. By focussing on mammalian species for which genome assemblies are available, MaTrics provides an interface between mammalogy and comparative genomics. MaTrics was developed within a project aimed to find genetic causes of phenotypic traits of mammals using Forward Genomics. This approach requires genomes and comprehensive and recorded information on homologous phenotypes that are coded as discrete categories in a matrix. MaTrics is an evolving online resource providing information on phenotypic traits in numeric code; traits are coded either as absent/present or with several states as multistate. The state record for each species is linked to at least one reference (e.g., literature, photographs, histological sections, CT scans, or museum specimens) and so MaTrics contributes to digitalization of museum collections. Currently, MaTrics covers 147 mammalian species and includes 231 characters related to structure, morphology, physiology, ecology, and ethology and available in a machine actionable NEXUS-format*. Filling MaTrics revealed substantial knowledge gaps, highlighting the need for phenotyping efforts. Studies based on selected data from MaTrics and using Forward Genomics identified associations between genes and certain phenotypes ranging from lifestyles (e.g., aquatic) to dietary specializations (e.g., herbivory, carnivory). These findings motivate the expansion of phenotyping in MaTrics by filling research gaps and by adding taxa and traits. Only databases like MaTrics will provide machine actionable information on phenotypic traits, an important limitation to genomics. MaTrics is available within the data repository Morph.D.Base (www.morphdbase.de).

Victor Girard, Florence Jollivet, Oskar Knittelfelder, Marion Celle, Jean-Noel Arsac, Gilles Chatelain, Daan M Van den Brink, Thierry Baron, Andrej Shevchenko, Ronald P Kühnlein, Nathalie Davoust^#, Bertrand Mollereau^#
Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.
PLoS Genet, 17(11) Art. No. e1009921 (2021)
Open Access DOI

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.

Lars Hubatsch, Louise Jawerth, Celina Love, Jonathan Bauermann, Ty Dora Tang, Stefano Bo, Anthony Hyman, Christoph A. Weber
Quantitative theory for the diffusive dynamics of liquid condensates.
Elife, 10 Art. No. e68620 (2021)
Open Access DOI

Key processes of biological condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date, a physics-based, quantitative framework for the dynamics of labeled condensate components is lacking. Here we derive the corresponding dynamic equations, building on the physics of phase separation, and quantitatively validate the related framework via experiments. We show that by using our framework we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Interestingly, our theory can also be used to determine a relationship between the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This enables us to investigate the effect of salt addition on partitioning and bypasses recently described quenching artifacts in the dense phase. Our approach opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities, and quantifying rates of biochemical reactions in liquid condensates.

Fabio Da Silva^*, Kaiqing Zhang^*, Anneline Pinson, Edoardo Fatti, Michaela Wilsch-Bräuninger, Jessica Herbst, Valerie Vidal, Andreas Schedl, Wieland Huttner^#, Christof Niehrs^#
Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex.
EMBO J, 40(19) Art. No. e108041 (2021)
Open Access DOI

The role of WNT/β-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/β-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.

B Duygu Özpolat^#, Nadine Randel^#, Elizabeth A Williams^#, Luis Alberto Bezares-Calderón^#, Gabriele Andreatta, Guillaume Balavoine, Paola Y Bertucci, David E K Ferrier, Maria Cristina Gambi, Eve Gazave, Mette Handberg-Thorsager, Jörg Hardege, Cameron Hird, Yu-Wen Hsieh, Jerome Hui, Kevin Nzumbi Mutemi, Stephan Q Schneider, Oleg Simakov, Hernando M Vergara, Michel Vervoort, Gáspár Jékely^#, Kristin Tessmar-Raible^#, Florian Raible^#, Detlev Arendt^#
The Nereid on the rise: Platynereis as a model system.
Evodevo, 12(1) Art. No. 10 (2021)
Open Access DOI

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community.

Dipannita Sarkar, Mohammad Shariq, Deepanjali Dwivedi, Nirmal Krishnan, Ronald Naumann, Upinder Singh Bhalla, Hiyaa Singhee Ghosh
Adult brain neurons require continual expression of the schizophrenia-risk gene Tcf4 for structural and functional integrity.
Transl Psychiatry, 11(1) Art. No. 494 (2021)
Open Access DOI

The schizophrenia-risk gene Tcf4 has been widely studied in the context of brain development using mouse models of haploinsufficiency, in utero knockdown and embryonic deletion. However, Tcf4 continues to be abundantly expressed in adult brain neurons where its functions remain unknown. Given the importance of Tcf4 in psychiatric diseases, we investigated its role in adult neurons using cell-specific deletion and genetic tracing in adult animals. Acute loss of Tcf4 in adult excitatory neurons in vivo caused hyperexcitability and increased dendritic complexity of neurons, effects that were distinct from previously observed effects in embryonic-deficiency models. Interestingly, transcriptomic analysis of genetically traced adult-deleted FACS-sorted Tcf4-knockout neurons revealed that Tcf4 targets in adult neurons are distinct from those in the embryonic brain. Meta-analysis of the adult-deleted neuronal transcriptome from our study with the existing datasets of embryonic Tcf4 deficiencies revealed plasma membrane and ciliary genes to underlie Tcf4-mediated structure-function regulation specifically in adult neurons. The profound changes both in the structure and excitability of adult neurons upon acute loss of Tcf4 indicates that proactive regulation of membrane-related processes underlies the functional and structural integrity of adult neurons. These findings not only provide insights for the functional relevance of continual expression of a psychiatric disease-risk gene in the adult brain but also identify previously unappreciated gene networks underpinning mature neuronal regulation during the adult lifespan.

Jenna L Wingfield, Betlehem Mekonnen, Ilaria Mengoni, Peiwei Liu, Mareike A Jordan, Dennis R Diener, Gaia Pigino, Karl F Lechtreck
In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.
J Cell Sci, 134(18) Art. No. jcs.259010 (2021)
DOI

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella.

Stefano Suzzi, Reiner Ahrendt, Stefan Hans, Svetlana A Semenova, Avinash Chekuru, Paul Wirsching, Volker Kroehne, Saygın Bilican, Shady Sayed, Sylke Winkler, Sandra Spieß, Anja Machate, Jan Kaslin, Pertti Panula, Michael Brand
Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.
PLoS Genet, 17(9) Art. No. 1009794 (2021)
Open Access DOI

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Josef Ecker^*, Elisa Benedetti^*, Alida S D Kindt^*, Marcus Höring, Markus Perl, Andrea Christel Machmüller, Anna Sichler, Johannes Plagge, Yuting Wang, Sebastian Zeissig, Andrej Shevchenko, Ralph Burkhardt, Jan Krumsiek, Gerhard Liebisch, Klaus-Peter Janssen
The Colorectal Cancer Lipidome: Identification of a Robust Tumor-Specific Lipid Species Signature.
Gastroenterology, 161(3) 910-923 (2021)
DOI

Lipidomic changes were causally linked to metabolic diseases, but the scenario for colorectal cancer (CRC) is less clear. We investigated the CRC lipidome for putative tumor-specific alterations through analysis of 3 independent retrospective patient cohorts from 2 clinical centers, to derive a clinically useful signature.

Harald C Köfeler, Thomas O Eichmann, R Ahrends, John A Bowden, Niklas Danne-Rasche, Edward A Dennis, Maria Fedorova, William J Griffiths, Xianlin Han, Jürgen Hartler, Michal Holčapek, Robert Jirásko, Jeremy P Koelmel, Christer S. Ejsing, Gerhard Liebisch, Zuyao Ni, Valerie B O'Donnell, Oswald Quehenberger, Dominik Schwudke, Andrej Shevchenko, Michael J O Wakelam, Markus R Wenk, Denise Wolrab, Kim Ekroos
Quality control requirements for the correct annotation of lipidomics data.
Nat Commun, 12(1) Art. No. 4771 (2021)
Open Access DOI

Manon Benjdir, Étienne Audureau, Ariel Beresniak, Patrice Coll, Ralph Epaud, Klaus Fiedler, Bénédicte Jacquemin, Laurent Niddam, Spyros N Pandis, Gerhard Pohlmann, Torkjel M Sandanger, Kai Simons, Mette Sørensen, Patrick Wagner, Sophie Lanone
Assessing the impact of exposome on the course of chronic obstructive pulmonary disease and cystc fibrosis: The REMEDIA European Project Approach.
Environ Epidemiol, 5(4) Art. No. e165 (2021)
Open Access DOI

Because of the direct interaction of lungs with the environment, respiratory diseases are among the leading causes of environment-related deaths in the world. Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are two highly debilitating diseases that are of particular interest in the context of environmental studies; they both are characterized by a similar progressive loss of lung function with small bronchi alterations, and a high phenotypic variability of unknown origin, which prevents a good therapeutic efficacy. In the last years, there has been an evolution in the apprehension of the study of diseases going from a restricted "one exposure, one disease" approach to a broader concept with other associating factors, the exposome. The overall objective of the REMEDIA project is to extend the understanding of the contribution of the exposome to COPD and CF diseases. To achieve our aim, we will (1) exploit data from existing cohorts and population registries to create a unified global database gathering phenotype and exposome information; (2) develop a flexible individual sensor device combining environmental and biomarker toolkits; (3) use a versatile atmospheric simulation chamber to simulate the health effects of complex exposomes; (4) use machine learning supervised analyses and causal inference models to identify relevant risk factors; and (5) develop econometric and cost-effectiveness models to assess the costs, performance, and cost-effectiveness of a selection of prevention strategies. The results will be used to develop guidelines to better predict disease risks and constitute the elements of the REMEDIA toolbox. The multidisciplinary approach carried out by the REMEDIA European project should represent a major breakthrough in reducing the morbidity and mortality associated with COPD and CF diseases.

Till Korten, Stefan Diez, Heiner Linke, Dan V Nicolau Jr, Hillel Kugler
Design of network-based biocomputation circuits for the exact cover problem.
New J Phys, 23(8) Art. No. 085004 (2021)
Open Access DOI

Exact cover is a non-deterministic polynomial time (NP)—complete problem that is central to optimization challenges such as airline fleet planning and allocation of cloud computing resources. Solving exact cover requires the exploration of a solution space that increases exponentially with cardinality. Hence, it is time- and energy consuming to solve large instances of exact cover by serial computers. One approach to address these challenges is to utilize the inherent parallelism and high energy efficiency of biological systems in a network-based biocomputation (NBC) device. NBC is a parallel computing paradigm in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. The network is then explored in parallel using a large number of biological agents, such as molecular-motor-propelled protein filaments. The answer to the combinatorial problem can then be inferred by measuring the positions through which the agents exit the network. Here, we (i) show how exact cover can be encoded and solved in an NBC device, (ii) define a formalization that allows to prove the correctness of our approach and provides a mathematical basis for further studying NBC, and (iii) demonstrate various optimizations that significantly improve the computing performance of NBC. This work lays the ground for fabricating and scaling NBC devices to solve significantly larger combinatorial problems than have been demonstrated so far.

Buqing Yi, Ansgar Poetsch, Marlena Stadtmüller, Fabian Rost, Sylke Winkler, Alexander Dalpke
Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany in 2020: insights into the cause of the second wave.
Epidemiol Infect, 149 Art. No. e177 (2021)
Open Access DOI

In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.

Sarah Fischer, Rohan Jain^#, Thomas Krause, Purvi Jain, Satoru Tsushima, Anna Shevchenko, René Hübner, Norbert Jordan^#
Impact of the Microbial Origin and Active Microenvironment on the Shape of Biogenic Elemental Selenium Nanomaterials.
Environ Sci Technol, 55(13) 9161-9171 (2021)
DOI

The shape of nanomaterials affects their colloidal properties, cellular uptake, and fate in the environment. The microbial origin and microenvironment can play a role in altering the shape of the nanomaterial. However, such studies have never been conducted. Here, we demonstrate that the selenium nanomaterials produced by Escherichia coli K-12 are stable and remain as BioSe-Nanospheres under thermophilic conditions, while those produced by anaerobic granular sludge transform to BioSe-Nanorods, due to a lower quantity of proteins coating these nanoparticles, which has been verified by proteomics analysis as well as using chemically synthesized selenium nanomaterials. Furthermore, the presence of Bacillus safensis JG-B5T transform the purified BioSe-Nanospheres produced by E. coli K-12 to BioSe-Nanorods, though they are not transformed in the absence of B. safensis JG-B5T. This is due to the production of peptidases by B. safensis JG-B5T that cleaves the protein coating the BioSe-Nanospheres produced by E. coli K-12, leading to their transformation to trigonal BioSe-Nanorods, which is the thermodynamically more stable state. These findings suggest that the fate of selenium and probably other redox-active elements released from the biological wastewater treatment units needs to be reevaluated and improved by including microbial criteria for better accuracy.

Nesrine Khouzami, Lars Schütze, Pietro Incardona, Landfried Kraatz, Tina Subic, Jeronimo Castrillon, Ivo F. Sbalzarini
The OpenPME Problem Solving Environment for Numerical Simulations.
In: Computational Science – ICCS 2021 21st International Conference, Krakow, Poland, June 16–18, 2021, Proceedings, Part I (2021) (Lecture Notes in Computer Science ; 12742), Cham, Springer International Publishing (2021), 614-627
DOI

Mareike A Jordan, Gaia Pigino
The structural basis of intraflagellar transport at a glance.
J Cell Sci, 134(12) Art. No. jcs247163 (2021)
DOI

The intraflagellar transport (IFT) system is a remarkable molecular machine used by cells to assemble and maintain the cilium, a long organelle extending from eukaryotic cells that gives rise to motility, sensing and signaling. IFT plays a critical role in building the cilium by shuttling structural components and signaling receptors between the ciliary base and tip. To provide effective transport, IFT-A and IFT-B adaptor protein complexes assemble into highly repetitive polymers, called IFT trains, that are powered by the motors kinesin-2 and IFT-dynein to move bidirectionally along the microtubules. This dynamic system must be precisely regulated to shuttle different cargo proteins between the ciliary tip and base. In this Cell Science at a Glance article and the accompanying poster, we discuss the current structural and mechanistic understanding of IFT trains and how they function as macromolecular machines to assemble the structure of the cilium.

Cristina Subiran Adrados, Qinghao Yu, Lizbeth Airais Bolaños Castro, Luis Alberto Rodriguez Cabrera, Maximina H Yun
Salamander-Eci: An optical clearing protocol for the three-dimensional exploration of regeneration.
Dev Dyn, 250(6) 902-915 (2021)
Open Access DOI

Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms.

Leticia Colyn, Marina Bárcena-Varela, Gloria Álvarez-Sola, M Ujue Latasa, Iker Uriarte, Eva Santamaría, Jose M Herranz, Alvaro Santos-Laso, Maria Arechederra, Mikel Ruiz de Gauna, Patricia Aspichueta, Matteo Canale, Andrea Casadei-Gardini, Maria Francesconi, Simone Carotti, Sergio Morini, Leonard J Nelson, Maria J Iraburu, Chaobo Chen, Bruno Sangro, Jose Jg Marin, Maria L Martinez-Chantar, Jesus M Banales, Robert Arnes-Benito, Meritxell Huch, John Patino, Altaf A Dar, Mehdi Nosrati, Julen Oyarzábal, Felipe Prósper, Jesus Urman, Francisco Javier Cubero, Christian Trautwein, Carmen Berasain^#, Maite G Fernandez-Barrena^#, Matias A Avila^#
Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.
Hepatology, 73(6) 2380-2396 (2021)
DOI

Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors.

Takashi Namba, Christiane Haffner, Wieland Huttner
Ex vivo Tissue Culture Protocols for Studying the Developing Neocortex.
Bio Protoc, 11(10) Art. No. e4031 (2021)
Open Access DOI

The size of the neocortex and its morphology are highly divergent across mammalian species. Several approaches have been utilized for the analysis of neocortical development and comparison among different species. In the present protocol (Note: This protocol requires basic knowledge of brain anatomy), we describe three ex vivo neocortical slice/tissue culture methods: (i) organotypic slice culture (mouse, ferret, human); (ii) hemisphere rotation culture (mouse, ferret); and (iii) free-floating tissue culture (mouse, ferret, human). Each of these three culture methods offers distinct features with regard to the analyses to be performed and can be combined with genetic manipulation by electroporation and treatment with specific inhibitors. These three culture methods are therefore powerful techniques to examine the function of genes involved in neocortical development.

Tatiana Tiago, Barbara Hummel, Federica F Morelli, Valentina Basile, Jonathan Vinet, Veronica Galli, Laura Mediani, Francesco Antoniani, Silvia Pomella, Matteo Cassandri, Maria Giovanna Garone, Beatrice Silvestri, Marco Cimino, Giovanna Cenacchi, Roberta Costa, Vincent Mouly, Ina Poser, Esti Yeger-Lotem, Alessandro Rosa, Simon Alberti, Rossella Rota, Anat Ben-Zvi, Ritwick Sawarkar, Serena Carra
Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor.
Cell Death Dis, 12(5) Art. No. 452 (2021)
Open Access DOI

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

Stephanie Spannl, Tomasz Buhl, Ioannis Nellas, Salma A Zeidan, K Venkatesan Iyer, Helena Khaliullina, Carsten Schultz, André Nadler, Natalie Dye, Suzanne Eaton
Glycolysis regulates Hedgehog signalling via the plasma membrane potential : Erratum ; Correction to EMBO J. 39 (2020) 21: e101767
EMBO J, 40(7) Art. No. 107925 (2021)
DOI

Marko Popović^*^#, Valentin Druelle^*, Natalie Dye, Frank Jülicher, Matthieu Wyart^#
Inferring the flow properties of epithelial tissues from their geometry.
New J Phys, 23(3) Art. No. 033004 (2021)
Open Access DOI

Amorphous materials exhibit complex material properties with strongly nonlinear behaviors. Below a yield stress they behave as plastic solids, while they start to yield above a critical stress sigma(c). A key quantity controlling plasticity which is, however, hard to measure is the density P(x) of weak spots, where x is the additional stress required for local plastic failure. In the thermodynamic limit P(x) similar to x(theta) is singular at x = 0 in the solid phase below the yield stress sigma(c). This singularity is related to the presence of system spanning avalanches of plastic events. Here we address the question if the density of weak spots and the flow properties of a material can be determined from the geometry of an amorphous structure alone. We show that a vertex model for cell packings in tissues exhibits the phenomenology of plastic amorphous systems. As the yield stress is approached from above, the strain rate vanishes and the avalanches size S and their duration tau diverge. We then show that in general, in materials where the energy functional depends on topology, the value x is proportional to the length L of a bond that vanishes in a plastic event. For this class of models P(x) is therefore readily measurable from geometry alone. Applying this approach to a quantification of the cell packing geometry in the developing wing epithelium of the fruit fly, we find that in this tissue P(L) exhibits a power law with exponents similar to those found numerically for a vertex model in its solid phase. This suggests that this tissue exhibits plasticity and non-linear material properties that emerge from collective cell behaviors and that these material properties govern developmental processes. Our approach based on the relation between topology and energetics suggests a new route to outstanding questions associated with the yielding transition.

Rafał Bazan^*, Adam Schröfel^*, Ewa Joachimiak, Martyna Poprzeczko, Gaia Pigino^#, Dorota Wloga^#
Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.
PLoS Genet, 17(3) Art. No. e1009388 (2021)
Open Access DOI

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

Benjamin Dalton, Ivo F. Sbalzarini, Itsuo Hanasaki
Fundamentals of the logarithmic measure for revealing multimodal diffusion.
Biophys J, 120(5) 829-843 (2021)
Open Access PDF DOI

We develop a theoretical foundation for a time-series analysis method suitable for revealing the spectrum of diffusion coefficients in mixed Brownian systems, where no prior knowledge of particle distinction is required. This method is directly relevant for particle tracking in biological systems, where diffusion processes are often non-uniform. We transform Brownian data onto the logarithmic domain, where the coefficients for individual modes of diffusion appear as distinct spectral peaks in the probability density. We refer to the method as the logarithmic measure of diffusion, or simply as the logarithmic measure. We provide a general protocol for deriving analytical expressions for the probability densities on the logarithmic domain. The protocol is applicable for any number of spatial dimensions with any number of diffusive states. The analytical form can be fitted to data to reveal multiple diffusive modes. We validate the theoretical distributions and benchmark the accuracy and sensitivity of the method by extracting multi-modal diffusion coefficients from 2D Brownian simulations of poly-disperse filament bundles. Bundling the filaments allows us to control the system non-uniformity and hence quantify the sensitivity of the method. By exploiting the anisotropy of the simulated filaments, we generalize the logarithmic measure to rotational diffusion. By fitting the analytical forms to simulation data, we confirm the method's theoretical foundation. An error analysis in the single-mode regime shows that the proposed method is comparable in accuracy to the standard mean squared displacement approach for evaluating diffusion coefficients. For the case of multi-modal diffusion, we compare the logarithmic measure against other more sophisticated methods, showing that both model selectivity and extraction accuracy are comparable for small data sets. Therefore we suggest that the logarithmic measure, as a method for multi-modal diffusion coefficient extraction, is ideally suited for small data sets, a condition often confronted in the experimental context. Finally, we critically discuss the proposed benefits of the method and its information content.

Szabolcs Horvát^*, Adeeba Fathima^*, Stefan Goerlich, Michael Schlierf^#, Carl D. Modes^#, Nils Kroeger^#
Computational analysis of the effects of nitrogen source and sin1 knockout on biosilica morphology in the model diatom Thalassiosira pseudonana.
Discover Materials, 1 Art. No. 8 (2021)
Open Access DOI

Morphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as the sole nitrogen source. Fur- thermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work will be invaluable for unraveling genotype–phenotype correlations in diatom biosilica morphogenesis.

Anne Seifert, Hauke Drechsler, Julia Japtok, Till Korten, Stefan Diez^#, Andreas Hermann^#
The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility.
Int J Mol Sci, 22(5) Art. No. 2422 (2021)
Open Access DOI

Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.

Koichiro Uriu^*, Bo-Kai Liao^*, Andrew C. Oates, Luis G Morelli
From local resynchronization to global pattern recovery in the zebrafish segmentation clock.
Elife, 10 Art. No. e61358 (2021)
Open Access DOI

Integrity of rhythmic spatial gene expression patterns in the vertebrate segmentation clock requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition causes defective segment boundaries. Whether deformation of the oscillating tissue complements local synchronization during patterning and segment formation is not understood. We combine theory and experiment to investigate this question in the zebrafish segmentation clock. We remove a Notch inhibitor, allowing resynchronization, and analyze embryonic segment recovery. We observe unexpected intermingling of normal and defective segments, and capture this with a new model combining coupled oscillators and tissue mechanics. Intermingled segments are explained in the theory by advection of persistent phase vortices of oscillators. Experimentally observed changes in recovery patterns are predicted in the theory by temporal changes in tissue length and cell advection pattern. Thus, segmental pattern recovery occurs at two length and time scales: rapid local synchronization between neighboring cells, and the slower transport of the resulting patterns across the tissue through morphogenesis.

Tobias Pietzsch, Lorenzo Duso, Christoph Zechner
Compartor: A toolbox for the automatic generation of moment equations for dynamic compartment populations.
Bioinformatics, 37(17) 2782-2784 (2021)
Open Access DOI

Many biochemical processes in living organisms take place inside compartments that can interact with each other and remodel over time. In a recent work (Duso and Zechner, 2020), we have shown how the stochastic dynamics of a compartmentalized biochemical system can be effectively studied using moment equations. With this technique, the time evolution of a compartment population is summarized using a finite number of ordinary differential equations, which can be analyzed very efficiently. However, the derivation of moment equations by hand can become time-consuming for systems comprising multiple reactants and interactions. Here we present Compartor, a toolbox that automatically generates the moment equations associated with a user-defined compartmentalized system. Through the moment equation method, Compartor renders the analysis of stochastic population models accessible to a broader scientific community.

Simon Alberti^#, Anthony Hyman^#
Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.
Nat Rev Mol Cell Biol, 22(3) 196-213 (2021)
DOI

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.

Sarita Hebbar^*, Malte Lehmann^*, Sarah Behrens, Catrin Hälsig, Weihua Leng, Michaela Yuan, Sylke Winkler, Elisabeth Knust
Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.
Biol Open, 10(1) Art. No. bio052332 (2021)
Open Access DOI

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

Benjamin Dalton^*, David Oriola^*, Franziska Decker^*, Frank Jülicher, Jan Brugués
A gelation transition enables the self-organization of bipolar metaphase spindles
bioRxiv, Art. No. https://doi.org/10.1101/2021.01.15.426844 (2021)
Open Access DOI

Nicolas T Chartier^*, Arghyadip Mukherjee^*, Julia Pfanzelter^*, Sebastian Fürthauer, Ben T Larson, Anatol Fritsch, Rana Amini, Moritz Kreysing, Frank Jülicher^#, Stephan W. Grill^#
A hydraulic instability drives the cell death decision in the nematode germline.
Nat Phys, 17(8) 920-925 (2021)
Open Access PDF DOI

Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow-typically at the expense of others that undergo cell death2-4. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability5. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping6. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.

Barbara Stepien^*, Samir Vaid^*, Wieland Huttner
Length of the Neurogenic Period-A Key Determinant for the Generation of Upper-Layer Neurons During Neocortex Development and Evolution.
Front Cell Dev Biol, 9 Art. No. 676911 (2021)
Open Access DOI

The neocortex, a six-layer neuronal brain structure that arose during the evolution of, and is unique to, mammals, is the seat of higher order brain functions responsible for human cognitive abilities. Despite its recent evolutionary origin, it shows a striking variability in size and folding complexity even among closely related mammalian species. In most mammals, cortical neurogenesis occurs prenatally, and its length correlates with the length of gestation. The evolutionary expansion of the neocortex, notably in human, is associated with an increase in the number of neurons, particularly within its upper layers. Various mechanisms have been proposed and investigated to explain the evolutionary enlargement of the human neocortex, focussing in particular on changes pertaining to neural progenitor types and their division modes, driven in part by the emergence of human-specific genes with novel functions. These led to an amplification of the progenitor pool size, which affects the rate and timing of neuron production. In addition, in early theoretical studies, another mechanism of neocortex expansion was proposed-the lengthening of the neurogenic period. A critical role of neurogenic period length in determining neocortical neuron number was subsequently supported by mathematical modeling studies. Recently, we have provided experimental evidence in rodents directly supporting the mechanism of extending neurogenesis to specifically increase the number of upper-layer cortical neurons. Moreover, our study examined the relationship between cortical neurogenesis and gestation, linking the extension of the neurogenic period to the maternal environment. As the exact nature of factors promoting neurogenic period prolongation, as well as the generalization of this mechanism for evolutionary distinct lineages, remain elusive, the directions for future studies are outlined and discussed.

Basusree Ghosh^*, Rudrarup Bose^*, T-Y Dora Tang
Can coacervation unify disparate hypotheses in the origin of cellular life?
Curr Opin Colloid Interface Sci, 52 Art. No. 101415 (2021)
Open Access DOI

Anatol W Fritsch^*, Andrés Diaz Delgadillo^*, Omar Adame-Arana^*, Carsten Hoege, Matthäus Mittasch, Moritz Kreysing, Mark Leaver, Anthony A. Hyman, Frank Jülicher, Christoph A. Weber
Local thermodynamics governs the formation and dissolution of protein condensates in living cells.
bioRxiv, Art. No. https://doi.org/10.1101/2021.02.11.430794 (2021)
Open Access PDF DOI

Wilhelm Palm, Jonathan Rodenfels
Understanding the role of lipids and lipoproteins in development.
Development, 147(24) Art. No. dev186411 (2020)
DOI

Lipids exert diverse functions in living organisms. They form cellular membranes, store and transport energy and play signalling roles. Some lipid species function in all of these processes, making them ideal candidates to coordinate metabolism with cellular homeostasis and animal development. This theme was central to Suzanne Eaton's research in the fruit fly, Drosophila Here, we discuss her work on membrane lipid homeostasis in changing environments and on functions for lipids in the Hedgehog signalling pathway. We further highlight lipoproteins as inter-organ carriers of lipids and lipid-linked morphogens, which communicate dietary and developmental signals throughout the organism.

Peiwen Xiong, C Darrin Hulsey, Carmelo Fruciano, Wai Y Wong, Alexander Nater, Andreas F Kautt, Oleg Simakov, Martin Pippel, Shigehiro Kuraku, Axel Meyer, Paolo Franchini
The comparative genomic landscape of adaptive radiation in crater lake cichlid fishes.
Mol Ecol, 30(4) 955-972 (2020)
Open Access DOI

Factors ranging from ecological opportunity to genome composition might explain why only some lineages form adaptive radiations. While being rare, particular systems can provide natural experiments within an identical ecological setting where species numbers and phenotypic divergence in two closely related lineages are notably different. We investigated one such natural experiment using two de novo assembled and 40 resequenced genomes and asked why two closely related Neotropical cichlid fish lineages, the Amphilophus citrinellus species complex (Midas cichlids; radiating) and Archocentrus centrarchus (Flyer cichlid; nonradiating), have resulted in such disparate evolutionary outcomes. Although both lineages inhabit many of the same Nicaraguan lakes, whole-genome inferred demography suggests that priority effects are not likely to be the cause of the dissimilarities. Also, genome-wide levels of selection, transposable element dynamics, gene family expansion, major chromosomal rearrangements and the number of genes under positive selection were not markedly different between the two lineages. To more finely investigate particular subsets of the genome that have undergone adaptive divergence in Midas cichlids, we also examined if there was evidence for 'molecular pre-adaptation' in regions identified by QTL mapping of repeatedly diverging adaptive traits. Although most of our analyses failed to pinpoint substantial genomic differences, we did identify functional categories containing many genes under positive selection that provide candidates for future studies on the propensity of Midas cichlids to radiate. Our results point to a disproportionate role of local, rather than genome-wide factors underlying the propensity for these cichlid fishes to adaptively radiate.

Petra Kiesel^*, Gonzalo Alvarez Viar^*, Nikolai Tsoy, Riccardo Maraspini, Peter Gorilak, Vladimir Varga, Alf Honigmann, Gaia Pigino
The molecular structure of mammalian primary cilia revealed by cryo-electron tomography.
Nat Struct Mol Biol, 27(12) 1115-1124 (2020)
DOI

Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook '9 + 0' arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles.

Alexandra B Schroeder, Ellen T A Dobson, Curtis Rueden, Pavel Tomancak, Florian Jug, Kevin W Eliceiri
The ImageJ ecosystem: Open-source software for image visualization, processing, and analysis.
Protein Sci, 30(1) 234-249 (2020)
DOI

For decades, biologists have relied on software to visualize and interpret imaging data. As techniques for acquiring images increase in complexity, resulting in larger multidimensional datasets, imaging software must adapt. ImageJ is an open-source image analysis software platform that has aided researchers with a variety of image analysis applications, driven mainly by engaged and collaborative user and developer communities. The close collaboration between programmers and users has resulted in adaptations to accommodate new challenges in image analysis that address the needs of ImageJ's diverse user base. ImageJ consists of many components, some relevant primarily for developers and a vast collection of user-centric plugins. It is available in many forms, including the widely used Fiji distribution. We refer to this entire ImageJ codebase and community as the ImageJ ecosystem. Here we review the core features of this ecosystem and highlight how ImageJ has responded to imaging technology advancements with new plugins and tools in recent years. These plugins and tools have been developed to address user needs in several areas such as visualization, segmentation, and tracking of biological entities in large, complex datasets. Moreover, new capabilities for deep learning are being added to ImageJ, reflecting a shift in the bioimage analysis community towards exploiting artificial intelligence. These new tools have been facilitated by profound architectural changes to the ImageJ core brought about by the ImageJ2 project. Therefore, we also discuss the contributions of ImageJ2 to enhancing multidimensional image processing and interoperability in the ImageJ ecosystem.

Elisa Nerli, Mauricio Rocha-Martins, Caren Norden
Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway.
Elife, 9 Art. No. e60462 (2020)
Open Access DOI

During brain development, progenitor cells need to balanceproliferation and differentiation in order to generate different neurons in the correct numbers and proportions. Currently, the patterns of multipotent progenitor divisions that lead to neurogenic entry and the factors that regulate them are not fully understood. We here use the zebrafish retina to address this gap, exploiting its suitability for quantitative live-imaging. We show that early neurogenic progenitors arise from asymmetric divisions. Notch regulates this asymmetry, as when inhibited, symmetric divisions producing two neurogenic progenitors occur. Surprisingly however, Notch does not act through an apicobasal activity gradient as previously suggested, but through asymmetric inheritance of Sara-positive endosomes. Further, the resulting neurogenic progenitors show cell biological features different from multipotent progenitors, raising the possibility that an intermediate progenitor state exists in the retina. Our study thus reveals new insights into the regulation of proliferative and differentiative events during central nervous system development.

Francisca M Real, Stefan A Haas, Paolo Franchini, Peiwen Xiong, Oleg Simakov, Heiner Kuhl, Robert Schöpflin, David Heller, M-Hossein Moeinzadeh, Verena Heinrich, Thomas Krannich, Annkatrin Bressin, Michaela F Hartmann, Stefan A Wudy, Dina K N Dechmann, Alicia Hurtado, Francisco J Barrionuevo, Magdalena Schindler, Izabela Harabula, Marco Osterwalder, Michael Hiller, Lars Wittler, Axel Visel, Bernd Timmermann, Axel Meyer, Martin Vingron, Rafael Jiménez, Stefan Mundlos^#, Darío G Lupiáñez^#
The mole genome reveals regulatory rearrangements associated with adaptive intersexuality.
Science, 370(6513) 208-214 (2020)
DOI

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Lei Xing, Nereo Kalebic, Takashi Namba, Samir Vaid, Pauline Wimberger, Wieland Huttner
Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex.
Neuron, 108(6) 1113-1129 (2020)
DOI

Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.

Stephanie Spannl^#, Tomasz Buhl, Ioannis Nellas, Salma A Zeidan, K Venkatesan Iyer, Helena Khaliullina, Carsten Schultz, André Nadler, Natalie Dye^#, Suzanne Eaton
Glycolysis regulates Hedgehog signalling via the plasma membrane potential.
EMBO J, 39(21) Art. No. e101767 (2020)
Open Access DOI

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

Jovana Vasiljević, Juha M. Torkko, Klaus-Peter Knoch, Michele Solimena
The making of insulin in health and disease.
Diabetologia, 63(10) 1981-1989 (2020)
Open Access DOI

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes. Graphical abstract.

Giulia Serafini, Giorgia Giordani, Luca Grillini, Davide Andrenacci, Giuseppe Gargiulo, Valeria Cavaliere
The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.
Int J Mol Sci, 21(19) Art. No. 7257 (2020)
Open Access DOI

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Gaelle R Carrat, Elizabeth Haythorne, Alejandra Tomas, Leena Haataja, Andreas Müller, Peter Arvan, Alexandra Piunti, Kaiying Cheng, Mutian Huang, Timothy J Pullen, Eleni Georgiadou, Theodoros Stylianides, Nur Shabrina Amirruddin, Victoria Salem, Walter Distaso, Andrew Cakebread, Kate J Heesom, Philip A Lewis, David J Hodson, Linford J Briant, Annie C H Fung, Richard B Sessions, Fabien Alpy, Alice P S Kong, Peter I Benke, Federico Torta, Adrian Kee Keong Teo, Isabelle Leclerc, Michele Solimena, Dale B Wigley, Guy A Rutter
The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis.
Mol Metab, 40 Art. No. 101015 (2020)
Open Access DOI

Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation.

Damla Kaptan, Sider Penkov, Xingyu Zhang, Vamshidhar Gade, Bharath Kumar Raghuraman, Roberta Galli, Júlio L Sampaio, Robert Haase, Edmund Koch, Andrej Shevchenko, Vasily Zaburdaev, Teymuras V. Kurzchalia
Exogenous ethanol induces a metabolic switch that prolongs the survival of Caenorhabditis elegans dauer larva and enhances its resistance to desiccation.
Aging Cell, 19(10) Art. No. e13214 (2020)
Open Access DOI

The dauer larva of Caenorhabditis elegans, destined to survive long periods of food scarcity and harsh environment, does not feed and has a very limited exchange of matter with the exterior. It was assumed that the survival time is determined by internal energy stores. Here, we show that ethanol can provide a potentially unlimited energy source for dauers by inducing a controlled metabolic shift that allows it to be metabolized into carbohydrates, amino acids, and lipids. Dauer larvae provided with ethanol survive much longer and have greater desiccation tolerance. On the cellular level, ethanol prevents the deterioration of mitochondria caused by energy depletion. By modeling the metabolism of dauers of wild-type and mutant strains with and without ethanol, we suggest that the mitochondrial health and survival of an organism provided with an unlimited source of carbon depends on the balance between energy production and toxic product(s) of lipid metabolism.

Gautam Dey, Sian Culley, Scott Curran, Uwe Schmidt, Ricardo Henriques, Wanda Kukulski, Buzz Baum
Closed mitosis requires local disassembly of the nuclear envelope.
Nature, 585(7823) 119-123 (2020)
DOI

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Steffen Dietzel, Elisa Ferrando-May, Hans-Ulrich Fried, Christian Kukat, Angela Naumann, Roland Nitschke, Pawel Pasierbek, Jan Peychl, Tobias Manuel Rasse, Britta Schroth-Diez, Martin Thomas Stöckl, Stefan Terjung, Roland Thuenauer, Silke Tulok, Stefanie Weidtkamp-Peters, Stefanie null
A Joint Action in Times of Pandemic: The German BioImaging Recommendations for Operating Imaging Core Facilities During the SARS-Cov-2 Emergency.
Cytometry A, 97(9) 882-886 (2020)
Open Access DOI

Operating shared resource laboratories (SRLs) in times of pandemic is a challenge for research institutions. In a multiuser, high-turnover working space, the transmission of infectious agents is difficult to control. To address this challenge, imaging core facility managers being members of German BioImaging discussed how shared microscopes could be operated with minimal risk of spreading SARS-CoV-2 between users and staff. Here, we describe the resulting guidelines and explain their rationale, with a focus on separating users in space and time, protective face masks, and keeping surfaces virus-free. These recommendations may prove useful for other types of SRLs. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Juliana G. Roscito, Kaushikaram Subramanian, Ronald Naumann, Mihail Sarov, Anna Shevchenko, Aliona Bogdanova, Thomas Kurth, Leo Foerster, Moritz Kreysing, Michael Hiller
Recapitulating evolutionary divergence in a single cis-regulatory element is sufficient to cause expression changes of the lens gene Tdrd7.
Mol Biol Evol, 38(2) 380-392 (2020)
Open Access PDF DOI

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than two-fold up-regulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

Robert W Fernandez, Kimberly Wei, Erin Y Wang, Deimante Mikalauskaite, Andrew Olson, Judy Pepper, Nakeirah Christie, Seongseop Kim, Susanne Weissenborn, Mihail Sarov, Michael R Koelle
Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit.
J Neurosci, 40(39) 7475-7488 (2020)
DOI

Maps of the synapses made and neurotransmitters released by all neurons in model systems such as C. elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter G protein coupled receptors (GPCRs) of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard lab conditions, suggesting the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.SIGNIFICANCE STATEMENTNeurotransmitters signal through G protein coupled receptors (GPCRs) to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism C. elegans. We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

Laura Christin Trautenberg, Oskar Knittelfelder, Carla Hofmann, Andrej Shevchenko, Marko Brankatschk, Elodie Prince
How to use the development of individual Drosophila larvae as a metabolic sensor.
J. Insect Physiol., 126 Art. No. 104095 (2020)
DOI

Metabolic research is a challenge because of the variety of data within experimental series and the difficulty of replicating results among scientific groups. The fruit fly, Drosophila melanogaster, is a cost-effective and reliable pioneer model to screen dietary variables for metabolic research. One of the main reasons for problems in this field are differences in food recipes, diet-associated microbial environments and the pharmacokinetic behavior of nutrients across the gut-blood barrier. To prevent such experimental shortcomings, a common strategy is to pool scores of subjects into one sample to create an average statement. However, this approach lacks information about the biological spread and may provoke misleading interpretations. We propose to use the developmental rate of individual Drosophila larvae as a metabolic sensor. To do so, we introduce here a 96-well plate-based assay, which allows screening for multiple variables including food quality, microbial load, and genetic differences. We demonstrate that on a diet that is rich in calories, pupation is sensitive to the variation of dietary lipid compounds and that genotypes considered as wild-types/controls produce different developmental profiles. Our platform is suited for later automation and represents a potent high-throughput screening tool for the pharmacology and food industry. If used systematically, our assay could become a powerful reference tool to compare the quality of used dietary configurations with published benchmark recipes.

Quentin Vagne^*, Jean-Patrick Vrel^*, Pierre Sens
A minimal self-organisation model of the Golgi apparatus.
Elife, 9 Art. No. e47318 (2020)
Open Access DOI

The design principles dictating the spatio-temporal organisation of eukaryotic cells, and in particular the mechanisms controlling the self-organisation and dynamics of membrane-bound organelles such as the Golgi apparatus, remain elusive. Although this organelle was discovered 120 years ago, such basic questions as whether vesicular transport through the Golgi occurs in an anterograde (from entry to exit) or retrograde fashion are still strongly debated. Here, we address these issues by studying a quantitative model of organelle dynamics that includes: de-novo compartment generation, inter-compartment vesicular exchange, and biochemical conversion of membrane components. We show that anterograde or retrograde vesicular transports are asymptotic behaviors of a much richer dynamical system. Indeed, the structure and composition of cellular compartments and the directionality of vesicular exchange are intimately linked. They are emergent properties that can be tuned by varying the relative rates of vesicle budding, fusion and biochemical conversion.

Michael Heide^#, Christiane Haffner, Ayako Y Murayama, Yoko Kurotaki, Haruka Shinohara, Hideyuki Okano, Erika Sasaki, Wieland Huttner^#
Human-specific ARHGAP11B increases size and folding of primate neocortex in the fetal marmoset.
Science, 369(6503) 546-550 (2020)
DOI

The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.

Hannah E Walters, Maximina H Yun
Rising from the ashes: cellular senescence in regeneration.
Curr Opin Genet Dev, 64 94-100 (2020)
DOI

Cellular senescence has recently become causally implicated in pathological ageing. Hence, a great deal of research is currently dedicated towards developing senolytic agents to selectively kill senescent cells. However, senescence also plays important roles in a range of physiological processes including during organismal development, providing a barrier to tumorigenesis and in limiting fibrosis. Recent evidence also suggests a role for senescence in coordinating tissue remodelling and in the regeneration of complex structures. Through its non-cell-autonomous effects, a transient induction of senescence may create a permissive environment for remodelling or regeneration through promoting local proliferation, cell plasticity, tissue patterning, balancing growth, or indirectly through finely tuned interactions with infiltrating immune mediators. A careful analysis of the beneficial roles of cellular senescence may provide insights into important physiological processes as well as informing strategies to counteract its detrimental consequences in ageing and disease.

Dror Sever^#, Anne Grapin-Botton^#
Regeneration of the pancreas: proliferation and cellular conversion of surviving cells.
Curr Opin Genet Dev, 64 84-93 (2020)
DOI

The most common pancreas-related disorders are diabetes, pancreatitis and different types of pancreatic cancers. Diabetes is a chronic condition which results from insufficient functional β-cell mass, either as a result of an autoimmune destruction of insulin producing β-cells, or as their death or de-differentiation following years of hyperactivity to compensate for insulin resistance. Chronic pancreatitis leads to cell death and can develop into diabetes or pancreatic cancer. To stimulate regeneration in such pathologies, it is of high importance to evaluate the endogenous regeneration capacity of the pancreas, to understand the conditions needed to trigger it, and to investigate the cellular and molecular regenerative responses. This short review focuses on observations made in the last 2 years on the mechanisms enhancing pancreatic cell proliferation, notably new combinations of pharmacological agents, as well as those triggering cellular conversion.

Laura Mediani, Veronica Galli, Arianna D Carrà, Ilaria Bigi, Jonathan Vinet, Massimo Ganassi, Francesco Antoniani, Tatiana Tiago, Marco Cimino, Daniel Mateju, Cristina Cereda, Orietta Pansarasa, Simon Alberti, Jessica Mandrioli, Serena Carra
BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes.
Cell Stress Chaperones, 25(6) 1045-1058 (2020)
DOI

Stress granules (SGs) are dynamic ribonucleoprotein granules induced by environmental stresses. They play an important role in the stress response by integrating mRNA stability, translation, and signaling pathways. Recent work has connected SG dysfunction to neurodegenerative diseases. In these diseases, SG dynamics are impaired because of mutations in SG proteins or protein quality control factors. Impaired SG dynamics and delayed SG dissolution have also been observed for SGs that accumulate misfolding-prone defective ribosomal products (DRiPs). DRiP accumulation inside SGs is controlled by a surveillance system referred to as granulostasis and encompasses the molecular chaperones VCP and the HSPB8-BAG3-HSP70 complex. BAG3 is a member of the BAG family of proteins, which includes five additional members. One of these proteins, BAG6, is functionally related to BAG3 and able to assist degradation of DRiPs. However, whether BAG6 is involved in granulostasis is unknown. We report that BAG6 is not recruited into SGs induced by different types of stress, nor does it affect SG dynamics. BAG6 also does not replace BAG3's function in SG granulostasis. We show that BAG3 and BAG6 target different subsets of DRiPs, and BAG3 binding to DRiPs is mediated by HSPB8 and HSP70. Our data support the idea that SGs are sensitive to BAG3-HSP70-bound DRiPs but not to BAG6-bound DRiPs. Additionally, only BAG3 is strongly upregulated in the stress recovery phase, when SGs dissolve. These data exclude a role for BAG6 in granulostasis and point to a more specialized function in the clearance of a specific subset of DRiPs.

David Oriola, Frank Jülicher^#, Jan Brugués^#
Active forces shape the metaphase spindle through a mechanical instability.
Proc Natl Acad Sci U.S.A., 117(28) 16154-16159 (2020)
DOI

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

Bharath Kumar Raghuraman^*, Sarita Hebbar^*, Mukesh Kumar, HongKee Moon, Ian Henry, Elisabeth Knust, Andrej Shevchenko
Absolute Quantification of Proteins in the Eye of Drosophila melanogaster.
Proteomics, 20(23) Art. No. e1900049 (2020)
Open Access DOI

Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster. We used a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins were independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances of the components of the PT machinery and the rhabdomere, the photosensitive organelle of the fly eye, and how they changed when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb). Data are available via ProteomeXchange with identifier PXD018001 This article is protected by copyright. All rights reserved.

Elisabeth Nüske, Guendalina Marini, Doris Richter, Weihua Leng, Aliona Bogdanova, Titus Franzmann, Gaia Pigino, Simon Alberti
Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells.
Biol Open, 9(7) Art. No. bio.046391 (2020)
Open Access DOI

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments.

Jessica Guerra^*, Paola Chiodelli^*, Chiara Tobia, Claudia Gerri, Marco Presta
Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System.
Cancers (Basel), 12(7) Art. No. 1756 (2020)
Open Access DOI

Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Mohammadreza Bahadorian, Christoph Zechner^#, Carl D. Modes^#
Gift of gab: Probing the limits of dynamic concentration-sensing across a network of communicating cells.
Phys Rev Research, 2(2) Art. No. 023403 (2020)
Open Access DOI

Many systems in biology and other sciences employ collaborative, collective communication strategies for improved efficiency and adaptive benefit. One such paradigm of particular interest is the community estimation of a dynamic signal, when, for example, an epithelial tissue of cells must decide whether to react to a given dynamic external concentration of stress-signaling molecules. At the level of dynamic cellular communication, however, it remains unknown what effect, if any, arises from communication beyond the mean field level. What are the limits and benefits to communication across a network of neighbor interactions? What is the role of Poissonian versus super-Poissonian dynamics in such a setting? How does the particular topology of connections impact the collective estimation and that of the individual participating cells? In this article we construct a robust and general framework of signal estimation over continuous-time Markov chains in order to address and answer these questions. Our results show that in the case of Possonian estimators, the communication solely enhances convergence speed of the mean squared error (MSE) of the estimators to their steady-state values while leaving these values unchanged. However, in the super-Poissonian regime, the MSE of estimators significantly decreases by increasing the number of neighbors. Surprisingly, in this case, the clustering coefficient of an estimator does not enhance its MSE while still reducing the total MSE of the population.

Alice Cezanne, Janelle Lauer, Anastasia Solomatina, Ivo F. Sbalzarini, Marino Zerial
A non-linear system patterns Rab5 GTPase on the membrane.
Elife, 9 Art. No. e54434 (2020)
Open Access PDF DOI

Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a 'handover' of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

Stella Finkelstein^*, Sidney M Gospe^*, Kai Schuhmann, Andrej Shevchenko, Vadim Y Arshavsky, Ekaterina S Lobanova
Phosphoinositide Profile of the Mouse Retina.
Cells, 9(6) Art. No. E1417 (2020)
Open Access DOI

Phosphoinositides are known to play multiple roles in eukaryotic cells. Although dysregulation of phosphoinositide metabolism in the retina has been reported to cause visual dysfunction in animal models and human patients, our understanding of the phosphoinositide composition of the retina is limited. Here, we report a characterization of the phosphoinositide profile of the mouse retina and an analysis of the subcellular localization of major phosphorylated phosphoinositide forms in light-sensitive photoreceptor neurons. Using chromatography of deacylated phosphatidylinositol headgroups, we established PI(4,5)P2 and PI(4)P as two major phosphorylated phosphoinositides in the retina. Using high-resolution mass spectrometry, we revealed 18:0/20:4 and 16:0/20:4 as major fatty-acyl chains of retinal phosphoinositides. Finally, analysis of fluorescent phosphoinositide sensors in rod photoreceptors demonstrated distinct subcellular distribution patterns of major phosphoinositides. The PI(4,5)P2 reporter was enriched in the inner segments and synapses, but was barely detected in the light-sensitive outer segments. The PI(4)P reporter was mostly found in the outer and inner segments and the areas around nuclei, but to a lesser degree in the synaptic region. These findings provide support for future mechanistic studies defining the biological significance of major mono- (PI(4)P) and bisphosphate (PI(4,5)P2) phosphatidylinositols in photoreceptor biology and retinal health.

Nicolas T Chartier, Arghyadip Mukherjee, Julia Pfannzelter, Sebastian Fürthauer, Ben T Larson, Anatol W Fritsch, Moritz Kreysing, Frank Jülicher, Stephan W. Grill
A hydraulic instability drives the cell death decision in the nematode germline
bioRxiv, Art. No. https://doi.org/10.1101/2020.05.30.125864 (2020)
Open Access PDF DOI

Oocytes are large and resourceful. During oogenesis some germ cells grow, typically at the expense of others that undergo apoptosis. How germ cells are selected to live or die out of a homogeneous population remains unclear. Here we show that this cell fate decision in C. elegans is mechanical and related to tissue hydraulics. Germ cells become inflated when the pressure inside them is lower than in the common cytoplasmic pool. This condition triggers a hydraulic instability which amplifies volume differences and causes some germ cells to grow and others to shrink. Shrinking germ cells are extruded and die, as we demonstrate by reducing germ cell volumes via thermoviscous pumping. Together, this reveals a robust mechanism of mechanochemical cell fate decision making in the germline.

Loreen Knöbel, Corinna Breusing, Till Bayer, Virag Sharma, Michael Hiller, Frank Melzner, Heiko Stuckas
Comparative de novo assembly and annotation of mantle tissue transcriptomes from the Mytilus edulis species complex (M. edulis, M. galloprovincialis, M. trossulus)
Mar Genomics, 51 Art. No. 100700 (2020)
DOI

Mytilus mussels (Mytilus edulis (ME), M. trossulus (MT), and M. galloprovincialis (MG)) are of interest in many fields of marine science and have been used as model in evolutionary research. For instance, they form mosaic hybrid zones or hybrid swarms in areas of secondary contact and hence are suited to address questions related to the evolution of reproductive barriers, adaptive hybridization or speciation. While existing genomic information mostly focuses on single species (ME, MG), this project generated RNA seq data of all three species from allopatric populations, i.e. samples representing genetically pure specimens. We investigated adult mantle tissue (four specimens per species), which is functionally involved in processes such as reproduction or biomineralization. The project provides three assembled transcriptomes (post filtering total transcript numbers for ME: 353339, MT: 437827, MG: 290267) representing genes annotated to at least 40 level 2 GO-terms (number (percentage) of annotated transcripts for ME: 44434 (12.6%), MT: 43960 (10%), MG: 60064 (20.7%)). Annotation showed that the most abundant 40 GO-terms are equally well covered by contigs of the three Mytilus transcriptomes. Therefore, this project lays a basis for evolutionary research by providing candidate genes representing various molecular functions such as reproduction, cellular processes or immune response. The potential of the new transcriptomes to address evolutionary questions is further exemplified by a pilot study on ME and MT transcriptomes that used reciprocal blast to identify 7652 one-to-one orthologue pairs of transcripts.

Lydie Flasse^#, Siham Yennek, Cédric Cortijo, Irene Seijo Barandiaran, Marine R-C Kraus, Anne Grapin-Botton^#
Apical Restriction of the Planar Cell Polarity Component VANGL in Pancreatic Ducts Is Required to Maintain Epithelial Integrity.
Cell Rep, 31(8) Art. No. 107677 (2020)
Open Access DOI

Cell polarity is essential for the architecture and function of numerous epithelial tissues. Here, we show that apical restriction of planar cell polarity (PCP) components is necessary for the maintenance of epithelial integrity. Using the mammalian pancreas as a model, we find that components of the core PCP pathway, such as the transmembrane protein Van Gogh-like (VANGL), become apically restricted over a period of several days. Expansion of VANGL localization to the basolateral membranes of progenitors leads to their death and disruption of the epithelial integrity. VANGL basolateral expansion does not affect apico-basal polarity but acts in the cells where Vangl is mislocalized by reducing Dishevelled and its downstream target ROCK. This reduction in ROCK activity culminates in progenitor cell egression, death, and eventually pancreatic hypoplasia. Thus, precise spatiotemporal modulation of VANGL-dependent PCP signaling is crucial for proper pancreatic morphogenesis.

József Jászai^#, Kristina Thamm, Jana Karbanová, Peggy Janich, Christine A. Fargeas, Wieland Huttner, Denis Corbeil^#
Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3.
J Biol Chem, 295(18) 6007-6022 (2020)
Open Access DOI

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin-Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor-like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Rajasekaran Bhavna
Segmentation clock dynamics is strongly synchronized in the forming somite.
Dev Biol, 460(1) 55-69 (2020)
DOI

During vertebrate somitogenesis an inherent segmentation clock coordinates the spatiotemporal signaling to generate segmented structures that pattern the body axis. Using our experimental and quantitative approach, we study the cell movements and the genetic oscillations of her1 expression level at single-cell resolution simultaneously and scale up to the entire pre-somitic mesoderm (PSM) tissue. From the experimentally determined phases of PSM cellular oscillators, we deduced an in vivo frequency profile gradient along the anterior-posterior PSM axis and inferred precise mathematical relations between spatial cell-level period and tissue-level somitogenesis period. We also confirmed a gradient in the relative velocities of cellular oscillators along the axis. The phase order parameter within an ensemble of oscillators revealed the degree of synchronization in the tailbud and the posterior PSM being only partial, whereas synchronization can be almost complete in the presumptive somite region but with temporal oscillations. Collectively, the degree of synchronization itself, possibly regulated by cell movement and the synchronized temporal phase of the transiently expressed clock protein Her1, can be an additional control mechanism for making precise somite boundaries.

Rita Mateus^*, Laurent Holtzer^*, Carole Seum, Zena Hadjivasiliou, Marine Dubois, Frank Jülicher, Marcos Gonzalez-Gaitan
BMP Signaling Gradient Scaling in the Zebrafish Pectoral Fin.
Cell Rep, 30(12) 4292-4302 (2020)
Open Access DOI

Secreted growth factors can act as morphogens that form spatial concentration gradients in developing organs, thereby controlling growth and patterning. For some morphogens, adaptation of the gradients to tissue size allows morphological patterns to remain proportioned as the organs grow. In the zebrafish pectoral fin, we found that BMP signaling forms a two-dimensional gradient. The length of the gradient scales with tissue length and its amplitude increases with fin size according to a power-law. Gradient scaling and amplitude power-laws are signatures of growth control by time derivatives of morphogenetic signaling: cell division correlates with the fold change over time of the cellular signaling levels. We show that Smoc1 regulates BMP gradient scaling and growth in the fin. Smoc1 scales the gradient by means of a feedback loop: Smoc1 is a BMP agonist and BMP signaling represses Smoc1 expression. Our work uncovers a layer of morphogen regulation during vertebrate appendage development.

Jonathan Rodenfels, Pablo Sartori, Stefan Golfier, Kartikeya Nagendra, Karla M. Neugebauer, Jonathon Howard
Contribution of increasing plasma membrane to the energetic cost of early zebrafish embryogenesis.
Mol Biol Cell, 31(7) 520-526 (2020)
DOI

How do early embryos allocate the resources stored in the sperm and egg? Recently, we established isothermal calorimetry to measure heat dissipation by living zebra-fish embryos and to estimate the energetics of specific developmental events. During the reductive cleavage divisions, the rate of heat dissipation increases from ∼60 nJ · s-1 at the two-cell stage to ∼90 nJ · s-1 at the 1024-cell stage. Here we ask which cellular process(es) drive this increasing energetic cost. We present evidence that the cost is due to the increase in the total surface area of all the cells of the embryo. First, embryo volume stays constant during the cleavage stage, indicating that the increase is not due to growth. Second, the heat increase is blocked by nocodazole, which inhibits DNA replication, mitosis, and cell division; this suggests some aspect of cell proliferation contributes to these costs. Third, the heat increases in proportion to the total cell surface area rather than total cell number. Fourth, the heat increase falls within the range of the estimated costs of maintaining and assembling plasma membranes and associated proteins. Thus, the increase in total plasma membrane associated with cell proliferation is likely to contribute appreciably to the total energy budget of the embryo.

Sider Penkov^*, Bharath Kumar Raghuraman^*, Cihan Erkut, Jana Oertel, Roberta Galli, Eduardo Jacobo Miranda Ackerman, Daniela Vorkel, Jean-Marc Verbavatz, Edmund Koch, Karim Fahmy, Andrej Shevchenko, Teymuras V. Kurzchalia
A metabolic switch regulates the transition between growth and diapause in C. elegans.
BMC Biol, 18(1) Art. No. 31 (2020)
Open Access DOI

Metabolic activity alternates between high and low states during different stages of an organism's life cycle. During the transition from growth to quiescence, a major metabolic shift often occurs from oxidative phosphorylation to glycolysis and gluconeogenesis. We use the entry of Caenorhabditis elegans into the dauer larval stage, a developmentally arrested stage formed in response to harsh environmental conditions, as a model to study the global metabolic changes and underlying molecular mechanisms associated with growth to quiescence transition.

Ritika Giri, Dimitrios Papadopoulos, Diana M Posadas, Hemanth K Potluri, Pavel Tomancak, Madhav Mani^#, Richard W Carthew^#
Ordered patterning of the sensory system is susceptible to stochastic features of gene expression.
Elife, 9 Art. No. e53638 (2020)
Open Access DOI

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. We investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution.

Sarah Fischer, Thomas Krause, Franziska Lederer, Mohamed L Merroun, Anna Shevchenko, René Hübner, Tamas Firkala, Thorsten Stumpf, Norbert Jordan, Rohan Jain
Bacillus safensis JG-B5T affects the fate of selenium by extracellular production of colloidally less stable selenium nanoparticles.
J Hazard Mater, 384 Art. No. 121146 (2020)
DOI

Understanding the impact of microorganisms on the mobility of selenium (Se) is important for predicting the fate of toxic Se in the environment and improving wastewater treatment technologies. The bacteria strain Bacillus safensis JG-B5T, isolated from soil in a uranium mining waste pile, can influence the Se speciation in the environment and engineered systems. However, the mechanism and conditions of this process remain unknown. This study found that the B. safensis JG-B5T is an obligate aerobic microorganism with an ability to reduce 70% of 2.5 mM selenite to produce red spherical biogenic elemental selenium nanoparticles (BioSeNPs). Only extracellular production of BioSeNPs was observed using transmission electron microscopy. The two-chamber reactor experiments, genome analysis and corona proteins identified on BioSeNPs suggested that the selenite reduction process was primarily mediated through membrane-associated proteins, like succinate dehydrogenase. Extracellular presence and low colloidal stability of BioSeNPs as indicated by ζ-potential measurements, render B. safensis JG-B5T an attractive candidate in wastewater treatment as it provides easy way of recovering Se while maintaining low Se discharge. As this microorganism decreases Se mobility, it will affect Se bioavailability in the environment and decreases its toxicity.

David Oriola, Frank Jülicher, Jan Brugués
Active force generation shapes the metaphase spindle through a mechanical instability.
bioRxiv, Art. No. https://doi.org/10.1101/2020.02.08.939868 (2020)
Open Access DOI

The metaphase spindle is a dynamic structure that segregates chromosomes during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here, we combine theory and experiments to show that molecular motor driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity and motor-driven active forces. Our study reveals how active force generation can mold liquid crystal droplets and it has implications on the morphology of non-membrane bound compartments demixed from the cytoplasm.

Sissy E Wamaitha, Katarzyna J Grybel, Gregorio Alanis-Lobato, Claudia Gerri, Sugako Ogushi, Afshan McCarthy, Shantha K Mahadevaiah, Lyn Healy, Rebecca A Lea, Miriam Molina-Arcas, Liani G Devito, Kay Elder, Phil Snell, Leila Christie, Julian Downward, James M A Turner, Kathy K Niakan
IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.
Nat Commun, 11(1) Art. No. 764 (2020)
Open Access DOI

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

Kirstin Meyer, Hernán Morales-Navarrete, Sarah Seifert, Michaela Wilsch-Braeuninger, Uta Dahmen, Elly M. Tanaka, Lutz Brusch, Yannis Kalaidzidis, Marino Zerial
Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration.
Mol Syst Biol, 16(2) Art. No. e8985 (2020)
Open Access DOI

The mechanisms of organ size control remain poorly understood. A key question is how cells collectively sense the overall status of a tissue. We addressed this problem focusing on mouse liver regeneration. Using digital tissue reconstruction and quantitative image analysis, we found that the apical surface of hepatocytes forming the bile canalicular network expands concomitant with an increase in F-actin and phospho-myosin, to compensate an overload of bile acids. These changes are sensed by the Hippo transcriptional co-activator YAP, which localizes to apical F-actin-rich regions and translocates to the nucleus in dependence of the integrity of the actin cytoskeleton. This mechanism tolerates moderate bile acid fluctuations under tissue homeostasis, but activates YAP in response to sustained bile acid overload. Using an integrated biophysical-biochemical model of bile pressure and Hippo signaling, we explained this behavior by the existence of a mechano-sensory mechanism that activates YAP in a switch-like manner. We propose that the apical surface of hepatocytes acts as a self-regulatory mechano-sensory system that responds to critical levels of bile acids as readout of tissue status.

Laia Leria, Miquel Vila-Farré, Marta Álvarez-Presas, Alejandro Sánchez-Gracia, Julio Rozas, Ronald Sluys, Marta Riutort
Cryptic species delineation in freshwater planarians of the genus Dugesia (Platyhelminthes, Tricladida): Extreme intraindividual genetic diversity, morphological stasis, and karyological variability.
Mol Phylogenet Evol, 143 Art. No. 106496 (2020)
DOI

The keystone of planarian taxonomy traditionally has been the anatomy of the copulatory apparatus. However, many planarian species comprise asexual fissiparous populations, with the fissiparous animals not developing a copulatory apparatus, thus precluding their morphological identification. Incorporation of molecular data into planarian systematics has been of great value, not only in the identification of fissiparous individuals but also as an additional source of information for determining species boundaries. Nevertheless, the discrepancy between morphological and molecular data has highlighted the need for extra sources of taxonomic information. Moreover, a recent study has pointed out that fissiparous reproduction may lead to high levels of intraindividual genetic diversity in planarians, which may mislead molecular analyses. In the present study we aim to test a new up-to-date integrative taxonomic procedure for planarians, including intraindividual genetic data and additional sources of taxonomic information, besides morphology and DNA, using Dugesia subtentaculata sensu lato as a model organism, a species with an intricate taxonomic history. First, we used three different methods for molecular species delimitation on single locus datasets, both with and without intraindividual information, for formulating Primary Species Hypotheses (PSHs). Subsequently, Secondary Species Hypotheses (SSHs) were formulated on the basis of three types of information: (1) a coalescent-based species delimitation method applied to multilocus data, (2) morphology of the copulatory apparatus, and (3) karyological metrics. This resulted in the delimitation of four morphologically cryptic species within the nominal species D. subtentaculata. Our results provide evidence that the analysis of intraindividual genetic data is essential for properly developing PSHs in planarians. Our study reveals also that karyological differentiation, rather than morphological differentiation, may play an important role in speciation processes in planarians, thus suggesting that the currently known diversity of the group could be highly underestimated.

Irene Adrian-Kalchhauser, Anders Blomberg, Tomas Larsson, Zuzana Musilova, Claire R Peart, Martin Pippel, Monica Hongroe Solbakken, Jaanus Suurväli, Jean-Claude Walser, Joanna Yvonne Wilson, Magnus Alm Rosenblad, Demian Burguera, Silvia Gutnik, Nico Michiels, Mats Töpel, Kirill Pankov, Siegfried Schloissnig, Sylke Winkler
The round goby genome provides insights into mechanisms that may facilitate biological invasions.
BMC Biol, 18(1) Art. No. 11 (2020)
Open Access DOI

The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success.

Martin Pippel^*, David Jebb^*, Franziska Patzold, Sylke Winkler, Heiko Vogel, Gene Myers, Michael Hiller^#, Anna K Hundsdoerfer^#
A highly contiguous genome assembly of the bat hawkmoth Hyles vespertilio (Lepidoptera: Sphingidae).
GigaScience, 9(1) Art. No. giaa001 (2020)
Open Access DOI

Adapted to different ecological niches, moth species belonging to the Hyles genus exhibit a spectacular diversity of larval color patterns. These species diverged ∼7.5 million years ago, making this rather young genus an interesting system to study a wide range of questions including the process of speciation, ecological adaptation, and adaptive radiation.

Yuting Wang, Sebastian Hinz, Ortrud Uckermann, Pia Hönscheid, Witigo von Schönfels, Greta Burmeister, Alexander Hendricks, Jacobo Miranda Ackerman, Gustavo Baretton, Jochen Hampe, Mario Brosch, Clemens Schafmayer, Andrej Shevchenko^#, Sebastian Zeissig^#
Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer.
Biochim Biophys Acta Mol Cell Biol Lipids, 1865(3) Art. No. 158579 (2020)
DOI

Solid tumors are characterized by global metabolic alterations which contribute to their growth and progression. Altered gene expression profiles and plasma lipid composition suggested a role for metabolic reprogramming in colorectal cancer (CRC) development. However, a conclusive picture of CRC-associated lipidome alterations in the tumor tissue has not emerged. Here, we determined molar abundances of 342 species from 20 lipid classes in matched biopsies of CRC and adjacent normal mucosa. We demonstrate that in contrast to previous reports, CRC shows a largely preserved lipidome composition that resembles that of normal colonic mucosa. Important exceptions include increased levels of lyso-phosphatidylinositols in CRC and reduced abundance of ether phospholipids in advanced stages of CRC. As such, our observations challenge the concept of widespread alterations in lipid metabolism in CRC and rather suggest changes in the cellular lipid profile that are limited to selected lipids involved in signaling and the scavenging of reactive oxygen species.

Vladimír Soukup, Akira Tazaki, Yuji Yamazaki, Anna Pospisilova, Hans-Henning Epperlein, Elly M. Tanaka, Robert Cerny
Oral and Palatal Dentition of Axolotl Arises From a Common Tooth-Competent Zone Along the Ecto-Endodermal Boundary.
Front Cell Dev Biol, 8 Art. No. 622308 (2020)
Open Access DOI

Vertebrate dentitions arise at various places within the oropharyngeal cavity including the jaws, the palate, or the pharynx. These dentitions develop in a highly organized way, where new tooth germs are progressively added adjacent to the initiator center, the first tooth. At the same time, the places where dentitions develop house the contact zones between the outer ectoderm and the inner endoderm, and this colocalization has instigated various suggestions on the roles of germ layers for tooth initiation and development. Here, we study development of the axolotl dentition, which is a complex of five pairs of tooth fields arranged into the typically tetrapod outer and inner dental arcades. By tracking the expression patterns of odontogenic genes, we reason that teeth of both dental arcades originate from common tooth-competent zones, one present on the mouth roof and one on the mouth floor. Progressive compartmentalization of these zones and a simultaneous addition of new tooth germs distinct for each prospective tooth field subsequently control the final shape and composition of the axolotl dentition. Interestingly, by following the fate of the GFP-labeled oral ectoderm, we further show that, in three out of five tooth field pairs, the first tooth develops right at the ecto-endodermal boundary. Our results thus indicate that a single tooth-competent zone gives rise to both dental arcades of a complex tetrapod dentition. Further, we propose that the ecto-endodermal boundary running through this zone should be accounted for as a potential source of instruction factors instigating the onset of the odontogenic program.

Elena Taverna^#, Wieland Huttner^#
The Golgi Apparatus in Polarized Neuroepithelial Stem Cells and Their Progeny: Canonical and Noncanonical Features.
In: The Golgi apparatus and centriole : functions, interactions and role in disease. (Eds.) Malgorzata Kloc (Results and Problems in Cell Differentiation ; 67)., Cham, Springer International Publishing (2019), 359-375 Ch. 15
DOI

Neurons forming the central nervous system are generated by neural stem and progenitor cells, via a process called neurogenesis (Gotz and Huttner, Nat Rev Mol Cell Biol, 6:777-788, 2005). In this book chapter, we focus on neurogenesis in the dorsolateral telencephalon, the rostral-most region of the neural tube, which contains the part of the central nervous system that is most expanded in mammals (Borrell and Reillo, Dev Neurobiol, 72:955-971, 2012; Wilsch-Brauninger et al., Curr Opin Neurobiol 39:122-132, 2016). We will discuss recent advances in the dissection of the cell biological mechanisms of neurogenesis, with particular attention to the organization and function of the Golgi apparatus and its relationship to the centrosome.

Clemens Heissenberger, Lisa Liendl, Fabian Nagelreiter, Yulia Gonskikh, Guohuan Yang, Ernst H K Stelzer, Teresa L Krammer, Lucia Micutkova, Stefan Vogt, David P. Kreil, Gerhard Sekot, Emilio Siena, Ina Poser, Eva Harreither, Angela Linder, Viktoria Ehret, Thomas H Helbich, Regina Grillari-Voglauer, Pidder Jansen-Dürr, Martin Koš, Norbert Polacek, Johannes Grillari, Markus Schosserer
Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.
Nucleic Acids Res, 47(22) 11807-11825 (2019)
Open Access DOI

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

Kaushikaram Subramanian, Martin Weigert, Oliver Borsch, Heike Petzold, Alfonso Garcia-Ulloa, Eugene W Myers, Marius Ader, Irina Solovei, Moritz Kreysing
Rod nuclear architecture determines contrast transmission of the retina and behavioral sensitivity in mice.
Elife, 8 Art. No. e49542 (2019)
Open Access PDF DOI

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and the underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture at the expense of image detail. Here we show that retinal optical quality improves 2-fold during terminal development, and that this enhancement is caused by nuclear inversion. We further demonstrate that improved retinal contrast transmission, rather than photon-budget or resolution, enhances scotopic contrast sensitivity by 18-27%, and improves motion detection capabilities up to 10-fold in dim environments. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast transmission as a decisive determinant of mammalian visual perception.

Tatsunori Seki, Tomokatsu Hori, Hajime Miyata, Michiyo Maehara, Takashi Namba
Analysis of proliferating neuronal progenitors and immature neurons in the human hippocampus surgically removed from control and epileptic patients.
Sci Rep, 9(1) Art. No. 18194 (2019)
Open Access DOI

Adult neurogenesis in the mammalian hippocampus is a well-known phenomenon. However, it remains controversial as to what extent adult neurogenesis actually occurs in the adult human hippocampus, and how brain diseases, such as epilepsy, affect human adult neurogenesis. To address these questions, we analyzed immature neuronal marker-expressing (PSA-NCAM+) cells and proliferating neuronal progenitor (Ki67+/HuB+/DCX+) cells in the surgically removed hippocampus of epileptic patients. In control patients, a substantial number of PSA-NCAM+ cells were distributed densely below the granule cell layer. In epileptic patients with granule cell dispersion, the number of PSA-NCAM+ cells was reduced, and aberrant PSA-NCAM+ cells were found. However, the numbers of Ki67+/HuB+/DCX+ cells were very low in both control and epileptic patients. The large number of PSA-NCAM+ cells and few DCX+/HuB+/Ki-67+ cells observed in the controls suggest that immature-type neurons are not recently generated neurons, and that the level of hippocampal neuronal production in adult humans is low. These results also suggest that PSA-NCAM is a useful marker for analyzing the pathology of epilepsy, but different interpretations of the immunohistochemical results between humans and rodents are required.

Kaushikaram Subramanian, Martin Weigert, Oliver Borsch, Heike Petzold, Alfonso Garcia, Eugene Myers, Marius Ader, Irina Solovei, Moritz Kreysing
Rod nuclear architecture determines contrast transmission of the retina and behavioral sensitivity in mice
bioRxiv, Art. No. https://doi.org/10.1101/752444 (2019)
Open Access PDF DOI

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture, at the expense of image detail. Here we show that retinal optical quality improves 2-fold during terminal development, which, confirmed by a mouse model, happens due to nuclear inversion. We further reveal that improved retinal contrast-transmission, rather than photon-budget or resolution, leads to enhanced contrast sensitivity under low light condition. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast-transmission as a decisive determinant of mammalian visual perception

Claire Basquin, Dmitry Ershov, Noémie Gaudin, Hanh Thi-Kim Vu, Bruno Louis, Jean-François Papon, Anne-Marie Orfila, Sarah Mansour, Jochen Rink, Juliette Azimzadeh
Emergence of a Bilaterally Symmetric Pattern from Chiral Components in the Planarian Epidermis.
Dev Cell, 51(4) 516-525 (2019)
DOI

Most animals exhibit mirror-symmetric body plans, yet the molecular constituents from which they are formed are often chiral. In planarian flatworms, centrioles are arranged in a bilaterally symmetric pattern across the ventral epidermis. Here, we found that this pattern is generated by a network of centrioles with prominent chiral asymmetric properties. We identify centriole components required for establishing asymmetric connections between centrioles and balancing their effects to align centrioles along polarity fields. SMED-ODF2, SMED-VFL1, and SMED-VFL3 affect the assembly of centriole appendages that tether cytoskeletal connectors to position the centrioles. We further show that the medio-lateral polarization of centrioles relies on mechanisms that are partly distinct on the left and right sides of the planarian body. Our findings shed light on how bilaterally symmetrical patterns can emerge from chiral cellular organizations.

Hanh Thi-Kim Vu, Sarah Mansour, Michael Kücken, Corinna Blasse, Claire Basquin, Juliette Azimzadeh, Eugene W Myers, Lutz Brusch^#, Jochen Rink^#
Dynamic Polarization of the Multiciliated Planarian Epidermis between Body Plan Landmarks.
Dev Cell, 51(4) 526-542 (2019)
DOI

Polarity is a universal design principle of biological systems that manifests at all organizational scales, yet its coordination across scales remains poorly understood. Here, we make use of the extreme anatomical plasticity of planarian flatworms to probe the interplay between global body plan polarity and local cell polarity. Our quantitative analysis of ciliary rootlet orientation in the epidermis reveals a dynamic polarity field with head and tail as independent determinants of anteroposterior (A/P) polarization and the body margin as determinant of mediolateral (M/L) polarization. Mathematical modeling rationalizes the global polarity field and its response to experimental manipulations as superposition of separate A/P and M/L fields, and we identify the core PCP and Ft/Ds pathways as their molecular mediators. Overall, our study establishes a framework for the alignment of cellular polarity vectors relative to planarian body plan landmarks and establishes the core PCP and Ft/Ds pathways as evolutionarily conserved 2D-polarization module.

Johanna Lattner, Weihua Leng, Elisabeth Knust, Marko Brankatschk^#, David Flores-Benitez^#
Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila.
Elife, 8 Art. No. e50900 (2019)
Open Access DOI

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting βHeavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P2 and formation of a novel, Moesin- and PI(4,5)P2-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Christoph Stritt, Michele Wyler, Elena L Gimmi, Martin Pippel, Anne C Roulin
Diversity, dynamics and effects of long terminal repeat retrotransposons in the model grass Brachypodium distachyon.
New Phytol, 227(6) 1736-1748 (2019)
Open Access DOI

Transposable elements (TEs) are the main reason for the high plasticity of plant genomes, where they occur as communities of diverse evolutionary lineages. Because research has typically focused on single abundant families or summarized TEs at a coarse taxonomic level, our knowledge about how these lineages differ in their effects on genome evolution is still rudimentary. Here we investigate the community composition and dynamics of 32 long terminal repeat retrotransposon (LTR-RT) families in the 272-Mb genome of the Mediterranean grass Brachypodium distachyon. We find that much of the recent transpositional activity in the B. distachyon genome is due to centromeric Gypsy families and Copia elements belonging to the Angela lineage. With a half-life as low as 66 kyr, the latter are the most dynamic part of the genome and an important source of within-species polymorphisms. Second, GC-rich Gypsy elements of the Retand lineage are the most abundant TEs in the genome. Their presence explains > 20% of the genome-wide variation in GC content and is associated with higher methylation levels. Our study shows how individual TE lineages change the genetic and epigenetic constitution of the host beyond simple changes in genome size.

Pavel Vopalensky, Maria Antonietta Tosches, Kaia Achim, Mette Handberg-Thorsager, Detlev Arendt
From spiral cleavage to bilateral symmetry: the developmental cell lineage of the annelid brain.
BMC Biol, 17(1) Art. No. 81 (2019)
Open Access DOI

During early development, patterns of cell division-embryonic cleavage-accompany the gradual restriction of blastomeres to specific cell fates. In Spiralia, which include annelids, mollusks, and flatworms, "spiral cleavage" produces a highly stereotypic, spiral-like arrangement of blastomeres and swimming trochophore-type larvae with rotational (spiral) symmetry. However, starting at larval stages, spiralian larvae acquire elements of bilateral symmetry, before they metamorphose into fully bilateral juveniles. How this spiral-to-bilateral transition occurs is not known and is especially puzzling for the early differentiating brain and head sensory organs, which emerge directly from the spiral cleavage pattern. Here we present the developmental cell lineage of the Platynereis larval episphere.

Moritz Kreysing
Probing the Functional Role of Physical Motion in Development.
Dev Cell, 51(2) 135-144 (2019)
DOI

Spatiotemporal organization during development has frequently been proposed to be explainable by reaction-transport models, where biochemical reactions couple to physical motion. However, whereas genetic tools allow causality of molecular players to be dissected via perturbation experiments, the functional role of physical transport processes, such as diffusion and cytoplasmic streaming, frequently remains untestable. This Perspective explores the challenges of validating reaction-transport hypotheses and highlights new opportunities provided by perturbation approaches that specifically target physical transport mechanisms. Using these methods, experimental physics may begin to catch up with molecular biology and find ways to test roles of diffusion and flows in development.

Patryk Burek, Nico Scherf, Heinrich Herre
Ontology patterns for the representation of quality changes of cells in time.
J Biomed Semantics, 10(1) Art. No. 16 (2019)
Open Access DOI

Cell tracking experiments, based on time-lapse microscopy, have become an important tool in biomedical research. The goal is the reconstruction of cell migration patterns, shape and state changes, and, comprehensive genealogical information from these data. This information can be used to develop process models of cellular dynamics. However, so far there has been no structured, standardized way of annotating and storing the tracking results, which is critical for comparative analysis and data integration. The key requirement to be satisfied by an ontology is the representation of a cell's change over time. Unfortunately, popular ontology languages, such as Web Ontology Language (OWL), have limitations for the representation of temporal information. The current paper addresses the fundamental problem of modeling changes of qualities over time in biomedical ontologies specified in OWL.

Kaushikaram Subramanian
Optics of the mammalian retina: Role of rod nuclear architecture in retinal transparency & visual sensitivity.
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2019)

Seungmin Han, Juergen Fink, David J. Jörg, Eunmin Lee, Min Kyu Yum, Lemonia Chatzeli, Sebastian R Merker, Manon Josserand, Teodora Trendafilova, Amanda Andersson-Rolf, Catherine Dabrowska, Hyunki Kim, Ronald Naumann, Ji-Hyun Lee, Nobuo Sasaki, Richard Lester Mort, Onur Basak, Hans Clevers, Daniel E Stange, Anna Philpott, Jong Kyoung Kim, Benjamin D Simons, Bon-Kyoung Koo
Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells.
Cell Stem Cell, 25(3) 342-356 (2019)
Open Access DOI

The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.

Aleksandar J Krmpot, Stanko N Nikolić, Sho Oasa, Dimitrios Papadopoulos, Marco Vitali, Makoto Oura, Shintaro Mikuni, Per Thyberg, Simone Tisa, Masataka Kinjo, Lennart Nilsson, Lars Terenius, Rudolf Rigler^#, Vladana Vukojević^#
Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.
Anal Chem, 91(17) 11129-11137 (2019)
DOI

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 μs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Wei-Xuan Li, Ronald Sluys, Miquel Vila-Farré, Jia-Jia Chen, Ying Yang, Shuang-Fei Li, An-Tai Wang
A new continent in the geographic distribution of the genus Oregoniplana (Platyhelminthes: Tricladida: Maricola), its rediscovery in South Africa and its molecular phylogenetic position.
Zool J Linn Soc-Lond, 187(1) 82-99 (2019)
DOI

Here we describe a new species for the genus Oregoniplana from the coast of China, representing the third species for the genus and the first record of this genus for Asia. The other species are known from Oregon, USA, and from South Africa. Specimens of the South African species, Oregoniplana pantherina, were recently rediscovered, forming the second record for the species, which provided the basis for a necessary re-description. Molecular data (18S rDNA and 28S rDNA) obtained for the new Chinese species facilitated determination of the position of the genus Oregoniplana in the phylogenetic tree of the marine triclads. We report molecular data also for species previously discovered in China, viz. Miroplana shenzhensis and Pentacoelum sinensis, thus enabling us to determine for the first time the phylogenetic position of the genus Miroplana. These new molecular data revealed that Pentacoleum sinensis occupies an unexpected position in the phylogenetic tree. Laboratory cultures of the new Chinese species provided information on food preferences and reproduction. Oregoniplana pantherina exhibits a peculiar kind of locomotion and an unusual resting position in which the animal has three points of contact on either side.

Marko Barovic, Marius Distler, Eyke Schöniger, Nicole Radisch, Daniela Aust, Jürgen Weitz, Mark Ibberson, Anke Schulte, Michele Solimena
Metabolically phenotyped pancreatectomized patients as living donors for the study of islets in health and diabetes.
Mol Metab, 27S 1-6 (2019)
Open Access DOI

The availability of human pancreatic islets with characteristics closely resembling those present in vivo is instrumental for ex vivo studies in diabetes research.

Volker Hartenstein, Michaela Yuan, Amelia Younossi-Hartenstein, Aanavi Karandikar, F Javier Bernardo-Garcia, Simon Sprecher, Elisabeth Knust
Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes.
Dev Biol, 453(1) 56-67 (2019)
DOI

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 μm diameter) alternating with thin segments (>0.1 μm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

Matthias Huelsmann, Nikolai Hecker, Mark S Springer, John Gatesy, Virag Sharma, Michael Hiller
Genes lost during the transition from land to water in cetaceans highlight genomic changes associated with aquatic adaptations.
Sci Adv, 5(9) Art. No. eaaw6671 (2019)
Open Access PDF DOI

The transition from land to water in whales and dolphins (cetaceans) was accompanied by remarkable adaptations. To reveal genomic changes that occurred during this transition, we screened for protein-coding genes that were inactivated in the ancestral cetacean lineage. We found 85 gene losses. Some of these were likely beneficial for cetaceans, for example, by reducing the risk of thrombus formation during diving (F12 and KLKB1), erroneous DNA damage repair (POLM), and oxidative stress-induced lung inflammation (MAP3K19). Additional gene losses may reflect other diving-related adaptations, such as enhanced vasoconstriction during the diving response (mediated by SLC6A18) and altered pulmonary surfactant composition (SEC14L3), while loss of SLC4A9 relates to a reduced need for saliva. Last, loss of melatonin synthesis and receptor genes (AANAT, ASMT, and MTNR1A/B) may have been a precondition for adopting unihemispheric sleep. Our findings suggest that some genes lost in ancestral cetaceans were likely involved in adapting to a fully aquatic lifestyle.

Mauricio Rocha-Martins, Mariana Silveira
The people behind the papers - Maurício Rocha-Martins and Mariana Silveira.
Development, 146(16) Art. No. dev182998 (2019)
DOI

Retinal ganglion cells connect the retina to the brain, and their degeneration underlies glaucoma, which is the leading cause of irreversible blindness in humans and currently untreatable. Replacement of lost cells could be achieved by in vivo reprogramming of endogenous cells in the retina, a strategy explored by authors of a new paper in Development who focus on a transcription factor renowned for its reprogramming ability in other contexts. We caught up with first author Maurício Rocha-Martins and his former supervisor Mariana Silveira, Professor at the Federal University of Rio de Janeiro in Brazil, to find out more about the story.

Clyde Savio Pinto, Ameya Khandekar, Rajasekaran Bhavna, Petra Kiesel, Gaia Pigino, Mahendra Sonawane
Microridges are apical epithelial projections formed of F-actin networks that organize the glycan layer.
Sci Rep, 9(1) Art. No. 12191 (2019)
Open Access DOI

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

Agnes I Lukaszewicz, Cu Nguyen, Elizabeth Melendez, David P Lin, Jia-Ling Teo, Keane K Y Lai, Wieland Huttner, Song-Hai Shi, Michael Kahn
The Mode of Stem Cell Division Is Dependent on the Differential Interaction of β-Catenin with the Kat3 Coactivators CBP or p300.
Cancers (Basel), 11(7) Art. No. E962 (2019)
Open Access DOI

Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential β-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/β-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases β-catenin Kat3 coactivator usage and enhances β-catenin/CBP transcription at the expense of β-catenin/p300 transcription. Biased β-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced β-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/β-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of β-catenin Kat3 coactivator and the mode of stem cell division.

Steven D. Briscoe, Clifton W. Ragsdale
Evolution of the Chordate Telencephalon.
Curr Biol, 29(13) 647-662 (2019)
DOI

The dramatic evolutionary expansion of the neocortex, together with a proliferation of specialized cortical areas, is believed to underlie the emergence of human cognitive abilities. In a broader phylogenetic context, however, neocortex evolution in mammals, including humans, is remarkably conservative, characterized largely by size variations on a shared six-layered neuronal architecture. By contrast, the telencephalon in non-mammalian vertebrates, including reptiles, amphibians, bony and cartilaginous fishes, and cyclostomes, features a great variety of very different tissue structures. Our understanding of the evolutionary relationships of these telencephalic structures, especially those of basally branching vertebrates and invertebrate chordates, remains fragmentary and is impeded by conceptual obstacles. To make sense of highly divergent anatomies requires a hierarchical view of biological organization, one that permits the recognition of homologies at multiple levels beyond neuroanatomical structure. Here we review the origin and diversification of the telencephalon with a focus on key evolutionary innovations shaping the neocortex at multiple levels of organization.

Pavel Tomancak
Cell communication in the blink of an eye.
Nature, 571(7766) 484-485 (2019)
DOI

Aniruddha Mitra, Marc Suñé, Stefan Diez, José M Sancho, David Oriola, Jaume Casademunt
A Brownian Ratchet Model Explains the Biased Sidestepping of Single-Headed Kinesin-3 KIF1A.
Biophys J, 116(12) 2266-2274 (2019)
DOI

The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5μm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.

Jeshlee Vijayakumar, Charlène Perrois, Marjorie Heim, Luc Bousset, Simon Alberti, Florence Besse
The prion-like domain of Drosophila Imp promotes axonal transport of RNP granules in vivo.
Nat Commun, 10(1) Art. No. 2593 (2019)
Open Access DOI

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.

Nadine Vastenhouw^#, Wen Xi Cao, Howard D Lipshitz^#
The maternal-to-zygotic transition revisited.
Development, 146(11) Art. No. dev161471 (2019)
DOI

The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.

Nicole Prior, Christopher J Hindley, Fabian Rost, Elena Meléndez, Winnie W Y Lau, Berthold Göttgens, Steffen Rulands, Benjamin D Simons, Meritxell Huch
Lgr5+ stem and progenitor cells reside at the apex of a heterogeneous embryonic hepatoblast pool.
Development, 146(12) Art. No. dev174557 (2019)
Open Access DOI

During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.

João P L Castro^*, Michelle N Yancoskie^*, Marta Marchini, Stefanie Belohlavy, Layla Hiramatsu, Marek Kučka, William H Beluch, Ronald Naumann, Isabella Skuplik, John Cobb, Nick H Barton, Campbell Rolian^#, Yingguang Frank Chan^#
An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice.
Elife, 8 Art. No. e42014 (2019)
Open Access DOI

Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

Beatrice Senigagliesi, Carlotta Penzo, Luisa Ulloa Severino, Riccardo Maraspini, Sara Petrosino, Hernán Morales-Navarrete, Enrico Pobega, Elena Ambrosetti, Pietro Parisse, Silvia Pegoraro, Guidalberto Manfioletti, Loredana Casalis, Riccardo Sgarra
The High Mobility Group A1 (HMGA1) Chromatin Architectural Factor Modulates Nuclear Stiffness in Breast Cancer Cells.
Int J Mol Sci, 20(11) Art. No. 2733 (2019)
Open Access DOI

Plasticity is an essential condition for cancer cells to invade surrounding tissues. The nucleus is the most rigid cellular organelle and it undergoes substantial deformations to get through environmental constrictions. Nuclear stiffness mostly depends on the nuclear lamina and chromatin, which in turn might be affected by nuclear architectural proteins. Among these is the HMGA1 (High Mobility Group A1) protein, a factor that plays a causal role in neoplastic transformation and that is able to disentangle heterochromatic domains by H1 displacement. Here we made use of atomic force microscopy to analyze the stiffness of breast cancer cellular models in which we modulated HMGA1 expression to investigate its role in regulating nuclear plasticity. Since histone H1 is the main modulator of chromatin structure and HMGA1 is a well-established histone H1 competitor, we correlated HMGA1 expression and cellular stiffness with histone H1 expression level, post-translational modifications, and nuclear distribution. Our results showed that HMGA1 expression level correlates with nuclear stiffness, is associated to histone H1 phosphorylation status, and alters both histone H1 chromatin distribution and expression. These data suggest that HMGA1 might promote chromatin relaxation through a histone H1-mediated mechanism strongly impacting on the invasiveness of cancer cells.

Falk Zakrzewski, Walter de Back, Martin Weigert, Torsten Wenke, Silke Zeugner, Robert Mantey, Christian Sperling, Katrin Friedrich, Ingo Roeder, Daniela Aust, Gustavo Baretton, Pia Hönscheid
Automated detection of the HER2 gene amplification status in Fluorescence in situ hybridization images for the diagnostics of cancer tissues.
Sci Rep, 9(1) Art. No. 8231 (2019)
Open Access DOI

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.

Rana Amini^#, Anastasia Labudina, Caren Norden^#
Stochastic single cell migration leads to robust horizontal cell layer formation in the vertebrate retina.
Development, 146(12) Art. No. dev173450 (2019)
DOI

Developmental programs that arrange cells and tissues into patterned organs are remarkably robust. In the developing vertebrate retina, for example, neurons reproducibly assemble into distinct layers giving the mature organ its overall structured appearance. This stereotypic neuronal arrangement, termed lamination, is important for efficient neuronal connectivity. Although retinal lamination is conserved in many vertebrates, including humans, how it emerges from single cell behaviour is not fully understood. To shed light on this issue, we here investigated the formation of the retinal horizontal cell layer. Using in vivo light sheet imaging of the developing zebrafish retina, we generated a comprehensive quantitative analysis of horizontal single cell behaviour from birth to final positioning. Interestingly, we find that all parameters analysed, including cell cycle dynamics, migration paths and kinetics, as well as sister cell dispersal, are very heterogeneous. Thus, horizontal cells show individual non-stereotypic behaviour before final positioning. Yet these initially variable cell dynamics always generate the correct laminar pattern. Consequently, our data show that the extent of single cell stochasticity in the lamination of the vertebrate retina is underexplored.

Edgar Boczek, Giorgio Gaglia, Maya Olshina, Shireen Sarraf
The first Autumn School on Proteostasis: from molecular mechanisms to organismal consequences.
Cell Stress Chaperones, 24(3) 481-492 (2019)
DOI

The first Autumn School on Proteostasis was held at the Mediterranean Institute for Life Sciences (MedILS) in Split, Croatia, from November 12th-16th, 2018, bringing together 44 graduate students and postdoctoral fellows and 22 principal investigators from around the world. This meeting was geared towards providing students with an in-depth understanding of the field of proteostasis, with the aim of broadening their perspectives of the field. Session topics covered multiple aspects of cellular and organismal proteostasis, including fundamental principles, responses to heat shock, aging and disease, and protein folding, misfolding, and degradation. The structure of the meeting and the restricted number of participants afforded the students and postdocs the opportunity to interact with principal investigators to discuss not only their latest research, but also their career prospects and progression in a close, supportive environment.

Ioannis Giannios, Ioannis Serafimidis, Vivian Anastasiou, Daniela Pezzolla, Mathias Lesche, Cordula Andree, Marc Bickle, Anthony Gavalas
Protein Methyltransferase Inhibition Decreases Endocrine Specification Through the Upregulation of Aldh1b1 Expression.
Stem Cells, 37(5) 640-651 (2019)
Open Access DOI

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self-renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into β-cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient β-cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI-5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640-651.

Satu Kujawski, Mahendra Sonawane, Elisabeth Knust
penner/lgl2 is required for the integrity of the photoreceptor layer in the zebrafish retina.
Biol Open, 8(4) Art. No. bio041830 (2019)
Open Access DOI

The vertebrate retina is a complex tissue built from multiple neuronal cell types, which develop from a pseudostratified neuroepithelium. These cells are arranged into a highly organized and stereotypic pattern formed by nuclear and plexiform layers. The process of lamination as well as the maturation and differentiation of photoreceptor cells rely on the establishment and maintenance of apico-basal cell polarity and formation of adhesive junctions. Defects in any of these processes can result in impaired vision and are causally related to a variety of human diseases leading to blindness. While the importance of apical polarity regulators in retinal stratification and disease is well established, little is known about the function of basal regulators in retinal development. Here, we analyzed the role of Lgl2, a basolateral polarity factor, in the zebrafish retina. Lgl2 is upregulated in photoreceptor cells and in the retinal pigment epithelium by 72 h post fertilization. In both cell types, Lgl2 is localized basolaterally. Loss of zygotic Lgl2 does not interfere with retinal lamination or photoreceptor cell polarity or maturation. However, knockdown of both maternal and zygotic Lgl2 leads to impaired cell adhesion. As a consequence, severe layering defects occur in the distal retina, manifested by a breakdown of the outer plexiform layer and the outer limiting membrane. These results define zebrafish Lgl2 as an important regulator of retinal lamination, which, given the high degree of evolutionary conservation, may be preserved in other vertebrates, including human.

Milos Kostic, Judith Paridaen, Katherine S. Long, Nereo Kalebic, Barbara Langen, Nannette Grübling, Pauline Wimberger, Hiroshi Kawasaki, Takashi Namba^#, Wieland Huttner^#
YAP Activity Is Necessary and Sufficient for Basal Progenitor Abundance and Proliferation in the Developing Neocortex.
Cell Rep, 27(4) 1103-1118 (2019)
Open Access DOI

Neocortex expansion during mammalian evolution has been linked to an increase in proliferation of basal progenitors in the subventricular zone. Here, we explored a potential role of YAP, the major downstream effector of the Hippo pathway, in proliferation of basal progenitors. YAP expression and activity are high in ferret and human basal progenitors, which exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. Conditional expression of a constitutively active YAP in mouse basal progenitors resulted in increased proliferation of basal progenitor and promoted production of upper-layer neurons. Pharmacological and genetic interference with YAP function in ferret and human developing neocortex resulted in decreased abundance of cycling basal progenitors. Together, our data indicate that YAP is necessary and sufficient to promote the proliferation of basal progenitors and suggest that increases in YAP levels and presumably activity contributed to the evolutionary expansion of the neocortex.

Landi Sun, Yuan Gao, Jianfeng He, Lihong Cui, Jana Meissner, Jean-Marc Verbavatz, Bo Li, Xiqiao Feng, Xin Liang
Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors.
Proc Natl Acad Sci U.S.A., 116(15) 7343-7352 (2019)
DOI

Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Their ultrastructures are key to understanding the molecular nature and mechanics of mechanotransduction. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila Each campaniform sensillum has an MO located at the distal tip of its dendrite. Here we analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. We focused on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). We found that NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC 29+29ARs confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC3 suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end. Our analysis reveals an ultrastructural pattern of NompC that is structurally and mechanically optimized for the sensory functions of campaniform mechanoreceptors.

Barbara Stepien^#, Wieland Huttner^#
Transport, Metabolism, and Function of Thyroid Hormones in the Developing Mammalian Brain.
Front Endocrinol (Lausanne), 10 Art. No. 209 (2019)
Open Access DOI

Ever since the discovery of thyroid hormone deficiency as the primary cause of cretinism in the second half of the 19th century, the crucial role of thyroid hormone (TH) signaling in embryonic brain development has been established. However, the biological understanding of TH function in brain formation is far from complete, despite advances in treating thyroid function deficiency disorders. The pleiotropic nature of TH action makes it difficult to identify and study discrete roles of TH in various aspect of embryogenesis, including neurogenesis and brain maturation. These challenges notwithstanding, enormous progress has been achieved in understanding TH production and its regulation, their conversions and routes of entry into the developing mammalian brain. The endocrine environment has to adjust when an embryo ceases to rely solely on maternal source of hormones as its own thyroid gland develops and starts to produce endogenous TH. A number of mechanisms are in place to secure the proper delivery and action of TH with placenta, blood-brain interface, and choroid plexus as barriers of entry that need to selectively transport and modify these hormones thus controlling their active levels. Additionally, target cells also possess mechanisms to import, modify and bind TH to further fine-tune their action. A complex picture of a tightly regulated network of transport proteins, modifying enzymes, and receptors has emerged from the past studies. TH have been implicated in multiple processes related to brain formation in mammals-neuronal progenitor proliferation, neuronal migration, functional maturation, and survival-with their exact roles changing over developmental time. Given the plethora of effects thyroid hormones exert on various cell types at different developmental periods, the precise spatiotemporal regulation of their action is of crucial importance. In this review we summarize the current knowledge about TH delivery, conversions, and function in the developing mammalian brain. We also discuss their potential role in vertebrate brain evolution and offer future directions for research aimed at elucidating TH signaling in nervous system development.

Stefan Münster, Akanksha Jain, Alexander Mietke, Anastasios Pavlopoulos, Stephan W. Grill^#, Pavel Tomancak^#
Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.
Nature, 568(7752) 395-399 (2019)
DOI

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the gastrulating tissue is surrounded by a rigid protective shell4. Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility5,6, it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle (Tribolium castaneum) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly (Drosophila melanogaster) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects.

Shashank Rai^*, Maryam Arasteh^*, Matthew Jefferson^*, Timothy Pearson, Yingxue Wang, Weijiao Zhang, Bertalan Bicsak, Devina Divekar, Penny P Powell, Ronald Naumann, Naiara Beraza, Simon R Carding, Oliver Florey, Ulrike Mayer^#, Thomas Wileman^#
The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.
Autophagy, 15(4) 599-612 (2019)
Open Access DOI

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.

Mirna Bilus, Maja Semanjski, Marko Mocibob, Igor Zivkovic, Nevena Cvetesic, Dan S Tawfik, Agnes Toth-Petroczy, Boris Macek, Ita Gruic-Sovulj
On the Mechanism and Origin of Isoleucyl-tRNA Synthetase Editing against Norvaline.
J Mol Biol, 431(6) 1284-1297 (2019)
DOI

Aminoacyl-tRNA synthetases (aaRSs), the enzymes responsible for coupling tRNAs to their cognate amino acids, minimize translational errors by intrinsic hydrolytic editing. Here, we compared norvaline (Nva), a linear amino acid not coded for protein synthesis, to the proteinogenic, branched valine (Val) in their propensity to mistranslate isoleucine (Ile) in proteins. We show that in the synthetic site of isoleucyl-tRNA synthetase (IleRS), Nva and Val are activated and transferred to tRNA at similar rates. The efficiency of the synthetic site in pre-transfer editing of Nva and Val also appears to be similar. Post-transfer editing was, however, more rapid with Nva and consequently IleRS misaminoacylates Nva-tRNAIle at slower rate than Val-tRNAIle. Accordingly, an Escherichia coli strain lacking IleRS post-transfer editing misincorporated Nva and Val in the proteome to a similar extent and at the same Ile positions. However, Nva mistranslation inflicted higher toxicity than Val, in agreement with IleRS editing being optimized for hydrolysis of Nva-tRNAIle. Furthermore, we found that the evolutionary-related IleRS, leucyl- and valyl-tRNA synthetases (I/L/VRSs), all efficiently hydrolyze Nva-tRNAs even when editing of Nva seems redundant. We thus hypothesize that editing of Nva-tRNAs had already existed in the last common ancestor of I/L/VRSs, and that the editing domain of I/L/VRSs had primarily evolved to prevent infiltration of Nva into modern proteins.

Naomi D Chrispijn, Dei M Elurbe, Michaela Mickoleit, Marco Aben, Dennis E M de Bakker, Karolina M Andralojc, Jan Huisken, Jeroen Bakkers, Leonie M Kamminga
Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development.
Sci Rep, 9(1) Art. No. 4327 (2019)
Open Access DOI

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

Jonathan Rodenfels, Karla M. Neugebauer, Jonathon Howard
Heat Oscillations Driven by the Embryonic Cell Cycle Reveal the Energetic Costs of Signaling.
Dev Cell, 48(5) 646-658 (2019)
DOI

All living systems function out of equilibrium and exchange energy in the form of heat with their environment. Thus, heat flow can inform on the energetic costs of cellular processes, which are largely unknown. Here, we have repurposed an isothermal calorimeter to measure heat flow between developing zebrafish embryos and the surrounding medium. Heat flow increased over time with cell number. Unexpectedly, a prominent oscillatory component of the heat flow, with periods matching the synchronous early reductive cleavage divisions, persisted even when DNA synthesis and mitosis were blocked by inhibitors. Instead, the heat flow oscillations were driven by the phosphorylation and dephosphorylation reactions catalyzed by the cell-cycle oscillator, the biochemical network controlling mitotic entry and exit. We propose that the high energetic cost of cell-cycle signaling reflects the significant thermodynamic burden of imposing accurate and robust timing on cell proliferation during development.

Lydia Reinhardt, Susanne Kordes, Peter Reinhardt, Michael Glatza, Michael Baumann, Hannes C A Drexler, Sascha Menninger, Gunther Zischinsky, Jan Eickhoff, Claudia Fröb, Prabesh Bhattarai, Guruchandar Arulmozhivarman, Lara Marrone, Antje Janosch, Kenjiro Adachi, Martin Stehling, Eric D Anderson, Masin Abo-Rady, Marc Bickle, Udai Pandey, Michell M Reimer, Caghan Kizil, Hans R Schöler, Peter Nussbaumer, Bert Klebl, Jared Sterneckert
Dual Inhibition of GSK3β and CDK5 Protects the Cytoskeleton of Neurons from Neuroinflammatory-Mediated Degeneration In Vitro and In Vivo.
Stem Cell Rep, 12(3) 502-517 (2019)
Open Access DOI

Neuroinflammation is a hallmark of neurological disorders and is accompanied by the production of neurotoxic agents such as nitric oxide. We used stem cell-based phenotypic screening and identified small molecules that directly protected neurons from neuroinflammation-induced degeneration. We demonstrate that inhibition of CDK5 is involved in, but not sufficient for, neuroprotection. Instead, additional inhibition of GSK3β is required to enhance the neuroprotective effects of CDK5 inhibition, which was confirmed using short hairpin RNA-mediated knockdown of CDK5 and GSK3β. Quantitative phosphoproteomics and high-content imaging demonstrate that neurite degeneration is mediated by aberrant phosphorylation of multiple microtubule-associated proteins. Finally, we show that our hit compound protects neurons in vivo in zebrafish models of motor neuron degeneration and Alzheimer's disease. Thus, we demonstrate an overlap of CDK5 and GSK3β in mediating the regulation of the neuronal cytoskeleton and that our hit compound LDC8 represents a promising starting point for neuroprotective drugs.

Marc Bickle
The Academic Pill: How Academia Contributes to Curing Diseases.
SLAS Discov, 24(3) 203-212 (2019)
Open Access DOI

Sara Caviglia, David Flores-Benitez, Johanna Lattner, Stefan Luschnig, Marko Brankatschk
Rabs on the fly: Functions of Rab GTPases during development.
Small GTPases, 10(2) 89-98 (2019)
Open Access DOI

The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.

Germán Camargo Ortega, Sven Falk, Pia A Johansson, Elise Peyre, Loïc Broix, Sanjeeb Kumar Sahu, William Hirst, Thomas Schlichthaerle, Camino De Juan Romero, Kalina Draganova, Stanislav Vinopal, Kaviya Chinnappa, Anna Gavranovic, Tugay Karakaya, Thomas Steininger, Juliane Merl-Pham, Regina Feederle, Wei Shao, Song-Hai Shi, Stefanie M Hauck, Ralf Jungmann, Frank Bradke, Víctor Borrell, Arie Geerlof, Simone Reber, Vijay K Tiwari, Wieland Huttner, Michaela Wilsch-Bräuninger, Laurent Nguyen, Magdalena Götz
The centrosome protein AKNA regulates neurogenesis via microtubule organization.
Nature, 567(7746) 113-117 (2019)
DOI

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Elodie Prince, Benjamin Kroeger, Dragan Gligorov, Clive Wilson, Suzanne Eaton, François Karch, Marko Brankatschk, Robert K Maeda
Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity.
Traffic, 20(2) 137-151 (2019)
Open Access DOI

The male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only ~4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, we investigated their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these organelles require Rab6 for their formation and many are essential in the process of creating the long-term postmating behavior of females. In order to better serve the intracellular membrane and protein trafficking communities, we have created a searchable, online, open-access imaging resource to display our complete findings regarding Rab localization in the accessory gland.

Katherine S. Long^#, Wieland Huttner^#
How the extracellular matrix shapes neural development.
Open Biol, 9(1) Art. No. 180216 (2019)
Open Access DOI

During development, both cells and tissues must acquire the correct shape to allow their proper function. This is especially relevant in the nervous system, where the shape of individual cell processes, such as the axons and dendrites, and the shape of entire tissues, such as the folding of the neocortex, are highly specialized. While many aspects of neural development have been uncovered, there are still several open questions concerning the mechanisms governing cell and tissue shape. In this review, we discuss the role of the extracellular matrix (ECM) in these processes. In particular, we consider how the ECM regulates cell shape, proliferation, differentiation and migration, and more recent work highlighting a key role of ECM in the morphogenesis of neural tissues.

Kassiani Skouloudaki^*^#, Dimitrios Papadopoulos^*, Pavel Tomancak, Elisabeth Knust^#
The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation.
PLoS Genet, 15(1) Art. No. e1007852 (2019)
Open Access DOI

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.

Egon Deyaert, Margaux Leemans, Ranjan Kumar Singh, Rodrigo Gallardo, Jan Steyaert, Arjan Kortholt, Janelle Lauer^#, Wim Versées^#
Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein.
Biochem J, 476(1) 51-66 (2019)
DOI

The LRR (leucine-rich repeat)-Roc (Ras of complex proteins)-COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer-monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5'-diphosphate)-bound states and monomeric in the GTP (guanosine-5'-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR-Roc-COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the 'Roc dimerization loop'. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

Albert Thommen^*, Steffen Werner^*, Olga Frank^*, Jenny Philipp, Oskar Knittelfelder, Yihui Quek, Karim Fahmy, Andrej Shevchenko, Benjamin Friedrich, Frank Jülicher^#, Jochen Rink^#
Body size-dependent energy storage causes Kleiber's law scaling of the metabolic rate in planarians.
Elife, 8 Art. No. e38187 (2019)
Open Access DOI

Kleiber's law, or the 3/4 -power law scaling of the metabolic rate with body mass, is considered one of the few quantitative laws in biology, yet its physiological basis remains unknown. Here, we report Kleiber's law scaling in the planarian Schmidtea mediterranea. Its reversible and life history-independent changes in adult body mass over 3 orders of magnitude reveal that Kleiber's law does not emerge from the size-dependent decrease in cellular metabolic rate, but from a size-dependent increase in mass per cell. Through a combination of experiment and theoretical analysis of the organismal energy balance, we further show that the mass allometry is caused by body size dependent energy storage. Our results reveal the physiological origins of Kleiber's law in planarians and have general implications for understanding a fundamental scaling law in biology.

Johannes Girstmair, Maximilian J Telford
Reinvestigating the early embryogenesis in the flatworm Maritigrella crozieri highlights the unique spiral cleavage program found in polyclad flatworms.
Evodevo, 10 Art. No. 12 (2019)
Open Access DOI

Spiral cleavage is a conserved, early developmental mode found in several phyla of Lophotrochozoans resulting in highly diverse adult body plans. While the cleavage pattern has clearly been broadly conserved, it has also undergone many modifications in various taxa. The precise mechanisms of how different adaptations have altered the ancestral spiral cleavage pattern are an important ongoing evolutionary question, and adequately answering this question requires obtaining a broad developmental knowledge of different spirally cleaving taxa. In flatworms (Platyhelminthes), the spiral cleavage program has been lost or severely modified in most taxa. Polyclad flatworms, however, have retained the pattern up to the 32-cell stage. Here we study early embryogenesis of the cotylean polyclad flatworm Maritigrella crozieri to investigate how closely this species follows the canonical spiral cleavage pattern and to discover any potential deviations from it.

Ulrik Günther, Tobias Pietzsch, Aryaman Gupta, Kyle Harrington, Pavel Tomancak, Stefan Gumhold, Ivo F. Sbalzarini
scenery: Flexible Virtual Reality Visualization on the Java VM.
In: 2019 IEEE Visualization Conference (VIS) (2019), Piscataway, N.J., IEEE (2019), 166-170

Kristina Thamm, Deimantė Šimaitė, Jana Karbanová, Vicente Bermúdez, Doreen Reichert, Anne Morgenstern, Martin Bornhäuser, Wieland Huttner, Michaela Wilsch-Bräuninger, Denis Corbeil
Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.
Traffic, 20(1) 39-60 (2019)
DOI

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Lakshmi Balasubramanian, Vanessa Zuzarte-Luís, Tabish Syed, Debakshi Mullick, Saptarathi Deb, Harish Ranga-Prasad, Jana Meissner, Ana Almeida, Tobias Furstenhaupt, Kaleem Siddiqi, Miguel Prudêncio, Cecilia M P Rodrigues, Maria M Mota, Varadharajan Sundaramurthy
Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.
Front Cell Infect Microbiol, 9 Art. No. 451 (2019)
Open Access DOI

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Elisabeth Zinser, Ronald Naumann, Andreas B Wild, Julia Michalski, Andrea Deinzer, Lena Stich, Christine Kuhnt, Alexander Steinkasserer, Ilka Knippertz
Endogenous Expression of the Human CD83 Attenuates EAE Symptoms in Humanized Transgenic Mice and Increases the Activity of Regulatory T Cells.
Front Immunol, 10 Art. No. 1442 (2019)
Open Access DOI

The CD83 is a type I membrane protein and part of the immunoglobulin superfamily of receptors. CD83 is involved in the regulation of antigen presentation and dendritic cell dependent allogeneic T cell proliferation. A soluble form of CD83 inhibits dendritic cell maturation and function. Furthermore, CD83 is expressed on activated B cells, T cells, and in particular on regulatory T cells. Previous studies on murine CD83 demonstrated this molecule to be involved in several immune-regulatory processes, comprising that CD83 plays a key role in the development und function of different immune cells. In order to get further insights into the function of the human CD83 and to provide preclinical tools to guide the function of CD83/sCD83 for therapeutic purposes we generated Bacterial Artificial Chromosomes (BAC) transgenic mice. BACs are excellent tools for manipulating large DNA fragments and are utilized to engineer transgenic mice by pronuclear injection. Two different founders of BAC transgenic mice expressing human CD83 (BAC-hCD83tg mice) were generated and were examined for the hCD83 expression on different immune cells as well as both the in vitro and in vivo role of human CD83 (hCD83) in health and disease. Here, we found the hCD83 molecule to be present on activated DCs, B cells and subtypes of CD4+ T cells. CD8+ T cells, on the other hand, showed almost no hCD83 expression. To address the function of hCD83, we performed in vitro mixed lymphocyte reactions (MLR) as well as suppression assays and we used the in vivo model of experimental autoimmune encephalomyelitis (EAE) comparing wild-type and hCD83-BAC mice. Results herein showed a clearly diminished capacity of hCD83-BAC-derived T cells to proliferate accompanied by an enhanced activation and suppressive activity of hCD83-BAC-derived Tregs. Furthermore, hCD83-BAC mice were found to recover faster from EAE-associated symptoms than wild-type mice, encouraging the relevance also of the hCD83 as a key molecule for the regulatory phenotype of Tregs in vitro and in vivo.

Malte Lehmann, Elisabeth Knust, Sarita Hebbar
Drosophila melanogaster: A Valuable Genetic Model Organism to Elucidate the Biology of Retinitis Pigmentosa.
Methods Mol Biol, 1834 221-249 (2019)
DOI

Retinitis pigmentosa (RP) is a complex inherited disease. It is associated with mutations in a wide variety of genes with many different functions. These mutations impact the integrity of rod photoreceptors and ultimately result in the progressive degeneration of rods and cone photoreceptors in the retina, leading to complete blindness. A hallmark of this disease is the variable degree to which symptoms are manifest in patients. This is indicative of the influence of the environment, and/or of the distinct genetic makeup of the individual.The fruit fly, Drosophila melanogaster, has effectively proven to be a great model system to better understand interconnected genetic networks. Unraveling genetic interactions and thereby different cellular processes is relatively easy because more than a century of research on flies has enabled the creation of sophisticated genetic tools to perturb gene function. A remarkable conservation of disease genes across evolution and the similarity of the general organization of the fly and vertebrate photoreceptor cell had prompted research on fly retinal degeneration. To date six fly models for RP, including RP4, RP11, RP12, RP14, RP25, and RP26, have been established, and have provided useful information on RP disease biology. In this chapter, an outline of approaches and experimental specifications are described to enable utilizing or developing new fly models of RP.

Rachel Kraut, Elisabeth Knust
Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina.
PLoS ONE, 14(12) Art. No. e220220 (2019)
Open Access DOI

Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. We therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented here show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. We propose that these changes may be early signs of the degeneration-prone condition in crb retinas.

Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila Compound Eye with Light and Electron Microscopy.
Methods Mol Biol, 1834 345-364 (2019)
DOI

The Drosophila compound eye is composed of about 750 units, called ommatidia, which are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their functions are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes, even those without a Drosophila counterpart, can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology and retinal histology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by light and electron microscopy.

Joan Sim, Kathleen Amy Osborne, Irene Argudo García, Artur Matysik, Rachel Kraut
The BEACH Domain Is Critical for Blue Cheese Function in a Spatial and Epistatic Autophagy Hierarchy.
Front Cell Dev Biol, 7 Art. No. 129 (2019)
Open Access DOI

Drosophila blue cheese (bchs) encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. bchs mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on bchs-related degeneration, nor bchs' placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in bchs mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation. Further, pharmacological rescue requires at least one intact BEACH-containing isoform of the two identified in this study. Genetically augmenting a late step in autophagy, however, rescues even a strong mutation which retains only a third, non-BEACH containing isoform. Using living primary larval brain neurons, we elucidate the primary defect in bchs to be an excess of early autophagic compartments and a deficit in mature compartments. Conversely, rescuing the mutants by full-length Bchs over-expression induces mature compartment proliferation and rescues neuronal death. Surprisingly, only the longest Bchs isoform colocalizes well with autophagosomes, and shuttles between different vesicular locations depending on the type of autophagic impetus applied. Our results are consistent with Bchs promoting autophagic maturation, and the BEACH domain being required for this function.

Michal Krumnikl, Petr Bainar, Jana Klímová, Jan Kožusznik, Pavel Moravec, Václav Svatoň, Pavel Tomančák
SciJava Interface for Parallel Execution in the ImageJ Ecosystem.
In: Computer Information Systems and Industrial Management (2018)(Eds.) Władysław Homenda Lecture Notes in Computer Science, Cham, Springer International Publishing (2018), 288-299
DOI

Gulce Itir Percin, Jiri Eitler, Andrea Kranz, Jun Fu, Jeffrey W Pollard, Ronald Naumann, Claudia Waskow
CSF1R regulates the dendritic cell pool size in adult mice via embryo-derived tissue-resident macrophages.
Nat Commun, 9(1) Art. No. 5279 (2018)
Open Access DOI

Regulatory mechanisms controlling the pool size of spleen dendritic cells (DC) remain incompletely understood. DCs are continuously replenished from hematopoietic stem cells, and FLT3-mediated signals cell-intrinsically regulate homeostatic expansion of spleen DCs. Here we show that combining FLT3 and CSF1R-deficiencies results in specific and complete abrogation of spleen DCs in vivo. Spatiotemporally controlled CSF1R depletion reveals a cell-extrinsic and non-hematopoietic mechanism for DC pool size regulation. Lack of CSF1R-mediated signals impedes the differentiation of spleen macrophages of embryonic origin, and the resulted macrophage depletion during development or in adult mice results in loss of DCs. Moreover, embryo-derived macrophages are important for the physiologic regeneration of DC after activation-induced depletion in situ. In summary, we show that the differentiation of DC and their regeneration relies on ontogenetically distinct spleen macrophages, thereby providing a novel regulatory principle that may also be important for the differentiation of other hematopoietic cell types.

Anna Czarkwiani, Maximina H Yun
Out with the old, in with the new: senescence in development.
Curr Opin Cell Biol, 55 74-80 (2018)
DOI

Cellular senescence is a ubiquitous stress response that restricts the proliferative capacity of cells. During ageing, senescent cells accumulate in various tissues leading to a number of age-related pathologies and physiological decline. Previously thought to be a process restricted to adult organisms, cellular senescence has been recently demonstrated to occur during embryonic development of animals ranging from fish to mammals. Together, these studies suggest that developmentally programmed senescence is a transient but intrinsic biological process that contributes to the remodelling of developing structures by promoting immune-mediated cell clearance of particular cell populations or modifying the tissue microenvironment. These observations have important implications for the evolutionary origins of this essential, yet paradoxical mechanism.

Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, Sian Culley, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, Ricardo Henriques, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W Myers
Content-aware image restoration: pushing the limits of fluorescence microscopy.
Nat Methods, 15(12) 1090-1097 (2018)
DOI

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

Juliana G. Roscito, Katrin Sameith, Martin Pippel, Kees-Jan Francoijs, Sylke Winkler, Andreas Dahl, Georg Papoutsoglou, Gene Myers, Michael Hiller
The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.
GigaScience, 7(12) Art. No. giy141 (2018)
Open Access PDF DOI

Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.

Dongcheng Zhang, James M Osborne, Kwaku Dad Abu-Bonsrah, Bevan Cheeseman, Kerry A Landman, Boaz Jurkowicz, Donald F Newgreen
Stochastic clonal expansion of "superstars" enhances the reserve capacity of enteric nervous system precursor cells.
Dev Biol, 444 Suppl 1 287-296 (2018)
DOI

We quantified cell population increase in the quail embryo enteric nervous system (ENS) from E2.5 (about 1500 cells) to E12 (about 8 million cells). We then probed ENS proliferative capacity by grafting to the chorio-allantoic membrane large (600 cells) and small (40 cells) populations of enteric neural crest (ENC) cells with aneural gut. This demonstrated that ENC cells show an extremely high capacity to regulate their proliferation while forming the ENS. Previous mathematical models and clonal label experiments revealed that a few dominant ENS "superstar" cell clones emerge but most clones are small. The model implied that "superstars" arise stochastically, but the same outcome could arise if "superstars" were pre-determined. We investigated these two modes mathematically and by grafting experiments with large and small numbers of ENCs, each including one EGFP-labelled ENC cell. The stochastic model predicts that the frequency of "superstar" detection increases as the ENC population decreases, the pre-determined model does not. Experimentally, as predicted by the stochastic model, the frequency of "superstar" detection increased with small ENC cell number. We conclude that ENS "superstar" clones achieve this status stochastically. Clonal dominance implies that clonal diversity is greatly reduced and in this case, somatic mutations may affect the phenotype. We suggest that somatic mutations coupled with loss of clonal diversity may contribute to variable penetrance and expressivity in individuals with genetically identical ENS pathologies.

Virag Sharma, Michael Hiller
Loss of Enzymes in the Bile Acid Synthesis Pathway Explains Differences in Bile Composition among Mammals.
Genome Biol Evol, 10(12) 3211-3217 (2018)
Open Access PDF DOI

Bile acids are important for absorbing nutrients. Most mammals produce cholic and chenodeoxycholic bile acids. Here, we investigated genes in the bile acid synthesis pathway in four mammals that deviate from the usual mammalian bile composition. First, we show that naked-mole rats, elephants, and manatees repeatedly inactivated CYP8B1, an enzyme uniquely required for cholic acid synthesis, which explains the absence of cholic acid in these species. Second, no gene-inactivating mutations were found in any pathway gene in the rhinoceros, a species that lacks bile acids, indicating an evolutionarily recent change in its bile composition. Third, elephants and/or manatees that also lack bile acids altogether have lost additional nonessential enzymes (SLC27A5, ACOX2). Apart from uncovering genomic differences explaining deviations in bile composition, our analysis of bile acid enzymes in bile acid-lacking species suggests that essentiality prevents gene loss, while loss of pleiotropic genes is permitted if their other functions are compensated by functionally related proteins.

Pierre A. Haas^#, Raymond E. Goldstein^#
Embryonic Inversion in Volvox carteri: The Flipping and Peeling of Elastic Lips.
Phys Rev E, 98(5) Art. No. 052415 (2018)
Open Access DOI

The embryos of the green alga Volvox carteri are spherical sheets of cells that turn themselves inside out at the close of their development through a programme of cell shape changes. This process of inversion is a model for morphogenetic cell sheet deformations; it starts with four lips opening up at the anterior pole of the cell sheet, flipping over and peeling back to invert the embryo. Experimental studies have revealed that inversion is arrested if some of these cell shape changes are inhibited, but the mechanical basis for these observations has remained unclear. Here, we analyse the mechanics of this inversion by deriving an averaged elastic theory for these lips and we interpret the experimental observations in terms of the mechanics and evolution of inversion.

Erika Cecon, Anna Ivanova, Marine Luka, Florence Gbahou, Anne Friederich, Jean-Luc Guillaume, Patrick Keller, Klaus-Peter Knoch, Raise Ahmad, Philippe Delagrange, Michele Solimena, Ralf Jockers
Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C-terminal domain.
J Pineal Res, 66(2) Art. No. e12540 (2018)
DOI

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24h sleep-wake disorders and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) super-family. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors prooved to be difficult to obtain. Here we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat and human MT1 and MT2 by western blot, immunoprecipitation, immunofluorescence and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR KO mice as control. MT2 expression was not detected instead in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies. This article is protected by copyright. All rights reserved.

Juliana G. Roscito^*, Katrin Sameith^*, Genis Parra^*, Bjoern Langer, Andreas Petzold, Claudia Moebius, Marc Bickle, Miguel Trefaut Rodrigues, Michael Hiller
Phenotype loss is associated with widespread divergence of the gene regulatory landscape in evolution.
Nat Commun, 9(1) Art. No. 4737 (2018)
Open Access PDF DOI

Detecting the genomic changes underlying phenotypic changes between species is a main goal of evolutionary biology and genomics. Evolutionary theory predicts that changes in cis-regulatory elements are important for morphological changes. We combined genome sequencing, functional genomics and genome-wide comparative analyses to investigate regulatory elements in lineages that lost morphological traits. We first show that limb loss in snakes is associated with widespread divergence of limb regulatory elements. We next show that eye degeneration in subterranean mammals is associated with widespread divergence of eye regulatory elements. In both cases, sequence divergence results in an extensive loss of transcription factor binding sites. Importantly, diverged regulatory elements are associated with genes required for normal limb patterning or normal eye development and function, suggesting that regulatory divergence contributed to the loss of these phenotypes. Together, our results show that genome-wide decay of the phenotype-specific cis-regulatory landscape is a hallmark of lost morphological traits.

Mareike A Jordan, Dennis R Diener, Ludek Stepanek, Gaia Pigino
The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia.
Nat Cell Biol, 20(11) 1250-1255 (2018)
DOI

Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself1. This bidirectional transport, known as intraflagellar transport (IFT)2, is driven by the anterograde motor kinesin-23 and the retrograde motor dynein-1b (dynein-2 in mammals)4,5. However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT6. Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos7, IFT is highly processive1,2,8. Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery.

Sundar Ram Naganathan^#, Sebastian Fürthauer, Josana Rodriguez, Bruno Thomas Fievet, Frank Jülicher, Julie Ahringer, Carlo Vittorio Cannistraci, Stephan W. Grill^#
Morphogenetic degeneracies in the actomyosin cortex.
Elife, 7 Art. No. e37677 (2018)
Open Access DOI

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

Samir Vaid, J Gray Camp, Lena Hersemann, Christina Eugster Oegema, Anne-Kristin Heninger, Sylke Winkler, Holger Brandl, Mihail Sarov, Barbara Treutlein, Wieland Huttner^#, Takashi Namba^#
A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.
Development, 145(20) Art. No. dev169276 (2018)
DOI

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

David Jebb, Michael Hiller
Recurrent loss of HMGCS2 shows that ketogenesis is not essential for the evolution of large mammalian brains.
Elife, 7 Art. No. e38906 (2018)
Open Access PDF DOI

Apart from glucose, fatty acid-derived ketone bodies provide metabolic energy for the brain during fasting and neonatal development. We investigated the evolution of HMGCS2, the key enzyme required for ketone body biosynthesis (ketogenesis). Unexpectedly, we found that three mammalian lineages, comprising cetaceans (dolphins and whales), elephants and mastodons, and Old World fruit bats have lost this gene. Remarkably, many of these species have exceptionally large brains and signs of intelligent behavior. While fruit bats are sensitive to starvation, cetaceans and elephants can still withstand periods of fasting. This suggests that alternative strategies to fuel large brains during fasting evolved repeatedly and reveals flexibility in mammalian energy metabolism. Furthermore, we show that HMGCS2 loss preceded brain size expansion in toothed whales and elephants. Thus, while ketogenesis was likely important for brain size expansion in modern humans, ketogenesis is not a universal precondition for the evolution of large mammalian brains.

Steven D. Briscoe, Clifton W. Ragsdale
Homology, neocortex, and the evolution of developmental mechanisms.
Science, 362(6411) 190-193 (2018)
DOI

The six-layered neocortex of the mammalian pallium has no clear homolog in birds or non-avian reptiles. Recent research indicates that although these extant amniotes possess a variety of divergent and nonhomologous pallial structures, they share a conserved set of neuronal cell types and circuitries. These findings suggest a principle of brain evolution: that natural selection preferentially preserves the integrity of information-processing pathways, whereas other levels of biological organization, such as the three-dimensional architectures of neuronal assemblies, are less constrained. We review the similarities of pallial neuronal cell types in amniotes, delineate candidate gene regulatory networks for their cellular identities, and propose a model of developmental evolution for the divergence of amniote pallial structures.

Thomas A Hopf, Anna G Green, Benjamin Schubert, Sophia Mersmann, Charlotta P I Schärfe, John Ingraham, Agnes Toth-Petroczy, Kelly Brock, Adam J Riesselman, Perry Palmedo, ChulHee Kang, Robert Sheridan, Eli J Draizen, Christian Dallago, Chris Sander, Debora S Marks
The EVcouplings Python framework for coevolutionary sequence analysis.
Bioinformatics, Art. No. doi: 10.1093/bioinformatics/bty862 (2018)
DOI

Coevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The combination of an easy to use, flexible command line interface and an underlying modular Python package makes the full power of coevolutionary analyses available to entry-level and advanced users.

Petra Schwille, Joachim P. Spatz, Katharina Landfester, Eberhard Bodenschatz, Stephan Herminghaus, Victor Sourjik, Tobias Erb, Philippe Bastiaens, Reinhard Lipowsky, Anthony Hyman, Peter Dabrock, Jean-Christophe Baret, Tanja Vidakovic-Koch, Peter Bieling, Rumiana Dimova, Hannes Mutschler, Tom Robinson, T-Y Dora Tang, Seraphine Wegner, Kai Sundmacher
MaxSynBio: Avenues Towards Creating Cells from the Bottom Up.
Angew Chem Int Ed Engl, 57(41) 13382-13392 (2018)
DOI

A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biology. MaxSynBio focuses on the detailed analysis and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnological processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnology.

Natalia Petersen, Thomas M Frimurer, Marianne Terndrup Pedersen, Kristoffer L Egerod, Nicolai J Wewer Albrechtsen, Jens J Holst, Anne Grapin-Botton, Kim B Jensen, Thue W Schwartz
Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine.
Gastroenterology, 155(4) 1164-1176 (2018)
DOI

Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation.

Marko Brankatschk^#, Theresia Gutmann, Oskar Knittelfelder, Alessandra Palladini, Elodie Prince, Michal Grzybek, Beate Brankatschk, Andrej Shevchenko, Ünal Coskun, Suzanne Eaton^#
A Temperature-Dependent Switch in Feeding Preference Improves Drosophila Development and Survival in the Cold.
Dev Cell, 46(6) 781-793 (2018)
DOI

Christiane Rennert, Sebastian Vlaic, Eugenia Marbach-Breitrück, Carlo Thiel, Susanne Sales, Andrej Shevchenko, Rolf Gebhardt, Madlen Matz-Soja
The Diurnal Timing of Starvation Differently Impacts Murine Hepatic Gene Expression and Lipid Metabolism - A Systems Biology Analysis Using Self-Organizing Maps.
Front Physiol, 9 Art. No. 1180 (2018)
DOI

Organisms adapt their metabolism and draw on reserves as a consequence of food deprivation. The central role of the liver in starvation response is to coordinate a sufficient energy supply for the entire organism, which has frequently been investigated. However, knowledge of how circadian rhythms impact on and alter this response is scarce. Therefore, we investigated the influence of different timings of starvation on global hepatic gene expression. Mice (n = 3 each) were challenged with 24-h food deprivation started in the morning or evening, coupled with refeeding for different lengths and compared with ad libitum fed control groups. Alterations in hepatocyte gene expression were quantified using microarrays and confirmed or complemented with qPCR, especially for lowly detectable transcription factors. Analysis was performed using self-organizing maps (SOMs), which bases on clustering genes with similar expression profiles. This provides an intuitive overview of expression trends and allows easier global comparisons between complex conditions. Transcriptome analysis revealed a strong circadian-driven response to fasting based on the diurnal expression of transcription factors (e.g., Ppara, Pparg). Starvation initiated in the morning produced known metabolic adaptations in the liver; e.g., switching from glucose storage to consumption and gluconeogenesis. However, starvation initiated in the evening produced a different expression signature that was controlled by yet unknown regulatory mechanisms. For example, the expression of genes involved in gluconeogenesis decreased and fatty acid and cholesterol synthesis genes were induced. The differential regulation after morning and evening starvation were also reflected at the lipidome level. The accumulation of hepatocellular storage lipids (triacylglycerides, cholesteryl esters) was significantly higher after the initiation of starvation in the morning compared to the evening. Concerning refeeding, the gene expression pattern after a 12 h refeeding period largely resembled that of the corresponding starvation state but approached the ad libitum control state after refeeding for 21 h. Some components of these regulatory circuits are discussed. Collectively, these data illustrate a highly time-dependent starvation response in the liver and suggest that a circadian influence cannot be neglected when starvation is the focus of research or medicine, e.g., in the case of treating victims of sudden starvation events.

Bianca S Heinrich^*, Zoltan Maliga^*, David A Stein, Anthony A. Hyman^#, Sean P J Whelan^#
Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments.
MBio, 9(5) Art. No. e02290-17 (2018)
Open Access DOI

RNA viruses that replicate in the cell cytoplasm typically concentrate their replication machinery within specialized compartments. This concentration favors enzymatic reactions and shields viral RNA from detection by cytosolic pattern recognition receptors. Nonsegmented negative-strand (NNS) RNA viruses, which include some of the most significant human, animal, and plant pathogens extant, form inclusions that are sites of RNA synthesis and are not circumscribed by a membrane. These inclusions share similarities with cellular protein/RNA structures such as P granules and nucleoli, which are phase-separated liquid compartments. Here we show that replication compartments of vesicular stomatitis virus (VSV) have the properties of liquid-like compartments that form by phase separation. Expression of the individual viral components of the replication machinery in cells demonstrates that the 3 viral proteins required for replication are sufficient to drive cytoplasmic phase separation. Therefore, liquid-liquid phase separation, previously linked to organization of P granules, nucleolus homeostasis, and cell signaling, plays a key role in host-pathogen interactions. This work suggests novel therapeutic approaches to the problem of combating NNS RNA viral infections.IMPORTANCE RNA viruses compartmentalize their replication machinery to evade detection by host pattern recognition receptors and concentrate the machinery of RNA synthesis. For positive-strand RNA viruses, RNA replication occurs in a virus-induced membrane-associated replication organelle. For NNS RNA viruses, the replication compartment is a cytoplasmic inclusion that is not circumscribed by a cellular membrane. Such structures were first observed in the cell bodies of neurons from humans infected with rabies virus and were termed Negri bodies. How the replication machinery that forms this inclusion remains associated in the absence of a membrane has been an enduring mystery. In this article, we present evidence that the VSV replication compartments form through phase separation. Phase separation is increasingly recognized as responsible for cellular structures as diverse as processing bodies (P-bodies) and nucleoli and was recently demonstrated for rabies virus. This article further links the fields of host-pathogen interaction with that of phase separation.

Katherine S. Long, Ben Newland, Marta Florio, Nereo Kalebic, Barbara Langen, Anna Kolterer, Pauline Wimberger, Wieland Huttner
Extracellular Matrix Components HAPLN1, Lumican, and Collagen I Cause Hyaluronic Acid-Dependent Folding of the Developing Human Neocortex.
Neuron, 99(4) 702-719 (2018)
DOI

Neocortical expansion, thought to underlie the cognitive traits unique to humans, is accompanied by cortical folding. This folding starts around gestational week (GW) 20, but what causes it remains largely unknown. Extracellular matrix (ECM) has been previously implicated in neocortical expansion and here we investigate the potential role of ECM in the formation of neocortical folds. We focus on three specific ECM components localized in the human fetal cortical plate (CP): hyaluronan and proteoglycan link protein 1 (HAPLN1), lumican and collagen I (collectively, HLC). Addition of HLC to cultures of human fetal neocortex (11-22 GW) caused local changes in tissue stiffness, induced CP folding, increased CP hyaluronic acid (HA), and required the HA-receptor CD168 and downstream ERK signaling. Importantly, loss of HA reduced HLC-induced and 22 GW physiological nascent folds. This was altered in samples with neurodevelopmental disorders, indicating it may be a useful system to study such disorders.

Lucas Lacasa, Inés P. Mariño, Joaquin Miguez, Vincenzo Nicosia, Édgar Roldán, Ana Lisica, Stephan W. Grill, Jesús Gómez-Gardeñes
Multiplex Decomposition of Non-Markovian Dynamics and the Hidden Layer Reconstruction Problem
Physical Review X, 8(3) Art. No. 031038 (2018)
Open Access DOI

Miroslav P Ivanov, Rene Ladurner, Ina Poser, Rebecca Beveridge, Evelyn Rampler, Otto Hudecz, Maria Novatchkova, Jean-Karim Hériché, Gordana Wutz, Petra van der Lelij, Emanuel Kreidl, James R A Hutchins, Heinz Axelsson-Ekker, Jan Ellenberg, Anthony Hyman, Karl Mechtler, Jan-Michael Peters
The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion.
EMBO J, 37(15) Art. No. e97150 (2018)
DOI

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.

Jie Wang, Jeong-Mo Choi, Alex S Holehouse, Hyun O. Lee, Xiaojie Zhang, Marcus Jahnel, Shovamayee Maharana, Regis P. Lemaitre, Andrei I. Pozniakovsky, David N. Drechsel, Ina Poser, Rohit V Pappu, Simon Alberti, Anthony Hyman
A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.
Cell, 174(3) 688-699 (2018)
DOI

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences.

Svend Bertel Dahl-Jensen, Siham Yennek, Lydie Flasse, Hjalte List Larsen, Dror Sever, Gopal Karremore, Ivana Novak, Kim Sneppen, Anne Grapin-Botton
Deconstructing the principles of ductal network formation in the pancreas.
PLoS Biol, 16(7) 2002842-2002842 (2018)
Open Access DOI

The mammalian pancreas is a branched organ that does not exhibit stereotypic branching patterns, similarly to most other glands. Inside branches, it contains a network of ducts that undergo a transition from unconnected microlumen to a mesh of interconnected ducts and finally to a treelike structure. This ductal remodeling is poorly understood, both on a microscopic and macroscopic level. In this article, we quantify the network properties at different developmental stages. We find that the pancreatic network exhibits stereotypic traits at each stage and that the network properties change with time toward the most economical and optimized delivery of exocrine products into the duodenum. Using in silico modeling, we show how steps of pancreatic network development can be deconstructed into two simple rules likely to be conserved for many other glands. The early stage of the network is explained by noisy, redundant duct connection as new microlumens form. The later transition is attributed to pruning of the network based on the flux of fluid running through the pancreatic network into the duodenum.

Pierre A. Haas^*, Stephanie S M H Höhn^*, Aurelia R Honerkamp-Smith, Julius B Kirkegaard, Raymond E. Goldstein
The noisy basis of morphogenesis: Mechanisms and mechanics of cell sheet folding inferred from developmental variability.
PLoS Biol, 16(7) Art. No. e2005536 (2018)
Open Access DOI

Variability is emerging as an integral part of development. It is therefore imperative to ask how to access the information contained in this variability. Yet most studies of development average their observations and, discarding the variability, seek to derive models, biological or physical, that explain these average observations. Here, we analyse this variability in a study of cell sheet folding in the green alga Volvox, whose spherical embryos turn themselves inside out in a process sharing invagination, expansion, involution, and peeling of a cell sheet with animal models of morphogenesis. We generalise our earlier, qualitative model of the initial stages of inversion by combining ideas from morphoelasticity and shell theory. Together with three-dimensional visualisations of inversion using light sheet microscopy, this yields a detailed, quantitative model of the entire inversion process. With this model, we show how the variability of inversion reveals that two separate, temporally uncoupled processes drive the initial invagination and subsequent expansion of the cell sheet. This implies a prototypical transition towards higher developmental complexity in the volvocine algae and provides proof of principle of analysing morphogenesis based on its variability.

Peter Dobrowolski, Melina Fischer, Ronald Naumann
Novel insights into the genetic background of genetically modified mice.
Transgenic Res, 27(3) 265-275 (2018)
Open Access DOI

Unclear or misclassified genetic background of laboratory rodents or a lack of strain awareness causes a number of difficulties in performing or reproducing scientific experiments. Until now, genetic differentiation between strains and substrains of inbred mice has been a challenge. We have developed a screening method for analyzing inbred strains regarding their genetic background. It is based on 240 highly informative short tandem repeat (STR) markers covering the 19 autosomes as well as X and Y chromosomes. Combination of analysis results for presence of known C57BL/6 substrain-specific mutations together with autosomal STR markers and the Y-chromosomal STR-haplotype provides a comprehensive snapshot of the genetic background of mice. In this study, the genetic background of 72 mouse lines obtained from 18 scientific institutions in Germany and Austria was determined. By analyzing only 3 individuals per genetically modified line it was possible to detect mixed genetic backgrounds frequently. In several lines presence of a mispairing Y chromosome was detected. At least every second genetically modified line displayed a mixed genetic background which could lead to unexpected and non-reproducible results, irrespective of the investigated gene of interest.

Virag Sharma, Thomas Lehmann, Heiko Stuckas, Liane Funke, Michael Hiller
Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.
PLoS Biol, 16(6) Art. No. e2005293 (2018)
Open Access PDF DOI

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Iwan Zimmermann^*, Pascal Egloff^*, Cedric Aj Hutter^*, Fabian M Arnold, Peter Stohler, Nicolas Bocquet, Melanie N Hug, Sylwia Huber, Martin Siegrist, Lisa Hetemann, Jennifer Gera, Samira Gmür, Peter Spies, Daniel Gygax, Eric R Geertsma^#, Roger Jp Dawson^#, Markus A Seeger^#
Synthetic single domain antibodies for the conformational trapping of membrane proteins.
Elife, 7 Art. No. e34317 (2018)
Open Access DOI

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Alessandro Muscoloni, Carlo Vittorio Cannistraci
Leveraging the nonuniform PSO network model as a benchmark for performance evaluation in community detection and link prediction
New J Phys, Art. No. doi: 10.1088/1367-2630/aac6f9 (2018)
Open Access DOI

Anastasia Felker, Karin D Prummel, Anne M Merks, Michaela Mickoleit, Eline C Brombacher, Jan Huisken, Daniela Panáková, Christian Mosimann
Continuous addition of progenitors forms the cardiac ventricle in zebrafish.
Nat Commun, 9(1) Art. No. 2001 (2018)
Open Access DOI

The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend the atrium and ventricle, and form inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we track zebrafish heart development using transgenics based on the cardiopharyngeal gene tbx1. Live imaging uncovers a tbx1 reporter-expressing cell sheath that continuously disseminates from the lateral plate mesoderm towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborates that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduces ventricle size and abolishes BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process.

David Oriola, Daniel Needleman, Jan Brugués
The Physics of the Metaphase Spindle.
Ann Rev Biophys, 47 655-673 (2018)
DOI

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Máté Pálfy
The role of transcription factors in shaping the chromatin landscape and regulating the timing of zygotic genome activation in zebrafish.
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2018)

Celina Galles, Gastón M Prez, Sider Penkov, Sebastian Boland, Exequiel O J Porta, Silvia G Altabe, Guillermo R Labadie, Ulrike Schmidt, Hans-Joachim Knölker, Teymuras V. Kurzchalia^#, Diego Mendoza^#
Endocannabinoids in Caenorhabditis elegans are essential for the mobilization of cholesterol from internal reserves.
Sci Rep, 8(1) Art. No. 6398 (2018)
Open Access DOI

Proper cholesterol transport is crucial for the functionality of cells. In C. elegans, certain cholesterol derivatives called dafachronic acids (DAs) govern the entry into diapause. In their absence, worms form a developmentally arrested dauer larva. Thus, cholesterol transport to appropriate places for DA biosynthesis warrants the reproductive growth. Recently, we discovered a novel class of glycosphingolipids, PEGCs, required for cholesterol mobilization/transport from internal storage pools. Here, we identify other components involved in this process. We found that strains lacking polyunsaturated fatty acids (PUFAs) undergo increased dauer arrest when grown without cholesterol. This correlates with the depletion of the PUFA-derived endocannabinoids 2-arachidonoyl glycerol and anandamide. Feeding of these endocannabinoids inhibits dauer formation caused by PUFAs deficiency or impaired cholesterol trafficking (e.g. in Niemann-Pick C1 or DAF-7/TGF-β mutants). Moreover, in parallel to PEGCs, endocannabinoids abolish the arrest induced by cholesterol depletion. These findings reveal an unsuspected function of endocannabinoids in cholesterol trafficking regulation.

Shovamayee Maharana, Jie Wang, Dimitrios Papadopoulos, Doris Richter, Andrei I. Pozniakovsky, Ina Poser, Marc Bickle, Sandra Rizk, Jordina Guillén-Boixet, Titus Franzmann, Marcus Jahnel, Lara Marrone, Young-Tae Chang, Jared Sterneckert, Pavel Tomancak, Anthony Hyman^#, Simon Alberti^#
RNA buffers the phase separation behavior of prion-like RNA binding proteins.
Science, 360(6391) 918-921 (2018)
DOI

Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are largely soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. What keeps these proteins soluble in the nucleus and promotes aggregation in the cytoplasm is still unknown. We report here that RNA critically regulates the phase behavior of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Reduction of nuclear RNA levels or genetic ablation of RNA binding causes excessive phase separation and the formation of cytotoxic solid-like assemblies in cells. We propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. Changes in RNA levels or RNA binding abilities of RBPs cause aberrant phase transitions.

Carlo Vittorio Cannistraci, Tuomo Nieminen, Masahiro Nishi, Levon M Khachigian, Juho Viikilä, Mika Laine, Domenico Cianflone, Attilio Maseri, Khung Keong Yeo, Ravinay Bhindi, Enrico Ammirati
"Summer Shift": A Potential Effect of Sunshine on the Time Onset of ST-Elevation Acute Myocardial Infarction.
J Am Heart Assoc, 7(8) Art. No. doi: 10.1161/JAHA.117.006878 (2018)
Open Access DOI

ST-elevation acute myocardial infarction (STEMI) represents one of the leading causes of death. The time of STEMI onset has a circadian rhythm with a peak during diurnal hours, and the occurrence of STEMI follows a seasonal pattern with a salient peak of cases in the winter months and a marked reduction of cases in the summer months. Scholars investigated the reason behind the winter peak, suggesting that environmental and climatic factors concur in STEMI pathogenesis, but no studies have investigated whether the circadian rhythm is modified with the seasonal pattern, in particular during the summer reduction in STEMI occurrence.

Carsten Wolff^#, Jean-Yves Tinevez, Tobias Pietzsch, Evangelia Stamataki, Benjamin Harich, Léo Guignard, Stephan Preibisch, Spencer Shorte, Patrick Keller, Pavel Tomancak^#, Anastasios Pavlopoulos^#
Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb.
Elife, 7 Art. No. e34410 (2018)
Open Access DOI

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustaceanParhyale hawaiensiswith multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT).In silicoclonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.

Virag Sharma, Nikolai Hecker, Juliana G. Roscito, Leo Foerster, Björn Langer, Michael Hiller
A genomics approach reveals insights into the importance of gene losses for mammalian adaptations.
Nat Commun, 9(1) Art. No. 1215 (2018)
Open Access PDF DOI

Identifying the genomic changes that underlie phenotypic adaptations is a key challenge in evolutionary biology and genomics. Loss of protein-coding genes is one type of genomic change with the potential to affect phenotypic evolution. Here, we develop a genomics approach to accurately detect gene losses and investigate their importance for adaptive evolution in mammals. We discover a number of gene losses that likely contributed to morphological, physiological, and metabolic adaptations in aquatic and flying mammals. These gene losses shed light on possible molecular and cellular mechanisms that underlie these adaptive phenotypes. In addition, we show that gene loss events that occur as a consequence of relaxed selection following adaptation provide novel insights into species' biology. Our results suggest that gene loss is an evolutionary mechanism for adaptation that may be more widespread than previously anticipated. Hence, investigating gene losses has great potential to reveal the genomic basis underlying macroevolutionary changes.

Stefania Tavano, Elena Taverna, Nereo Kalebic, Christiane Haffner, Takashi Namba, Andreas Dahl, Michaela Wilsch-Bräuninger, Judith Paridaen^#, Wieland B. Huttner^#
Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.
Neuron, 97(6) 1299-1314 (2018)
DOI

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Pavel Vopalensky, Sabrina Pralow, Nadine Vastenhouw
Reduced expression of the Nodal co-receptor Oep causes loss of mesendodermal competence in zebrafish.
Development, 145(5) Art. No. dev158832 (2018)
DOI

The activation of specific gene expression programs depends on the presence of the appropriate signals and the competence of cells to respond to those signals. Although it is well established that cellular competence is regulated in space and time, the molecular mechanisms underlying the loss of competence remain largely unknown. Here, we determine the time window during which zebrafish prospective ectoderm loses its ability to respond to Nodal signals, and show that this coincides with a decrease in the levels of the Nodal co-receptor One-eyed pinhead (Oep). Bypassing Oep using a photoactivatable receptor, or an Oep-independent ligand, allows activation of Nodal target genes for an extended period of time. These results suggest that the reduced expression of Oep causes the loss of responsiveness to Nodal signals in the prospective ectoderm. Indeed, extending the presence of Oep prolongs the window of competence to respond to Nodal signals. Our findings suggest a simple mechanism in which the decreasing level of one component of the Nodal signaling pathway regulates the loss of mesendodermal competence in the prospective ectoderm.

Giorgos Tsoumpekos, Linda Nemetschke, Elisabeth Knust
Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension.
J Cell Biol, 217(3) 1033-1045 (2018)
DOI

Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. Here, we identify theDrosophila melanogasterscaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss ofbig bangresults in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits ofbig bangmutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth.

Renata Zuber, Michaela Norum, Yiwen Wang, Kathrin Oehl, Nicole Gehring, Davide Accardi, Slawomir Bartozsewski, Jürgen Berger, Matthias Flötenmeyer, Bernard Moussian
The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila.
Eur J Cell Biol, 97(2) 90-101 (2018)
DOI

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.

Elisa Monzón-Casanova, Michael Screen, Manuel D Díaz-Muñoz, Richard M R Coulson, Sarah E Bell, Greta Lamers, Michele Solimena, Christopher W J Smith, Martin Turner
The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.
Nat Immunol, 19(3) 267-278 (2018)
DOI

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

Michele Solimena, Anke M Schulte, Lorella Marselli, Florian Ehehalt, Doris Richter, Manuela Kleeberg, Hassan Mziaut, Klaus-Peter Knoch, Julia Parnis, Marco Bugliani, Afshan Siddiq, Anne Jörns, Frédéric Burdet, Robin Liechti, Mara Suleiman, Daniel Margerie, Farooq Syed, Marius Distler, Robert Grützmann, Enrico Petretto, Aida Moreno-Moral, Carolin Wegbrod, Anke Sönmez, Katja Pfriem, Anne Friedrich, Jörn Meinel, C B Wollheim, Gustavo Baretton, Raphael Scharfmann, Everson Nogoceke, Enzio Bonifacio, Dorothée Sturm, Birgit Meyer-Puttlitz, Ugo Boggi, Hans-Detlev Saeger, Franco Filipponi, Mathias Lesche, Paolo Meda, Andreas Dahl, Leonore Wigger, Ioannis Xenarios, Mario Falchi, Bernard Thorens, Jürgen Weitz, Krister Bokvist, Sigurd Lenzen, Guy A Rutter, Philippe Froguel, Manon von Bülow, Mark Ibberson, Piero Marchetti
Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.
Diabetologia, 61(3) 641-657 (2018)
Open Access DOI

Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP).

Santosh Phuyal, Mayes Kasem, Oskar Knittelfelder, Animesh Sharma, Davi de Miranda Fonseca, Vaineta Vebraite, Sergey Shaposhnikov, Geir Slupphaug, Vidar Skaug, Shanbeh Zienolddiny
Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes.
Nanotoxicology, 12(2) 138-152 (2018)
Open Access DOI

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

Emma Teeling, Sonja Vernes, Liliana M Dávalos, David A Ray, M Thomas P Gilbert, Eugene Myers, Bat1K Consortium
Bat Biology, Genomes, and the Bat1K Project: To Generate Chromosome-Level Genomes for All Living Bat Species.
Annu Rev Anim Biosci, 6 23-46 (2018)
DOI

Bats are unique among mammals, possessing some of the rarest mammalian adaptations, including true self-powered flight, laryngeal echolocation, exceptional longevity, unique immunity, contracted genomes, and vocal learning. They provide key ecosystem services, pollinating tropical plants, dispersing seeds, and controlling insect pest populations, thus driving healthy ecosystems. They account for more than 20% of all living mammalian diversity, and their crown-group evolutionary history dates back to the Eocene. Despite their great numbers and diversity, many species are threatened and endangered. Here we announce Bat1K, an initiative to sequence the genomes of all living bat species (n∼1,300) to chromosome-level assembly. The Bat1K genome consortium unites bat biologists (>148 members as of writing), computational scientists, conservation organizations, genome technologists, and any interested individuals committed to a better understanding of the genetic and evolutionary mechanisms that underlie the unique adaptations of bats. Our aim is to catalog the unique genetic diversity present in all living bats to better understand the molecular basis of their unique adaptations; uncover their evolutionary history; link genotype with phenotype; and ultimately better understand, promote, and conserve bats. Here we review the unique adaptations of bats and highlight how chromosome-level genome assemblies can uncover the molecular basis of these traits. We present a novel sequencing and assembly strategy and review the striking societal and scientific benefits that will result from the Bat1K initiative.

Sergej Nowoshilow, Siegfried Schloissnig, Jifeng Fei, Andreas Dahl, Andy W C Pang, Martin Pippel, Sylke Winkler, Alex R Hastie, George Young, Juliana G. Roscito, Francisco Falcon, Dunja Knapp, Sean Powell, Alfredo Cruz, Han Cao, Bianca Habermann, Michael Hiller, Elly M. Tanaka, Eugene W Myers
The axolotl genome and the evolution of key tissue formation regulators.
Nature, 554(7690) 50-55 (2018)
Open Access PDF DOI

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/-and Pax7-/-mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Markus Grohme^*, Siegfried Schloissnig^*, Andrei Rozanski, Martin Pippel, George Robert Young, Sylke Winkler, Holger Brandl, Ian Henry, Andreas Dahl, Sean Powell, Michael Hiller, Eugene Myers, Jochen Rink
The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.
Nature, 554(7690) 56-61 (2018)
Open Access PDF DOI

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

Mette Handberg-Thorsager, Juliana Gutierrez-Mazariegos, Stefan T Arold, Eswar Kumar Nadendla, Paola Y Bertucci, Pierre Germain, Pavel Tomançak, Keely Pierzchalski, Jace W Jones, Ricard Albalat, Maureen A Kane, William Bourguet, Vincent Laudet, Detlev Arendt, Michael Schubert
The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation.
Sci Adv, 4(2) Art. No. eaao1261 (2018)
Open Access DOI

Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation ofhoxgenes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelidPlatynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinearhoxgene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation ofhox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient.

Simon Alberti
Guilty by Association: Mapping Out the Molecular Sociology of Droplet Compartments.
Mol Cell, 69(3) 349-351 (2018)
DOI

The molecular interactions driving the formation of stress-inducible granules have largely remained unknown. In two recent papers, Youn et al. (2018) and Markmiller et al. (2018) use proximity labeling proteomics to map out the protein interactome of stress-inducible ribonucleoprotein granules.

Violeta Georgieva Tsonkova, Fredrik Wolfhagen Sand, Xenia Asbæk Wolf, Lars Groth Grunnet, Anna Kirstine Ringgaard, Camilla Ingvorsen, Louise Winkel, Mark Kalisz, Kevin Dalgaard, Christine Bruun, Johannes Fels, Charlotte Helgstrand, Sven Hastrup, Fredrik Kryh Öberg, Erik Vernet, Michael Paolo Bastner Sandrini, Allan Christian Shaw, Carsten Jessen, Mads Grønborg, Jacob Hald, Hanni Willenbrock, Dennis Madsen, Rasmus Wernersson, Lena Hansson, Jan Nygaard Jensen, Annette Plesner, Tomas Alanentalo, Maja Borup Kjær Petersen, Anne Grapin-Botton, Christian Honoré, Jonas Ahnfelt-Rønne, Jacob Hecksher-Sørensen, Philippe Ravassard, Ole D Madsen, Claude Rescan, Thomas Frogne
The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates.
Mol Metab, 8 144-157 (2018)
DOI

To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates.

Shradha Das, Elisabeth Knust
A dual role of the extracellular domain of DrosophilaCrumbs for morphogenesis of the embryonic neuroectoderm.
Biol Open, 7(1) Art. No. bio031435 (2018)
Open Access DOI

Epithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb), the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in theDrosophilaembryo. However, different epithelia exhibit different phenotypic severity upon loss ofcrbUsing a transgenomic approach allowed us to more accurately define the role ofcrbin different epithelia. In particular, we provide evidence that the loss of epithelial tissue integrity in the ventral epidermis ofcrbmutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. We demonstrate that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results we suggest a dual role of the extracellular domain of Crb in the ventral neuroectoderm. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.

Rana Amini, Mauricio Rocha-Martins, Caren Norden
Neuronal Migration and Lamination in the Vertebrate Retina.
Front Neurosci, 11 Art. No. 742 (2018)
Open Access PDF DOI

In the retina, like in most other brain regions, developing neurons are arranged into distinct layers giving the mature tissue its stratified appearance. This process needs to be highly controlled and orchestrated, as neuronal layering defects lead to impaired retinal function. To achieve successful neuronal layering and lamination in the retina and beyond, three main developmental steps need to be executed: First, the correct type of neuron has to be generated at a precise developmental time. Second, as most retinal neurons are born away from the position at which they later function, newborn neurons have to move to their final layer within the developing tissue, a process also termed neuronal lamination. Third, these neurons need to connect to their correct synaptic partners. Here, we discuss neuronal migration and lamination in the vertebrate retina and summarize our knowledge on these aspects of retinal development. We give an overview of how lamination emerges and discuss the different modes of neuronal translocation that occur during retinogenesis and what we know about the cell biological machineries driving them. In addition, retinal mosaics and their importance for correct retinal function are examined. We close by stating the open questions and future directions in this exciting field.

Franziska Decker, David Oriola, Benjamin Dalton, Jan Brugues
Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.
Elife, 7 Art. No. e31149 (2018)
Open Access DOI

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopuslaevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting.

Samata Chaudhuri, Till Korten, Slobodanka Korten, Gloria Milani, Tobia Lana, Geertruy Te Kronnie, Stefan Diez
Label-Free Detection of Microvesicles and Proteins by the Bundling of Gliding Microtubules.
Nano Lett, 18(1) 117-123 (2018)
DOI

Development of miniaturized devices for the rapid and sensitive detection of analyte is crucial for various applications across healthcare, pharmaceutical, environmental, and other industries. Here, we report on the detection of unlabeled analyte by using fluorescently labeled, antibody-conjugated microtubules in a kinesin-1 gliding motility assay. The detection principle is based on the formation of fluorescent supramolecular assemblies of microtubule bundles and spools in the presence of multivalent analytes. We demonstrate the rapid, label-free detection of CD45+ microvesicles derived from leukemia cells. Moreover, we employ our platform for the label-free detection of multivalent proteins at subnanomolar concentrations, as well as for profiling the cross-reactivity between commercially available secondary antibodies. As the detection principle is based on the molecular recognition between antigen and antibody, our method can find general application where it identifies any analyte, including clinically relevant microvesicles and proteins.

Alf Honigmann^#, André Nadler^#
The Next Frontier: Quantitative Biochemistry in Living Cells.
Biochemistry, 57(1) 47-55 (2018)
DOI

Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

Lucía Cordero-Espinoza, Meritxell Huch
The balancing act of the liver: tissue regeneration versus fibrosis.
J Clin Invest, 128(1) 85-96 (2018)
DOI

Epithelial cell loss alters a tissue's optimal function and awakens evolutionarily adapted healing mechanisms to reestablish homeostasis. Although adult mammalian organs have a limited regeneration potential, the liver stands out as one remarkable exception. Following injury, the liver mounts a dynamic multicellular response wherein stromal cells are activated in situ and/or recruited from the bloodstream, the extracellular matrix (ECM) is remodeled, and epithelial cells expand to replenish their lost numbers. Chronic damage makes this response persistent instead of transient, tipping the system into an abnormal steady state known as fibrosis, in which ECM accumulates excessively and tissue function degenerates. Here we explore the cellular and molecular switches that balance hepatic regeneration and fibrosis, with a focus on uncovering avenues of disease modeling and therapeutic intervention.

Miquel Vila-Farré, Jochen Rink
The Ecology of Freshwater Planarians.
Methods Mol Biol, 1774 173-205 (2018)
DOI

Planarians are on the rise as a model system for regeneration and stem cell dynamics. Almost in parallel the interest in planarian field biology has declined. Besides representing an independent research discipline in its own right, understanding of the natural habitat is also directly relevant to optimizing culture conditions in the laboratory. Moreover, the current laboratory models are but few of hundreds of planarian species worldwide. Their adaptation to a wide range of ecological niches has resulted in a fascinating diversity of regenerative abilities, body size, reproduction strategies, and life expectancy, to name just a few. With the currently ongoing establishment of large planarian species collections, such phenotypic diversity becomes accessible to comparative mechanistic analysis in the laboratory. Overall, we hope that this chapter inspires an integral view of the planarian model system that not only includes the molecular and cellular processes under investigation but also the evolutionary forces that shaped them in the first place.

Iskra Yanakieva^#, Marija Matejčić, Caren Norden^#
Choosing the right microscope to image mitosis in zebrafish embryos: A practical guide.
Methods Cell Biol, 145 107-127 (2018)
DOI

Tissue growth and organismal development require orchestrated cell proliferation. To understand how cell division guides development, it is important to explore mitosis at the tissue-wide, cellular, and subcellular scale. At the tissue level this includes determining a tissue's mitotic index, at the cellular level the tracing of cell lineages, and at the subcellular level the characterization of intracellular components. These different tasks can be addressed by different imaging approaches (e.g., laser-scanning confocal, spinning disk confocal, and light-sheet fluorescence microscopy). Here, we summarize three protocols for exploring different facets of mitosis in developing zebrafish embryos. Zebrafish embryos are transparent and their rapid external development greatly facilitates the study of cellular processes and developmental dynamics using microscopy. A critical step in all imaging studies of mitosis in development is to choose the most suitable microscope for each scientific question. This choice is important in order to ensure a balance between the required temporal and spatial resolution and minimal phototoxicity that could otherwise perturb the process of interest. The use of different microscopy techniques, best suited for the purpose of each experiment, thus permits to generate a comprehensive and unbiased view on how mitosis influences development.

Maria Winzi, Nuria Casas Vila, Maciej Paszkowski-Rogacz, Li Ding, Svenja Noack, Mirko Theis, Falk Butter, Frank Buchholz
The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells.
PLoS ONE, 13(1) Art. No. e191682 (2018)
Open Access DOI

RNA interference (RNAi) screens have been shown to be valuable to study embryonic stem cell (ESC) self-renewal and they have been successfully applied to identify coding as well as noncoding genes required for maintaining pluripotency. Here, we used an RNAi library targeting >640 long noncoding RNAs (lncRNA) to probe for their role in early cell differentiation. Utilizing a Sox1-GFP ESC reporter cell line, we identified the lncRNA lncR492 as lineage-specific inhibitor of neuroectodermal differentiation. Molecular characterization showed that lncR492 interacts with the mRNA binding protein HuR and facilitates its inhibitory function by activation of Wnt signaling. Thus, lncRNAs modulate the fate decision of pluripotent stem cells.

Jorge Kageyama, Damian Wollny, Barbara Treutlein, J Gray Camp
ShinyCortex: Exploring Single-Cell Transcriptome Data From the Developing Human Cortex.
Front Neurosci, 12 Art. No. 315 (2018)
Open Access DOI

Single-cell mRNA sequencing (scRNA-seq) is a powerful method to identify and classify cell types and reconstruct differentiation trajectories within complex tissues, such as the developing human cortex. scRNA-seq data also enables the discovery of cell type-specific marker genes and genes that regulate developmental transitions. Here we provide a brief overview of how scRNA-seq has been shaping the study of human cortex development, and present ShinyCortex, a resource that brings together data from recent scRNA-seq studies of the developing cortex for further analysis. ShinyCortex is based in R and displays recently published scRNA-seq data from the human and mouse cortex in a comprehensible, dynamic and accessible way, suitable for data exploration by biologists.

Olga Vvedenskaya, Yuting Wang, Jacobo Miranda Ackerman, Oskar Knittelfelder, Andrej Shevchenko
Analytical challenges in human plasma lipidomics: A winding path towards the truth.
Trends Analyt Chem, 120 Art. No. 115277 (2018)
DOI

Human plasma lipidome has been extensively studied in many pathophysiological contexts with the hope of identifying biomarkers for early diagnostics and monitoring the progression and treatment of a broad spectrum of diseases. However, despite remarkable progress in lipidomics technologies, the concordance of lipidomics measurements between independent laboratories remains limited and not fulfilling the criteria of common laboratory diagnostics. Here we highlighted a few critical aspects of epidemiological studies of the plasma lipidome, including the selection of study cohorts, collection of plasma samples as well as extraction, identification and quantification of lipids. We argue that reporting the abundances of plasma lipids as molar concentrations is a key turning point during the transition of research lipidomics into a common tool of clinical diagnostics.

Jochen Rink
Stem Cells, Patterning and Regeneration in Planarians: Self-Organization at the Organismal Scale.
Methods Mol Biol, 1774 57-172 (2018)
DOI

The establishment of size and shape remains a fundamental challenge in biological research that planarian flatworms uniquely epitomize. Planarians can regenerate complete and perfectly proportioned animals from tiny and arbitrarily shaped tissue pieces; they continuously renew all organismal cell types from abundant pluripotent stem cells, yet maintain shape and anatomy in the face of constant turnover; they grow when feeding and literally degrow when starving, while scaling form and function over as much as a 40-fold range in body length or an 800-fold change in total cell numbers. This review provides a broad overview of the current understanding of the planarian stem cell system, the mechanisms that pattern the planarian body plan and how the interplay between patterning signals and cell fate choices orchestrates regeneration. What emerges is a conceptual framework for the maintenance and regeneration of the planarian body plan on basis of the interplay between pluripotent stem cells and self-organizing patterns and further, the general utility of planarians as model system for the mechanistic basis of size and shape.

Michael Weber^*, Nico Scherf^*, Alexander M Meyer, Daniela Panáková, Peter Kohl, Jan Huisken
Cell-accurate optical mapping across the entire developing heart.
Elife, 6 Art. No. e28307 (2017)
Open Access DOI

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

Katharina S Schneider, Christina J Groß, Roland F Dreier, Benedikt S Saller, Ritu Mishra, Oliver Gorka, Rosalie Heilig, Etienne Meunier, Mathias S Dick, Tamara Ćiković, Jan Sodenkamp, Guillaume Médard, Ronald Naumann, Jurgen Ruland, Bernhard Kuster, Petr Broz, Olaf Groß
The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.
Cell Rep, 21(13) 3846-3859 (2017)
Open Access DOI

Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis.

Martin Weigert, Uwe Schmidt, Tobias Boothe, Andreas Müller, Alexandr Dibrov, Akanksha Jain, Benjamin Wilhelm, Deborah Schmidt, Coleman Broaddus, Sian Culley, Mauricio Rocha-Martins, Fabián Segovia-Miranda, Caren Norden, Ricardo Henriques, Marino Zerial, Michele Solimena, Jochen Rink, Pavel Tomancak, Loic Royer, Florian Jug, Eugene W Myers
Content-Aware Image Restoration: Pushing the Limits of Fluorescence Microscopy
bioRxiv, Art. No. https://doi.org/10.1101/236463 (2017)
Open Access DOI

B Duygu Özpolat, Mette Handberg-Thorsager, Michel Vervoort, Guillaume Balavoine
Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii.
Elife, 6 Art. No. e30463 (2017)
Open Access DOI

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos.

Felix Ruhnow, Linda Kloβ, Stefan Diez
Challenges in Estimating the Motility Parameters of Single Processive Motor Proteins.
Biophys J, 113(11) 2433-2443 (2017)
DOI

Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechano-systems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Toward this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods. Here, we investigate the movement of fluorescently labeled, processive, dimeric motor proteins and propose a unified algorithm to correct the measurements for finite filament length as well as photobleaching. Particular emphasis is put on estimating the statistical errors associated with the proposed evaluation method, as knowledge of these values is crucial when comparing measurements from different experiments. Testing our approach with simulated and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules, we show 1) that velocity distributions should be fitted by a t location-scale probability density function rather than by a normal distribution; 2) that the impossibility to measure events shorter than the image acquisition time needs to be taken into account; 3) that the interaction time and run length of the motors can be estimated independent of the filament length distribution; and 4) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. Moreover, our analysis reveals that controlling the temperature during the experiments with a precision below 1 K is of importance. We believe our method will not only improve the evaluation of experimental data, but also allow for better statistical comparisons between different populations of motor proteins (e.g., with distinct mutations or linked to different cargos) and filaments (e.g., in distinct nucleotide states or with different posttranslational modifications). Therefore, we include a detailed workflow for image processing and analysis (including MATLAB code), serving as a tutorial for the estimation of motility parameters in stepping-motility assays.

Oleksandr Ostrenko, Pietro Incardona, Rajesh Ramaswamy, Lutz Brusch, Ivo F. Sbalzarini
pSSAlib: The partial-propensity stochastic chemical network simulator.
PLoS Comput Biol, 13(12) Art. No. e1005865 (2017)
Open Access DOI

Chemical reaction networks are ubiquitous in biology, and their dynamics is fundamentally stochastic. Here, we present the software library pSSAlib, which provides a complete and concise implementation of the most efficient partial-propensity methods for simulating exact stochastic chemical kinetics. pSSAlib can import models encoded in Systems Biology Markup Language, supports time delays in chemical reactions, and stochastic spatiotemporal reaction-diffusion systems. It also provides tools for statistical analysis of simulation results and supports multiple output formats. It has previously been used for studies of biochemical reaction pathways and to benchmark other stochastic simulation methods. Here, we describe pSSAlib in detail and apply it to a new model of the endocytic pathway in eukaryotic cells, leading to the discovery of a stochastic counterpart of the cut-out switch motif underlying early-to-late endosome conversion. pSSAlib is provided as a stand-alone command-line tool and as a developer API. We also provide a plug-in for the SBMLToolbox. The open-source code and pre-packaged installers are freely available from http://mosaic.mpi-cbg.de.

Bevan Cheeseman
The Adaptive Particle Representation (APR) for Simple and Efficient Adaptive Resolution Processing, Storage and Simulations
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2017)

Jifeng Fei, Maritta Schuez, Dunja Knapp, Yuka Taniguchi, David N. Drechsel, Elly M. Tanaka
Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.
Proc Natl Acad Sci U.S.A., 114(47) 12501-12506 (2017)
DOI

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

Alessandro Muscoloni, Josephine Maria Thomas, Sara Ciucci, Ginestra Bianconi, Carlo Vittorio Cannistraci
Machine learning meets complex networks via coalescent embedding in the hyperbolic space.
Nat Commun, 8(1) 1615-1615 (2017)
Open Access DOI

Physicists recently observed that realistic complex networks emerge as discrete samples from a continuous hyperbolic geometry enclosed in a circle: the radius represents the node centrality and the angular displacement between two nodes resembles their topological proximity. The hyperbolic circle aims to become a universal space of representation and analysis of many real networks. Yet, inferring the angular coordinates to map a real network back to its latent geometry remains a challenging inverse problem. Here, we show that intelligent machines for unsupervised recognition and visualization of similarities in big data can also infer the network angular coordinates of the hyperbolic model according to a geometrical organization that we term "angular coalescence." Based on this phenomenon, we propose a class of algorithms that offers fast and accurate "coalescent embedding" in the hyperbolic circle even for large networks. This computational solution to an inverse problem in physics of complex systems favors the application of network latent geometry techniques in disciplines dealing with big network data analysis including biology, medicine, and social science.

Nikolai Hecker, Virag Sharma, Michael Hiller
Transition to an Aquatic Habitat Permitted the Repeated Loss of the Pleiotropic KLK8 Gene in Mammals.
Genome Biol Evol, 9(11) 3179-3188 (2017)
PDF DOI

Kallikrein related peptidase 8 (KLK8; also called neuropsin) is a serine protease that plays distinct roles in the skin and hippocampus. In the skin, KLK8 influences keratinocyte proliferation and desquamation, and activates antimicrobial peptides in sweat. In the hippocampus, KLK8 affects memory acquisition. Here, we examined the evolution of KLK8 in mammals and discovered that, out of 70 placental mammals, KLK8 is exclusively lost in three independent fully-aquatic lineages, comprising dolphin, killer whale, minke whale, and manatee. In addition, while the sperm whale has an intact KLK8 reading frame, the gene evolves neutrally in this species. We suggest that the distinct functions of KLK8 likely became obsolete in the aquatic environment, leading to the subsequent loss of KLK8 in several fully-aquatic mammalian lineages. First, the cetacean and manatee skin lacks sweat glands as an adaptation to the aquatic environment, which likely made the epidermal function of KLK8 obsolete. Second, cetaceans and manatees exhibit a proportionally small hippocampus, which may have rendered the hippocampal functions of KLK8 obsolete. Together, our results shed light on the genomic changes that correlate with skin and neuroanatomical differences of aquatic mammals, and show that even pleiotropic genes can be lost during evolution if an environmental change nullifies the need for the different functions of such genes.

Shai Joseph
Competition for DNA binding between histones and transcription factors regulates the timing of zygotic genome activation in zebrafish embryos
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2017)

Jacob Kruse, Carsten Rother, Uwe Schmidt
Learning to Push the Limits of Efficient FFT-based Image Deconvolution
In: 2017 IEEE International Conference on Computer Vision : ICCV 2017 : proceedings : 22-29 October 2017, Venice, Italy (2017), Piscataway, N.J., IEEE (2017), 4596-4604
DOI

This work addresses the task of non-blind image deconvolution. Motivated to keep up with the constant increase in image size, with megapixel images becoming the norm, we aim at pushing the limits of efficient FFT-based techniques. Based on an analysis of traditional and more recent learning-based methods, we generalize existing discriminative approaches by using more powerful regularization, based on convolutional neural networks. Additionally, we propose a simple, yet effective, boundary adjustment method that alleviates the problematic circular convolution assumption, which is necessary for FFT-based deconvolution. We evaluate our approach on two common non-blind deconvolution benchmarks and achieve state-of-the-art results even when including methods which are computationally considerably more expensive.

Natalie Dye, Marko Popović, Stephanie Spannl, Raphael Etournay, Dagmar Kainmüller, Suhrid Ghosh, Eugene W Myers, Frank Jülicher^#, Suzanne Eaton^#
Cell dynamics underlying oriented growth of the Drosophila wing imaginal disc.
Development, 144(23) 4406-4421 (2017)
DOI

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.

Karl Hoffmann, Anja Voss-Böhme, Jochen Rink, Lutz Brusch
A dynamically diluted alignment model reveals the impact of cell turnover on the plasticity of tissue polarity patterns.
J R Soc Interface, 14(135) Art. No. 20170466 (2017)
DOI

The polarization of cells and tissues is fundamental for tissue morphogenesis during biological development and regeneration. A deeper understanding of biological polarity pattern formation can be gained from the consideration of pattern reorganization in response to an opposing instructive cue, which we here consider using the example of experimentally inducible body axis inversions in planarian flatworms. We define a dynamically diluted alignment model linking three processes: entrainment of cell polarity by a global signal, local cell-cell coupling aligning polarity among neighbours, and cell turnover replacing polarized cells by initially unpolarized cells. We show that a persistent global orienting signal determines the final mean polarity orientation in this stochastic model. Combining numerical and analytical approaches, we find that neighbour coupling retards polarity pattern reorganization, whereas cell turnover accelerates it. We derive a formula for an effective neighbour coupling strength integrating both effects and find that the time of polarity reorganization depends linearly on this effective parameter and no abrupt transitions are observed. This allows us to determine neighbour coupling strengths from experimental observations. Our model is related to a dynamic 8-Potts model with annealed site-dilution and makes testable predictions regarding the polarization of dynamic systems, such as the planarian epithelium.

Sara Carvalhal, Michelle Stevense, Katrin Koehler, Ronald Naumann, Angela Huebner, Rolf Jessberger, Eric R Griffis
ALADIN is required for the production of fertile mouse oocytes.
Mol Biol Cell, 28(19) 2470-2478 (2017)
DOI

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Kamran Rizzolo, Jennifer Huen, Ashwani Kumar, Sadhna Phanse, James Vlasblom, Yoshito Kakihara, Hussein A Zeineddine, Zoran Minic, Jamie Snider, Wen Wang, Carles Pons, Thiago V Seraphim, Edgar Boczek, Simon Alberti, Michael Costanzo, Chad L Myers, Igor Stagljar, Charles Boone, Mohan Babu, Walid A Houry
Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.
Cell Rep, 20(11) 2735-2748 (2017)
Open Access DOI

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

Andreas Müller, Hassan Mziaut, Martin Neukam, Klaus-Peter Knoch, Michele Solimena
A 4D view on insulin secretory granule turnover in the β-cell.
Diabetes Obes Metab, 19 Suppl 1 107-114 (2017)
DOI

Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper β-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the β-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

Simon Alberti
The wisdom of crowds: regulating cell function through condensed states of living matter.
J Cell Sci, 130(17) 2789-2796 (2017)
DOI

Our understanding of cells has progressed rapidly in recent years, mainly because of technological advances. Modern technology now allows us to observe molecular processes in living cells with high spatial and temporal resolution. At the same time, we are beginning to compile the molecular parts list of cells. However, how all these parts work together to yield complex cellular behavior is still unclear. In addition, the established paradigm of molecular biology, which sees proteins as well-folded enzymes that undergo specific lock-and-key type interactions, is increasingly being challenged. In fact, it is now becoming clear that many proteins do not fold into three-dimensional structures and additionally show highly promiscuous binding behavior. Furthermore, proteins function in collectives and form condensed phases with different material properties, such as liquids, gels, glasses or filaments. Here, I examine emerging evidence that the formation of macromolecular condensates is a fundamental principle in cell biology. I further discuss how different condensed states of living matter regulate cellular functions and decision-making and ensure adaptive behavior and survival in times of cellular crisis.

Stephan W. Grill
The mechanics of positioning skin follicles.
Science, 357(6353) 750-751 (2017)
DOI

Sheik Pran Babu Sardar Pasha, Robert Münch, Patrick Schäfer, Peter Oertel, Alex Sykes, Yiqing Zhu, Mike Karl
Retinal cell death dependent reactive proliferative gliosis in the mouse retina.
Sci Rep, 7(1) Art. No. 9517 (2017)
Open Access DOI

Neurodegeneration is a common starting point of reactive gliosis, which may have beneficial and detrimental consequences. It remains incompletely understood how distinctive pathologies and cell death processes differentially regulate glial responses. Müller glia (MG) in the retina are a prime model: Neurons are regenerated in some species, but in mammals there may be proliferative disorders and scarring. Here, we investigated the relationship between retinal damage and MG proliferation, which are both induced in a reproducible and temporal order in organotypic culture of EGF-treated mouse retina: Hypothermia pretreatment during eye dissection reduced neuronal cell death and MG proliferation; stab wounds increased both. Combined (but not separate) application of defined cell death signaling pathway inhibitors diminished neuronal cell death and maintained MG mitotically quiescent. The level of neuronal cell death determined MG activity, indicated by extracellular signal-regulated kinase (ERK) phosphorylation, and proliferation, both of which were abolished by EGFR inhibition. Our data suggest that retinal cell death, possibly either by programmed apoptosis or necrosis, primes MG to be able to transduce the EGFR-ERK activity required for cell proliferation. These results imply that cell death signaling pathways are potential targets for future therapies to prevent the proliferative gliosis frequently associated with certain neurodegenerative conditions.

Valentina Botti, François McNicoll, Michaela Steiner, Florian M Richter, Anfisa Solovyeva, Marius Wegener, Oliver D Schwich, Ina Poser, Kathi Zarnack, Ilka Wittig, Karla M. Neugebauer, Michaela Müller-McNicoll
Cellular differentiation state modulates the mRNA export activity of SR proteins.
J Cell Biol, 216(7) 1993-2009 (2017)
DOI

SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.

Serena Carra, Simon Alberti, Patrick A Arrigo, Justin L Benesch, Ivor J Benjamin, Wilbert Boelens, Britta Bartelt-Kirbach, Bianca J J M Brundel, Johannes Buchner, Bernd Bukau, John A Carver, Heath Ecroyd, Cecilia Emanuelsson, Stephanie Finet, Nikola Golenhofen, Pierre Goloubinoff, Nikolai Gusev, Martin Haslbeck, Lawrence E Hightower, Harm H Kampinga, Rachel E Klevit, Krzysztof Liberek, Hassane S Mchaourab, Kathryn A McMenimen, Angelo Poletti, Roy Quinlan, Sergei V Strelkov, Melinda E Toth, Elizabeth Vierling, Robert M Tanguay
The growing world of small heat shock proteins: from structure to functions.
Cell Stress Chaperones, 22(4) 601-611 (2017)
DOI

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

Peter Steinbach, Matthias Werner
gearshifft – The FFT Benchmark Suite for Heterogeneous Platforms
In: High performance computing ; 32nd international conference, ISC High Performance 2017, Frankfurt, Germany, June 18-22, 2017 : proceedings (2017)(Eds.) Julian Kunkel (Lecture Notes in Computer Science ; 10266), Cham, Springer International Publishing (2017), 199-216
DOI

Christoph Metzendorf^*, Anja Zeigerer^*, Sarah Seifert, Richard Sparla, Bahar Najafi, François Canonne-Hergaux, Marino Zerial, Martina Muckenthaler
Acute loss of the hepatic endo-lysosomal system in vivo causes compensatory changes in iron homeostasis.
Sci Rep, 7(1) Art. No. 4023 (2017)
Open Access DOI

Liver cells communicate with the extracellular environment to take up nutrients via endocytosis. Iron uptake is essential for metabolic activities and cell homeostasis. Here, we investigated the role of the endocytic system for maintaining iron homeostasis. We specifically depleted the small GTPase Rab5 in the mouse liver, causing a transient loss of the entire endo-lysosomal system. Strikingly, endosome depletion led to a fast reduction of hepatic iron levels, which was preceded by an increased abundance of the iron exporter ferroportin. Compensatory changes in livers of Rab5-depleted mice include increased expression of transferrin receptor 1 as well as reduced expression of the iron-regulatory hormone hepcidin. Serum iron indices (serum iron, free iron binding capacity and total iron binding capacity) in Rab5-KD mice were increased, consistent with an elevated splenic and hepatic iron export. Our data emphasize the critical importance of the endosomal compartments in hepatocytes to maintain hepatic and systemic iron homeostasis in vivo. The short time period (between day four and five) upon which these changes occur underscore the fast dynamics of the liver iron pool.

Virag Sharma, Michael Hiller
Increased alignment sensitivity improves the usage of genome alignments for comparative gene annotation.
Nucleic Acids Res, 45(14) 8369-8377 (2017)
Open Access PDF DOI

Genome alignments provide a powerful basis to transfer gene annotations from a well-annotated reference genome to many other aligned genomes. The completeness of these annotations crucially depends on the sensitivity of the underlying genome alignment. Here, we investigated the impact of the genome alignment parameters and found that parameters with a higher sensitivity allow the detection of thousands of novel alignments between orthologous exons that have been missed before. In particular, comparisons between species separated by an evolutionary distance of >0.75 substitutions per neutral site, like human and other non-placental vertebrates, benefit from increased sensitivity. To systematically test if increased sensitivity improves comparative gene annotations, we built a multiple alignment of 144 vertebrate genomes and used this alignment to map human genes to the other 143 vertebrates with CESAR. We found that higher alignment sensitivity substantially improves the completeness of comparative gene annotations by adding on average 2382 and 7440 novel exons and 117 and 317 novel genes for mammalian and non-mammalian species, respectively. Our results suggest a more sensitive alignment strategy that should generally be used for genome alignments between distantly-related species. Our 144-vertebrate genome alignment and the comparative gene annotations (https://bds.mpi-cbg.de/hillerlab/144VertebrateAlignment_CESAR/) are a valuable resource for comparative genomics.

Verena Rauschenberger, Dominic B Bernkopf, Sabrina Krenn, Kowcee Jalal, Jens Heller, Jürgen Behrens, Marc Gentzel, Alexandra Schambony
The phosphatase Pgam5 antagonizes Wnt/β-Catenin signaling in embryonic anterior-posterior axis patterning.
Development, 144(12) 2234-2247 (2017)
DOI

The scaffold protein Dishevelled is a central intracellular component of Wnt signaling pathways. Various kinases have been described that regulate and modulate Wnt signaling through phosphorylation of Dishevelled. However, besides general protein phosphatases 1 and 2 (PP1 and PP2), no specific protein phosphatases have been identified. Here, we report on the identification and functional characterization of the protein phosphatase Pgam5 in vitro and in vivo in Xenopus Pgam5 is a novel antagonist of Wnt/β-Catenin signaling in human cells and Xenopus embryogenesis. In early development, Pgam5 is essential for head formation, and for establishing and maintaining the Wnt/β-Catenin signaling gradient that patterns the anterior-posterior body axis. Inhibition of Wnt/β-Catenin signaling and developmental function depend on Pgam5 phosphatase activity. We show that Pgam5 interacts with Dishevelled2 and that Dishevelled2 is a substrate of Pgam5. Pgam5 mediates a marked decrease in Dishevelled2 phosphorylation in the cytoplasm and in the nucleus, as well as decreased interaction between Dishevelled2, Tcf1 and β-Catenin, indicating that Pgam5 regulates Dishevelled function upstream and downstream of β-Catenin stabilization.

Patricia Heyn, Hanna Salmonowicz, Jonathan Rodenfels, Karla M. Neugebauer
Activation of transcription enforces the formation of distinct nuclear bodies in zebrafish embryos.
RNA Biol, 14(6) 752-760 (2017)
DOI

Nuclear bodies are cellular compartments that lack lipid bilayers and harbor specific RNAs and proteins. Recent proposals that nuclear bodies form through liquid-liquid phase separation leave the question of how different nuclear bodies maintain their distinct identities unanswered. Here we investigate Cajal bodies (CBs), histone locus bodies (HLBs) and nucleoli - involved in assembly of the splicing machinery, histone mRNA 3' end processing, and rRNA processing, respectively - in the embryos of the zebrafish, Danio rerio. We take advantage of the transcriptional silence of the 1-cell embryo and follow nuclear body appearance as zygotic transcription becomes activated. CBs are present from fertilization onwards, while HLB and nucleolar components formed foci several hours later when histone genes and rDNA became active. HLB formation was blocked by transcription inhibition, suggesting nascent histone transcripts recruit HLB components like U7 snRNP. Surprisingly, we found that U7 base-pairing with nascent histone transcripts was not required for localization to HLBs. Rather, the type of Sm ring assembled on U7 determined its targeting to HLBs or CBs; the spliceosomal Sm ring targeted snRNAs to CBs while the specialized U7 Sm-ring localized to HLBs, demonstrating the contribution of protein constituents to the distinction among nuclear bodies. Thus, nucleolar, HLB, and CB components can mix in early embryogenesis when transcription is naturally or artificially silenced. These data support a model in which transcription of specific gene loci nucleates nuclear body components with high specificity and fidelity to perform distinct regulatory functions.

David G Drubin, Anthony Hyman
Stem cells: the new "model organism".
Mol Biol Cell, 28(11) 1409-1411 (2017)
DOI

Human tissue culture cells have long been a staple of molecular and cell biology research. However, although these cells are derived from humans, they have often lost considerable aspects of natural physiological function. Here we argue that combined advances in genome editing, stem cell production, and organoid derivation from stem cells represent a revolution in cell biology. These advances have important ramifications for the study of basic cell biology mechanisms, as well as for the ways in which discoveries in mechanisms are translated into understanding of disease.

Hjalte List Larsen, Anne Grapin-Botton
The molecular and morphogenetic basis of pancreas organogenesis.
Semin Cell Dev Biol, 66 51-68 (2017)
DOI

The pancreas is an essential endoderm-derived organ that ensures nutrient metabolism via its endocrine and exocrine functions. Here we review the essential processes governing the embryonic and early postnatal development of the pancreas discussing both the mechanisms and molecules controlling progenitor specification, expansion and differentiation. We elaborate on how these processes are orchestrated in space and coordinated with morphogenesis. We draw mainly from experiments conducted in the mouse model but also from investigations in other model organisms, complementing a recent comprehensive review of human pancreas development (Jennings et al., 2015) [1]. The understanding of pancreas development in model organisms provides a framework to interpret how human mutations lead to neonatal diabetes and may contribute to other forms of diabetes and to guide the production of desired pancreatic cell types from pluripotent stem cells for therapeutic purposes.

Jeffrey Woodruff^#, Beatriz Ferreira Gomes, Per Widlund, Julia Mahamid, Alf Honigmann, Anthony Hyman^#
The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin.
Cell, 169(6) 1066-1077 (2017)
DOI

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.

Arturo Raya-Sandino, Alejandro Castillo-Kauil, Alaide Domínguez-Calderón, Lourdes Alarcón, David Flores-Benitez, Francisco Cuellar-Perez, Bruno López-Bayghen, Bibiana Chávez-Munguía, José Vázquez-Prado, Lorenza González-Mariscal
Zonula occludens-2 regulates Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.
Biochim Biophys Acta, 1864(10) 1714-1733 (2017)
PDF DOI

Silencing Zonula occludens 2 (ZO-2), a tight junctions (TJ) scaffold protein, in epithelial cells (MDCK ZO-2 KD) triggers: 1) Decreased cell to substratum attachment, accompanied by reduced expression of claudin-7 and integrin β1, and increased vinculin recruitment to focal adhesions and stress fibers formation; 2) Lowered cell-cell aggregation and appearance of wider intercellular spaces; 3) Increased RhoA/ROCK activity, mediated by GEF-HI recruitment to cell borders by cingulin; 4) Increased Cdc42 activity, mitotic spindle disorientation and the appearance of cysts with multiple lumens; 5) Increased Rac and cofilin activity, multiple lamellipodia formation and random cell migration but increased wound closure; 6) Diminished cingulin phosphorylation and disappearance of planar network of microtubules at the TJ region; and 7) Increased transepithelial electrical resistance at steady state, coupled to an increased expression of ZO-1 and claudin-4 and a decreased expression of claudin-2 and paracingulin. Hence, ZO-2 is a crucial regulator of Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.

Stefanie Redemann, Johannes Baumgart, Norbert Lindow, Michael Shelley^#, Ehssan Nazockdast, Andrea Kratz, Steffen Prohaska, Jan Brugués, Sebastian Fürthauer, Thomas Müller-Reichert^#
C. elegans chromosomes connect to centrosomes by anchoring into the spindle network.
Nat Commun, 8 Art. No. 15288 (2017)
Open Access DOI

The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

Shradha Das, Elisabeth Knust
Stardust, the Janus-faced partner of Crumbs.
J Cell Biol, 216(5) 1219-1221 (2017)
DOI

The Drosophila melanogaster scaffolding protein Stardust (Sdt) stabilizes the transmembrane protein Crumbs, a conserved regulator of apical-basal epithelial polarity. In this issue, Perez-Mockus et al. (2017. J. Cell Biol https://doi.org/10.1083/jcb.201611196) report that a subset of Sdt isoforms are targeted by the ubiquitin ligase Neuralized, thus fine tuning the endocytosis and activity of this apical determinant.

Shai Joseph, Máté Pálfy, Lennart Hilbert, Mukesh Kumar, Jens Karschau, Vasily Zaburdaev, Andrej Shevchenko, Nadine Vastenhouw
Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos.
Elife, 6 Art. No. e23326 (2017)
Open Access DOI

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

Jan Brugués
Cytoskeleton Dynamics: Mind the Gap!
Curr Biol, 27(7) 279-281 (2017)
DOI

A new study presents a quantitative biophysical model of microtubule aster growth with autocatalytic microtubule nucleation. The model accounts for asters that grow indefinitely, even when their microtubules are unstable.

Máté Pálfy, Shai Joseph, Nadine Vastenhouw
The timing of zygotic genome activation.
Curr Opin Genet Dev, 43 53-60 (2017)
DOI

After fertilization, the embryonic genome is inactive until transcription is initiated during the maternal-to-zygotic transition. How the onset of transcription is regulated in a precisely timed manner, however, is a long standing question in biology. Several mechanisms have been shown to contribute to the temporal regulation of genome activation but none of them can fully explain the general absence of transcription as well the gene specific onset that follows. Here we review the work that has been done toward elucidating the mechanisms underlying the temporal regulation of transcription in embryos.

Jakob Ruess^*^#, Heinz Koeppl, Christoph Zechner^*^#
Sensitivity estimation for stochastic models of biochemical reaction networks in the presence of extrinsic variability.
J Chem Phys, 146(12) Art. No. 124122 (2017)
DOI

Determining the sensitivity of certain system states or outputs to variations in parameters facilitates our understanding of the inner working of that system and is an essential design tool for the de novo construction of robust systems. In cell biology, the output of interest is often the response of a certain reaction network to some input (e.g., stressors or nutrients) and one aims to quantify the sensitivity of this response in the presence of parameter heterogeneity. We argue that for such applications, parametric sensitivities in their standard form do not paint a complete picture of a system's robustness since one assumes that all cells in the population have the same parameters and are perturbed in the same way. Here, we consider stochasticreaction networks in which the parameters are randomly distributed over the population and propose a new sensitivity index that captures the robustness of system outputs upon changes in the characteristics of the parameter distribution, rather than the parameters themselves. Subsequently, we make use of Girsanov's likelihood ratio method to construct a Monte Carlo estimator of this sensitivity index. However, it turns out that this estimator has an exceedingly large variance. To overcome this problem, we propose a novel estimation algorithm that makes use of a marginalization of the path distribution of stochasticreaction networks and leads to Rao-Blackwellized estimators with reduced variance.

Catherine Rabouille^#, Simon Alberti^#
Cell adaptation upon stress: the emerging role of membrane-less compartments.
Curr Opin Cell Biol, 47 34-42 (2017)
DOI

Cells under stress transition from a growth to a quiescent state. The conventional thinking is that this is achieved through transcriptional programs, translational regulation, protein degradation, and post-translational modifications. However, there is an increasing realization that stress adaptation also goes along with dramatic changes in the architecture and organization of cells. In particular, it seems to involve the formation of membrane-less compartments and macromolecular assemblies. We propose that cells make widespread use of this ability to change macromolecular organization to adapt to stress conditions and protect themselves. Here, we address what triggers the formation of these assemblies under stress conditions. We present examples illustrating that in some cases, sophisticated signaling pathways transmit environmental fluctuations from the outside to the inside and in others, that external fluctuations directly affect the internal conditions in cells. We further argue that changes in the organization of the cytoplasm and the formation of membrane-less compartments have many advantages over other ways of altering protein function, such as protein degradation, translation or transcription. Furthermore, membrane-less compartments may act as protective devices for key cellular components.

Meritxell Huch, Jürgen A. Knoblich, Matthias Lutolf, Alfonso Martinez-Arias
The hope and the hype of organoid research.
Development, 144(6) 938-941 (2017)
DOI

The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.

Svend Bertel Dahl-Jensen, Anne Grapin-Botton
The physics of organoids: a biophysical approach to understanding organogenesis.
Development, 144(6) 946-951 (2017)
DOI

Organoids representing a diversity of tissues have recently been created, bridging the gap between cell culture and experiments performed in vivo Being small and amenable to continuous monitoring, they offer the opportunity to scrutinize the dynamics of organ development, including the exciting prospect of observing aspects of human embryo development live. From a physicist's perspective, their ability to self-organize - to differentiate and organize cells in space - calls for the identification of the simple rules that underlie this capacity. Organoids provide tractable conditions to investigate the effects of the growth environment, including its molecular composition and mechanical properties, along with the initial conditions such as cell number and type(s). From a theoretical standpoint, different types of in silico modeling can complement the measurements performed in organoids to understand the role of chemical diffusion, contact signaling, differential cell adhesion and mechanical controls. Here, we discuss what it means to take a biophysical approach to understanding organogenesis in vitro and how we might expect such approaches to develop in the future.

Gaia Pigino, Antonina Roll-Mecak
Microtubule dynamics: 50 years after the discovery of tubulin and still going strong.
Mol Biol Cell, 28(6) 705-706 (2017)
DOI

Kirstin Meyer, Oleksandr Ostrenko, George Bourantas, Hernán Morales-Navarrete, Natalie Porat-Shliom, Fabián Segovia-Miranda, Hidenori Nonaka, Ali Ghaemi, Jean-Marc Verbavatz, Lutz Brusch, Ivo F. Sbalzarini, Yannis Kalaidzidis, Roberto Weigert, Marino Zerial
A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule.
Cell Syst, 4(3) 277-290 (2017)
Open Access PDF DOI

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Maximilian Krause
The role of Histone H3 Lysine 4 trimethylation in zebrafish embryonic development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2017)

Anne Grapin-Botton, Palle Serup
Parsing the Pancreas.
N. Engl. J. Med., 376(9) 886-888 (2017)
DOI

Matthias Merkel, Raphael Etournay, Marko Popović, Guillaume Salbreux, Suzanne Eaton, Frank Jülicher
Triangles bridge the scales: Quantifying cellular contributions to tissue deformation.
Phys Rev E, 95(3) Art. No. 032401 (2017)
DOI

In this article, we propose a general framework to study the dynamics and topology of cellular networks that capture the geometry of cell packings in two-dimensional tissues. Such epithelia undergo large-scale deformation during morphogenesis of a multicellular organism. Large-scale deformations emerge from many individual cellular events such as cell shape changes, cell rearrangements, cell divisions, and cell extrusions. Using a triangle-based representation of cellular network geometry, we obtain an exact decomposition of large-scale material deformation. Interestingly, our approach reveals contributions of correlations between cellular rotations and elongation as well as cellular growth and elongation to tissue deformation. Using this triangle method, we discuss tissue remodeling in the developing pupal wing of the fly Drosophila melanogaster.

Irene Adrian-Kalchhauser, Ola Svensson, Verena E Kutschera, Magnus Alm Rosenblad, Martin Pippel, Sylke Winkler, Siegfried Schloissnig, Anders Blomberg, Patricia Burkhardt-Holm
The mitochondrial genome sequences of the round goby and the sand goby reveal patterns of recent evolution in gobiid fish.
BMC Genomics, 18(1) Art. No. 177 (2017)
Open Access PDF DOI

Vertebrate mitochondrial genomes are optimized for fast replication and low cost of RNA expression. Accordingly, they are devoid of introns, are transcribed as polycistrons and contain very little intergenic sequences. Usually, vertebrate mitochondrial genomes measure between 16.5 and 17 kilobases (kb).

Stephanie Spannl^*, Alexandra Kumichel^*, Sarita Hebbar, Katja Kapp, Marcos Gonzalez-Gaitan, Sylke Winkler, Rosana Blawid, Gregor Jessberger, Elisabeth Knust
The Crumbs_C isoform of Drosophila shows tissue- and stage-specific expression and prevents light-dependent retinal degeneration.
Biol Open, 6(2) 165-175 (2017)
Open Access PDF DOI

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Jacqueline Tabler, Maggie M Rigney, Gordon J Berman, Swetha Gopalakrishnan, Eglantine Heude, Hadeel Adel Al-Lami, Basil Z Yannakoudakis, Rebecca D Fitch, Crystal N. Carter, Steven A Vokes, Karen J Liu, Shahragim Tajbakhsh, Se Roian Egnor, John Wallingford
Cilia-mediated Hedgehog signaling controls form and function in the mammalian larynx.
Elife, 6 Art. No. e19153 (2017)
Open Access PDF DOI

Acoustic communication is fundamental to social interactions among animals, including humans. In fact, deficits in voice impair the quality of life for a large and diverse population of patients. Understanding the molecular genetic mechanisms of development and function in the vocal apparatus is thus an important challenge with relevance both to the basic biology of animal communication and to biomedicine. However, surprisingly little is known about the developmental biology of the mammalian larynx. Here, we used genetic fate mapping to chart the embryological origins of the tissues in the mouse larynx, and we describe the developmental etiology of laryngeal defects in mice with disruptions in cilia-mediated Hedgehog signaling. In addition, we show that mild laryngeal defects correlate with changes in the acoustic structure of vocalizations. Together, these data provide key new insights in the molecular genetics of form and function in the mammalian vocal apparatus.

Ekaterina Korotkevich, Ritsuya Niwayama, Aurélien Courtois, Stefanie Friese, Nicolas Berger, Frank Buchholz, Takashi Hiiragi
The Apical Domain Is Required and Sufficient for the First Lineage Segregation in the Mouse Embryo.
Dev Cell, 40(3) 235-247 (2017)
Open Access PDF DOI

Mammalian development begins with segregation of the extra-embryonic trophectoderm from the embryonic lineage in the blastocyst. While cell polarity and adhesion play key roles, the decisive cue driving this lineage segregation remains elusive. Here, to study symmetry breaking, we use a reduced system in which isolated blastomeres recapitulate the first lineage segregation. We find that in the 8-cell stage embryo, the apical domain recruits a spindle pole to ensure its differential distribution upon division. Daughter cells that inherit the apical domain adopt trophectoderm fate. However, the fate of apolar daughter cells depends on whether their position within the embryo facilitates apical domain formation by Cdh1-independent cell contact. Finally, we develop methods for transplanting apical domains and show that acquisition of this domain is not only required but also sufficient for the first lineage segregation. Thus, we provide mechanistic understanding that reconciles previous models for symmetry breaking in mouse development.

Tom Stückemann^*, James Cleland^*, Steffen Werner, Hanh Thi-Kim Vu, Robert Bayersdorf, Shang-Yun Liu, Benjamin Friedrich, Frank Jülicher, Jochen Rink
Antagonistic Self-Organizing Patterning Systems Control Maintenance and Regeneration of the Anteroposterior Axis in Planarians.
Dev Cell, 40(3) 248-263 (2017)
DOI

Planarian flatworms maintain their body plan in the face of constant internal turnover and can regenerate from arbitrary tissue fragments. Both phenomena require self-maintaining and self-organizing patterning mechanisms, the molecular mechanisms of which remain poorly understood. We show that a morphogenic gradient of canonical Wnt signaling patterns gene expression along the planarian anteroposterior (A/P) axis. Our results demonstrate that gradient formation likely occurs autonomously in the tail and that an autoregulatory module of Wnt-mediated Wnt expression both shapes the gradient at steady state and governs its re-establishment during regeneration. Functional antagonism between the tail Wnt gradient and an unknown head patterning system further determines the spatial proportions of the planarian A/P axis and mediates mutually exclusive molecular fate choices during regeneration. Overall, our results suggest that the planarian A/P axis is patterned by self-organizing patterning systems deployed from either end that are functionally coupled by mutual antagonism.

Takashi Namba, Wieland B. Huttner
Neural progenitor cells and their role in the development and evolutionary expansion of the neocortex.
Wiley Interdiscip Rev Dev Biol, 6(1) Art. No. e256 (2017)
DOI

The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors). Cortical expansion in development and evolution is linked to an increased abundance and proliferative capacity of BPs, notably basal radial glial cells, a recently characterized type of secondary progenitor derived from apical radial glial cells, the primary progenitors. To gain insight into the molecular basis underlying the prolonged and increased proliferation of NPCs and in particular BPs, comparative genomic and transcriptomic approaches, mostly for human versus mouse, have been employed and applied to specific NPC types and subpopulations. These have revealed two principal sets of molecular changes. One concerns differences in the expression of common genes between species with different degrees of cortical expansion. The other comprises human-specific genes or genomic regulatory sequences. Various systems that allow functional testing of these genomic and gene expression differences between species have emerged, including transient and stable transgenesis, genome editing, cerebral organoids, and organotypic slice cultures. These provide future avenues for uncovering the molecular basis of cortical expansion. For further resources related to this article, please visit the WIREs website.

Yung-Ning Chang, Eric R Geertsma
The novel class of seven transmembrane segment inverted repeat carriers.
Biol Chem, 398(2) 165-174 (2017)
DOI

Solute carriers from the SLC4, SLC23, and SLC26 families are involved in pH regulation, vitamin C transport and ion homeostasis. While these families do not share any obvious sequence relationship, they are united by their unique and novel architecture. Each member of this structural class is organized into two structurally related halves of seven transmembrane segments each. These halves span the membrane with opposite orientations and form an intricately intertwined structure of two inverted repeats. This review highlights the general design principles of this fold and reveals the diversity between the different families. We discuss their domain architecture, structural framework and transport mode and detail an initial transport mechanism for this fold inferred from the recently solved structures of different members.

Masatoshi Nishikawa, Sundar Naganathan, Frank Jülicher, Stephan W. Grill
Controlling contractile instabilities in the actomyosin cortex.
Elife, 6 Art. No. e19595 (2017)
Open Access DOI

The actomyosin cell cortex is an active contractile material for driving cell- and tissue morphogenesis. The cortex has a tendency to form a pattern of myosin foci, which is a signature of potentially unstable behavior. How a system that is prone to such instabilities can rveliably drive morphogenesis remains an outstanding question. Here, we report that in the Caenorhabditis elegans zygote, feedback between active RhoA and myosin induces a contractile instability in the cortex. We discover that an independent RhoA pacemaking oscillator controls this instability, generating a pulsatory pattern of myosin foci and preventing the collapse of cortical material into a few dynamic contracting regions. Our work reveals how contractile instabilities that are natural to occur in mechanically active media can be biochemically controlled to robustly drive morphogenetic events.

Nereo Kalebic, Katherine S. Long, Wieland B. Huttner
Neocortex expansion in development and evolution: the cell biology of neural stem and progenitor cells and the impact of human-specific gene expression.
In: Evolution of Nervous Systems . Vol. 3 : The nervous systems of non-human primates. (Eds.) Jon H Kaas, Amsterdam, Netherlands, Elsevier (2017), 73-89

Stefania Tavano, Wieland B. Huttner
The Cell Biology of Neural Stem and Progenitor Cells and Neocortex Expansion in Development and Evolution
In: Essentials of noncoding RNA in neuroscience : ontogenetics, plasticity of the vertebrate brain. (Eds.) Davide De Pietri Tonelli, Amsterdam, Netherlands, Academic Press (2017), 81-99
DOI

David Martin, Anne Grapin-Botton
The Importance of REST for Development and Function of Beta Cells.
Front Cell Dev Biol, 5 12-12 (2017)
Open Access DOI

Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST is expressed in the progenitors of both neurons and beta cells during development, but it is down-regulated as the cells differentiate. Although REST mutations and deregulation have yet to be connected to diabetes in humans, REST activation during both development and in adult beta cells leads to diabetes in mice.

Mingsi Xie, Anna Shevchenko, Binghua Wang, Andrej Shevchenko, Changsui Wang, Yimin Yang
Identification of a dairy product in the grass woven basket from Gumugou Cemetery (3800 BP, northwestern China)
Quat Int, 426 158-165 (2016)

Lora Winters
Mechanism of spindle assembly in Schizosaccharomyces pombe - The role of microtubule pivoting in spindle assembly
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Ayse Güven
The role of Sox9 and extracellular matrix in the development and evolutionary expansion of the neocortex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Alf Honigmann, A Pralle
Compartmentalization of the Cell Membrane.
J Mol Biol, 428(24 Pt A) 4739-4748 (2016)
DOI

Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.

Ilaria Visco, Carsten Hoege, Anthony Hyman, Petra Schwille
In vitro Reconstitution of a Membrane Switch Mechanism for the Polarity Protein LGL.
J Mol Biol, 428(24 Pt A) 4828-4842 (2016)
DOI

Cell polarity arises from a combination of interactions between biological molecules, such as activation, inhibition, and positive or negative feedback between specific polarity units. Activation and inhibition often take place in the form of a membrane binding switch. Lethal giant larvae (LGL), a conserved regulator of cell polarity in animals, was suggested to function as such a switch. LGL localizes to both the cytoplasm and, asymmetrically, the membrane. However, the spatial regulation mechanism of LGL membrane localization has remained unclear. For systematic elucidation, we set out to reconstitute a minimal polarity unit using a model membrane, Caenorhabditis elegans LGL (LGL-1), and atypical protein kinase C (aPKC) supposed to activate the membrane switch. We identified a membrane binding sequence (MBS) in LGL-1 by a screen in vivo, reconstituted LGL-1 membrane binding in vitro, and successfully implemented the membrane switch by aPKC phosphorylation activity, detaching LGL from membranes. Upon membrane binding, LGL-1 MBS folds into an alpha-helix in which three regions can be identified: a positively charged patch, a switch area containing the three aPKC phosphorylation sites, and a hydrophobic area probably buried in the membrane. Phosphorylation by aPKC dramatically reduces the binding affinity of the LGL-1 MBS to negatively charged model membranes, inducing its detachment. Specific residues in the MBS are critical for LGL-1 function in C. elegans.

Liliana Malinovska, Simon Alberti
Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging.
J Vis Exp, (118) Art. No. e54730 (2016)
DOI

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Fabian Rost, Aida Rodrigo Albors, Vladimir Mazurov, Lutz Brusch, Andreas Deutsch, Elly M. Tanaka, Osvaldo Chara
Accelerated cell divisions drive the outgrowth of the regenerating spinal cord in axolotls.
Elife, 5 Art. No. e20357 (2016)
Open Access PDF DOI

Axolotls are unique in their ability to regenerate the spinal cord. However, the mechanisms that underlie this phenomenon remain poorly understood. Previously, we showed that regenerating stem cells in the axolotl spinal cord revert to a molecular state resembling embryonic neuroepithelial cells and functionally acquire rapid proliferative divisions (Rodrigo Albors et al., 2015). Here, we refine the analysis of cell proliferation in space and time and identify a high-proliferation zone in the regenerating spinal cord that shifts posteriorly over time. By tracking sparsely-labeled cells, we also quantify cell influx into the regenerate. Taking a mathematical modeling approach, we integrate these quantitative datasets of cell proliferation, neural stem cell activation and cell influx, to predict regenerative tissue outgrowth. Our model shows that while cell influx and neural stem cell activation play a minor role, the acceleration of the cell cycle is the major driver of regenerative spinal cord outgrowth in axolotls.

Sandra Scharaw, Murat Iskar, Alessandro Ori, Gaelle Boncompain, Vibor Laketa, Ina Poser, Emma Lundberg, Franck Perez, Mike Beck, Peer Bork, Rainer Pepperkok
The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR.
J Cell Biol, 215(4) 543-558 (2016)
DOI

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.

Cordula Reuther, Alejandra Laguillo Diego, Stefan Diez
Kinesin-1 motors can increase the lifetime of taxol-stabilized microtubules.
Nat Nanotechnol, 11(11) 914-915 (2016)
DOI

Maksim V Baranov, Natalia H Revelo, Ilse Dingjan, Riccardo Maraspini, Martin Ter Beest, Alf Honigmann, Geert van den Bogaart
SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis.
Cell Rep, 17(6) 1518-1531 (2016)
Open Access PDF DOI

Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.

Bert Nitzsche, Elzbieta Dudek, Lukasz Hajdo, Andrzej A Kasprzak, Andrej Vilfan, Stefan Diez
Working stroke of the kinesin-14, ncd, comprises two substeps of different direction.
Proc Natl Acad Sci U.S.A., 113(43) Art. No. E6582-E6589 (2016)
DOI

Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]- and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.

Jaroslav Icha, Christiane Kunath, Mauricio Rocha-Martins, Caren Norden
Independent modes of ganglion cell translocation ensure correct lamination of the zebrafish retina.
J Cell Biol, 215(2) 259-275 (2016)
PDF DOI

The arrangement of neurons into distinct layers is critical for neuronal connectivity and function. During development, most neurons move from their birthplace to the appropriate layer, where they polarize. However, kinetics and modes of many neuronal translocation events still await exploration. In this study, we investigate retinal ganglion cell (RGC) translocation across the embryonic zebrafish retina. After completing their translocation, RGCs establish the most basal retinal layer where they form the optic nerve. Using in toto light sheet microscopy, we show that somal translocation of RGCs is a fast and directed event. It depends on basal process attachment and stabilized microtubules. Interestingly, interference with somal translocation induces a switch to multipolar migration. This multipolar mode is less efficient but still leads to successful RGC layer formation. When both modes are inhibited though, RGCs fail to translocate and induce lamination defects. This indicates that correct RGC translocation is crucial for subsequent retinal lamination.

Jacques Pécréaux^*^#, Stefanie Redemann^*, Zahraa Alayan, Benjamin Mercat, Sylvain Pastezeur, Carlos Garzon-Coral, Anthony Hyman, Jonathon Howard^#
The Mitotic Spindle in the One-Cell C. elegans Embryo Is Positioned with High Precision and Stability.
Biophys J, 111(8) 1773-1784 (2016)
Open Access PDF DOI

Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents of the mother cell are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early Caenorhabditis elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is ∼1000, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed before the onset of anaphase.

Helge Knoop, Tobias Gronemeier, Christoph Knigge, Peter Steinbach
Porting the MPI Parallelized LES Model PALM to Multi-GPU Systems – An Experience Report
In: High performance computing : ISC High Performance 2016 international workshops ExaComm, E-MuCoCoS, HPC-IODC, IXPUG, IWOPH, P^3MA, VHPC, WOPSSS, Frankfurt, Germany, June 19-23, 2016 : revised selected papers (2016) Lecture Notes in Computer Science ; 9945, Cham, Springer International Publishing (2016), 508-523
DOI

Kristina Thamm, Sylvi Graupner, Carsten Werner, Wieland B. Huttner, Denis Corbeil
Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133).
PLoS ONE, 11(10) Art. No. e0164079 (2016)
Open Access PDF DOI

The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

Christian Eggeling, Alf Honigmann
Closing the gap: The approach of optical and computational microscopy to uncover biomembrane organization.
Biochim Biophys Acta, 1858(10) 2558-2568 (2016)
DOI

Biological membranes are complex composites of lipids, proteins and sugars, which catalyze a myriad of vital cellular reactions in a spatiotemporal tightly controlled manner. Our understanding of the organization principles of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. With the recent advent of super-resolution optical microscopy (or nanoscopy) one now has approached the molecular scale regime with non-invasive live cell fluorescence observation techniques. Since in silico molecular dynamics (MD) simulation techniques are also improving to study larger and more complex systems we can now start to integrate live-cell and in silico experiments to develop a deeper understanding of biomembranes. In this review we summarize recent progress to measure lipid-protein interactions in living cells and give examples how MD simulations can complement and upgrade the experimental data. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Maria Montoya, Thierry Dorval, Marc Bickle
SLAS Europe High-Content Screening Conference in Dresden: A Glimpse of the Future?
J Biomol Screen, 21(9) 883-886 (2016)
DOI

L Carine Stapel
The road to uniform gene expression
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

German Tischler
Low Space External Memory Construction of the Succinct Permuted Longest Common Prefix Array
In: String Processing and Information Retrieval : 23rd International Symposium, SPIRE 2016, Beppu, Japan, October 18-20, 2016, Proceedings (2016) Lecture notes in computer science ; 9954, Cham, Springer International Publishing (2016), 178-190
DOI

Massimo Ganassi, Daniel Mateju, Ilaria Bigi, Laura Mediani, Ina Poser, Hyun-Ok Kate Lee, Samuel J Seguin, Federica F Morelli, Jonathan Vinet, Giuseppina Leo, Orietta Pansarasa, Cristina Cereda, Angelo Poletti, Simon Alberti, Serena Carra
A Surveillance Function of the HSPB8-BAG3-HSP70 Chaperone Complex Ensures Stress Granule Integrity and Dynamism.
Mol Cell, 63(5) 796-810 (2016)
DOI

Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS.

M Gai, Federico Bianchi, Cristiana Vagnoni, Fiammetta Vernì, Silvia Bonaccorsi, Selina Pasquero, G E Berto, F Sgrò, A M A Chiotto, Laura Annaratone, Anna Sapino, Anna Bergo, Nicoletta Landsberger, Jacqueline Bond, Wieland B. Huttner, F Di Cunto
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules.
EMBO Rep, 17(10) 1396-1409 (2016)
DOI

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT-stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral-MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.

Elisabeth Fischer-Friedrich, Yusuke Toyoda, Cedric J Cattin, Daniel J. Müller, Anthony Hyman, Frank Jülicher
Rheology of the Active Cell Cortex in Mitosis.
Biophys J, 111(3) 589-600 (2016)
DOI

The cell cortex is a key structure for the regulation of cell shape and tissue organization. To reach a better understanding of the mechanics and dynamics of the cortex, we study here HeLa cells in mitosis as a simple model system. In our assay, single rounded cells are dynamically compressed between two parallel plates. Our measurements indicate that the cortical layer is the dominant mechanical element in mitosis as opposed to the cytoplasmic interior. To characterize the time-dependent rheological response, we extract a complex elastic modulus that characterizes the resistance of the cortex against area dilation. In this way, we present a rheological characterization of the cortical actomyosin network in the linear regime. Furthermore, we investigate the influence of actin cross linkers and the impact of active prestress on rheological behavior. Notably, we find that cell mechanics values in mitosis are captured by a simple rheological model characterized by a single timescale on the order of 10 s, which marks the onset of fluidity in the system.

Gerard W Dougherty, Niki T Loges, Judith A Klinkenbusch, Heike Olbrich, Petra Pennekamp, Tabea Menchen, Johanna Raidt, Julia Wallmeier, Claudius Werner, Cordula Westermann, Christian Ruckert, Virginia Mirra, Rim Hjeij, Yasin Memari, Richard Durbin, Anja Kolb-Kokocinski, Kavita Praveen, Mohammad A Kashef, Sara Kashef, Fardin Eghtedari, Karsten Häffner, Pekka Valmari, György Baktai, Micha Aviram, Lea Bentur, Israel Amirav, Erica E Davis, Nicholas Katsanis, Martina Brueckner, Artem Shaposhnykov, Gaia Pigino, Bernd Dworniczak, Heymut Omran
DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.
Am J Respir Cell Mol Biol, 55(2) 213-224 (2016)
DOI

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Meritxell Huch^#, Laurent Dollé^#
The plastic cellular states of liver cells: Are EpCAM and Lgr5 fit for purpose?
Hepatology, 64(2) 652-662 (2016)
Open Access DOI

Adult liver cells have been considered restricted regarding their fate and lineage potential. That is, hepatocytes have been thought able only to generate hepatocytes and duct cells, only duct cells. While this may be the case for the majority of scenarios in a state of quiescence or homeostasis, evidence suggests that liver cells are capable of interconverting between cellular states of distinct phenotypic traits. This interconversion or plasticity had been suggested by classical studies using cellular markers, but recently lineage tracing approaches have proven that cells are highly plastic and retain an extraordinary ability to respond differently to normal tissue homeostasis, to tissue repair, or when challenged to expand ex vivo or to differentiate upon transplantation. Stemness, as "self-renewal and multipotency," seems not to be limited to a particular cell type but rather to a cellular state in which cells exhibit a high degree of plasticity and can move back and forth in different phenotypic states. For instance, upon damage cells can dedifferentiate to acquire stem cell potential that allows them to self-renew, repopulate a damaged tissue, and then undergo differentiation. In this review, we will discuss the evidence on cellular plasticity in the liver, focusing our attention on two markers, epithelial cell adhesion molecule and leucine-rich repeat-containing G protein-coupled receptor 5, which identify cells with stem cell potential. (Hepatology 2016;64:652-662).

Hassan Mziaut, Bernard Mulligan, Peter Hoboth, Oliver Otto, Anna Ivanova, Maik Herbig, Desiree Schumann, Tobias Hildebrandt, J Dehghany, Anke Sönmez, Carla Münster, M Meyer-Hermann, Jochen Guck, Yannis Kalaidzidis, Michele Solimena
The F-actin modifier villin regulates insulin granule dynamics and exocytosis downstream of islet cell autoantigen 512.
Mol Metab, 5(8) 656-668 (2016)
Open Access PDF DOI

Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis.

Xavier Prudent, Genis Parra, Peter Schwede, Juliana G. Roscito, Michael Hiller
Controlling for Phylogenetic Relatedness and Evolutionary Rates Improves the Discovery of Associations Between Species' Phenotypic and Genomic Differences.
Mol Biol Evol, 33(8) 2135-2150 (2016)
PDF DOI

The growing number of sequenced genomes allows us now to address a key question in genetics and evolutionary biology: which genomic changes underlie particular phenotypic changes between species? Previously, we developed a computational framework called Forward Genomics that associates phenotypic to genomic differences by focusing on phenotypes that are independently lost in different lineages. However, our previous implementation had three main limitations. Here, we present two new Forward Genomics methods that overcome these limitations by (1) directly controlling for phylogenetic relatedness, (2) controlling for differences in evolutionary rates, and (3) computing a statistical significance. We demonstrate on large-scale simulated data and on real data that both new methods substantially improve the sensitivity to detect associations between phenotypic and genomic differences. We applied these new methods to detect genomic differences involved in the loss of vision in the blind mole rat and the cape golden mole, two independent subterranean mammals. Forward Genomics identified several genes that are enriched in functions related to eye development and the perception of light, as well as genes involved in the circadian rhythm. These new Forward Genomics methods represent a significant advance in our ability to discover the genomic basis underlying phenotypic differences between species. Source code: https://github.com/hillerlab/ForwardGenomics/.

Doris Meder, Mònica Morales, Rainer Pepperkok, Ralph Schlapbach, Andreas Tiran, Geert Van Minnebruggen
Institutional core facilities: prerequisite for breakthroughs in the life sciences: Core facilities play an increasingly important role in biomedical research by providing scientists access to sophisticated technology and expertise.
EMBO Rep, 17(8) 1088-1093 (2016)
Open Access PDF DOI

Michaela Mickoleit
Imaging and Reconstructing the beating Heart and its Morphogenesis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Dan V Nicolau^*, Mercy Lard, Till Korten, Falco C M J M van Delft, Malin Persson, Elina Bengtsson, Alf Månsson, Stefan Diez, Heiner Linke, Dan V Nicolau^*
Reply to Einarsson: The computational power of parallel network exploration with many bioagents.
Proc Natl Acad Sci U.S.A., 113(23) Art. No. E3188 (2016)
Open Access PDF DOI

Sandra Soukup, Jussi Helppi, Dennis R Müller, Oliver Zierau, Bernhard Watzl, Günter Vollmer, Patrick Diel, Achim Bub, Sabine E Kulling
Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison.
Arch Toxicol, 90(6) 1335-1347 (2016)
DOI

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC-MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4'-glucuronides (39-49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33-41 %) and monoglucuronides (30-40 %) predominated. In male rats the disulfates (23-62 %) and 7-sulfo-4'-glucuronides (19-54 %) were predominant, while in female rats the 7-glucuronides (81-93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5-1.3 %) and rats (0.5-3.1 %) but comparatively high in mice (3.1-26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

Carlos Garzon-Coral, Horatiu Fantana, Jonathon Howard
A force-generating machinery maintains the spindle at the cell center during mitosis.
Science, 352(6289) 1124-1127 (2016)
DOI

The position and orientation of the mitotic spindle is precisely regulated to ensure the accurate partition of the cytoplasm between daughter cells and the correct localization of the daughters within growing tissue. Using magnetic tweezers to perturb the position of the spindle in intact cells, we discovered a force-generating machinery that maintains the spindle at the cell center during metaphase and anaphase in one- and two-cell Caenorhabditis elegans embryos. The forces increase with the number of microtubules and are larger in smaller cells. The machinery is rigid enough to suppress thermal fluctuations to ensure precise localization of the mitotic spindle, yet compliant enough to allow molecular force generators to fine-tune the position of the mitotic spindle to facilitate asymmetric division.

Gopi Shah
Pan-embryo Cell Dynamics - Multidimensional imaging uncovers the blueprint of zebrafish development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Alexander von Appen, Martin Beck
Structure Determination of the Nuclear Pore Complex with Three-Dimensional Cryo electron Microscopy.
J Mol Biol, 428(10 Pt A) 2001-2010 (2016)
Open Access DOI

Determining the structure of the nuclear pore complex (NPC) imposes an enormous challenge due to its size, intricate composition and membrane-embedded nature. In vertebrates, about 1000 protein building blocks assemble into a 110-MDa complex that fuses the inner and outer membranes of a cell's nucleus. Here, we review the recent progress in understanding the in situ architecture of the NPC with a specific focus on approaches using three-dimensional cryo electron microscopy. We discuss technological benefits and limitations and give an outlook toward obtaining a high-resolution structure of the NPC.

Anne Morbach
The Role of Cdep in the Embryonic Morphogenesis of Drosophila melanogaster
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Cihan Erkut, Vamshidhar Gade, Sunil Laxman, Teymuras V. Kurzchalia
The glyoxylate shunt is essential for desiccation tolerance in C. elegans and budding yeast.
Elife, 5 Art. No. e13614 (2016)
Open Access PDF DOI

Many organisms, including species from all kingdoms of life, can survive desiccation by entering a state with no detectable metabolism. To survive, C. elegans dauer larvae and stationary phase S. cerevisiae require elevated amounts of the disaccharide trehalose. We found that that dauer larvae and stationary phase yeast switched into a gluconeogenic mode in which metabolism was reoriented toward production of sugars from non-carbohydrate sources. This mode depended on full activity of the glyoxylate shunt (GS), which enables synthesis of trehalose from acetate. The GS was especially critical during preparation of worms for harsh desiccation (preconditioning) and during the entry of yeast into stationary phase. Loss of the GS dramatically decreased desiccation tolerance in both organisms. Our results reveal a novel physiological role for the GS and elucidate a conserved metabolic rewiring that confers desiccation tolerance on organisms as diverse as worm and yeast.

Jan Kosinski, Shyamal Mosalaganti, Alexander von Appen, Roman Teimer, Amanda L DiGuilio, William Wan, Khanh Huy Bui, Wim Hagen, John A G Briggs, Joseph S Glavy, Ed Hurt, Martin Beck
Molecular architecture of the inner ring scaffold of the human nuclear pore complex.
Science, 352(6283) 363-365 (2016)
DOI

Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Y complex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar.

David Flores-Benitez, Elisabeth Knust
Dynamics of epithelial cell polarity in Drosophila: how to regulate the regulators?
Curr Opin Cell Biol, 42 13-21 (2016)
PDF DOI

Apico-basal polarity is a hallmark of epithelial tissues. The integrated activity of several evolutionarily conserved protein complexes is essential to control epithelial polarity during development and homeostasis. Many components of these protein complexes were originally identified in genetic screens performed in Drosophila or Caenorhabditis elegans due to defects in cell polarity. With time, it became obvious that these protein complexes not only control various aspects of apico-basal polarity, but also perform a plethora of other functions, such as growth control, organization of endocytic activity, regulation of signaling and asymmetric cell division, to mention just a few. Here we summarize some results mostly obtained from studies in Drosophila to elucidate how variation in protein composition and modification of individual components contribute to make polarity complexes versatile platforms to fulfill a variety of functions.

Tom Stückemann
Patterning of the Planarian Anterior-Posterior Axis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Dragomir Milovanovic, Mitja Platen, Meike Junius, Ulf Diederichsen, Iwan A T Schaap, Alf Honigmann, Reinhard Jahn, Geert van den Bogaart
Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate.
J Biol Chem, 291(15) 7868-7876 (2016)
Open Access PDF DOI

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.

Marta Florio, Takashi Namba, Katherine Long
The Tortuous Routes of Migrating Neurons in the Folding Neocortex.
J Neurosci, 36(14) 3887-3889 (2016)
DOI

Konstantin Thierbach, Gopi Shah, Ingmar Glauche, Jan Huisken, Ingo Roeder, Nico Scherf
Biology-inspired visualization of morphogenetic motion in the zebrafish endoderm
In: 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI) (2016), Piscataway, N.J., IEEE (2016), 925-929
DOI

Effective visualization of large, multidimensional image data is a key instrument for data exploration in biological and medical applications. We propose a method to visualize motion patterns on a spherical surface, illustrated for the example of the zebrafish embryo. In particular, we analyze cellular motion by integrating prior knowledge about the underlying biological process and show how this improves visualization and connects to the conceptional description of the gastrulation process. We conclude that specialized visualizations are necessary to match data representation with conceptional ideas and to get a more intuitive understanding of the design principles of embryonic tissue formation.

Hao Yin, Roman L Bogorad, Carmen Barnes, Stephen Walsh, Iris Zhuang, Hidenori Nonaka, Vera M Ruda, Satya Kuchimanchi, Lubomir Nechev, Akin Akinc, Wen Xue, Marino Zerial, Robert Langer, Daniel G Anderson, Victor Koteliansky
RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver.
J Hepatol, 64(4) 899-907 (2016)
PDF DOI

The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models.

Radhika A Varier, Enrique Carrillo de Santa Pau, Petra van der Groep, Rik G H Lindeboom, Filomena Matarese, Anneloes Mensinga, Arne H Smits, Raghu Ram Edupuganti, Marijke P Baltissen, Pascal W T C Jansen, Natalie Ter Hoeve, Danny R van Weely, Ina Poser, Paul J van Diest, Hendrik G Stunnenberg, Michiel Vermeulen
Recruitment of the Mammalian Histone-modifying EMSY Complex to Target Genes Is Regulated by ZNF131.
J Biol Chem, 291(14) 7313-7324 (2016)
DOI

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.

Janet Karpinski, Ilona Hauber, Jan Chemnitz, Carola Schäfer, Maciej Paszkowski-Rogacz, Debojyoti Chakraborty, Niklas Beschorner, Helga Hofmann-Sieber, Ulrike C Lange, Adam Grundhoff, Karl Hackmann, Evelin Schrock, Josephine Abi-Ghanem, Maria Teresa Pisabarro, Vineeth Surendranath, Axel Schambach, Christoph Lindner, Jan van Lunzen, Joachim Hauber, Frank Buchholz
Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.
Nat Biotechnol, 34(4) 401-409 (2016)
DOI

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells, we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently, precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo, including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy.

Michal Levin, Leon Anavy, Alison G Cole, Eitan Winter, Natalia Mostov, Sally Khair, Naftalie Senderovich, Ekaterina Kovalev, David H Silver, Martin Feder, Selene L Fernandez-Valverde, Nagayasu Nakanishi, David Simmons, Oleg Simakov, Tomas Larsson, Shang-Yun Liu, Ayelet Jerafi-Vider, Karina Yaniv, Joseph F Ryan, Mark Q Martindale, Jochen Rink, Detlev Arendt, Sandie M Degnan, Bernard M Degnan, Tamar Hashimshony, Itai Yanai
The mid-developmental transition and the evolution of animal body plans.
Nature, 531(7596) 637-641 (2016)
DOI

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

Arnab Saha, Masatoshi Nishikawa, Martin Behrndt, Carl-Philipp Heisenberg, Frank Jülicher, Stephan W. Grill
Determining Physical Properties of the Cell Cortex.
Biophys J, 110(6) 1421-1429 (2016)
Open Access PDF DOI

Actin and myosin assemble into a thin layer of a highly dynamic network underneath the membrane of eukaryotic cells. This network generates the forces that drive cell- and tissue-scale morphogenetic processes. The effective material properties of this active network determine large-scale deformations and other morphogenetic events. For example, the characteristic time of stress relaxation (the Maxwell time τM) in the actomyosin sets the timescale of large-scale deformation of the cortex. Similarly, the characteristic length of stress propagation (the hydrodynamic length λ) sets the length scale of slow deformations, and a large hydrodynamic length is a prerequisite for long-ranged cortical flows. Here we introduce a method to determine physical parameters of the actomyosin cortical layer in vivo directly from laser ablation experiments. For this we investigate the cortical response to laser ablation in the one-cell-stage Caenorhabditis elegans embryo and in the gastrulating zebrafish embryo. These responses can be interpreted using a coarse-grained physical description of the cortex in terms of a two-dimensional thin film of an active viscoelastic gel. To determine the Maxwell time τM, the hydrodynamic length λ, the ratio of active stress ζΔμ, and per-area friction γ, we evaluated the response to laser ablation in two different ways: by quantifying flow and density fields as a function of space and time, and by determining the time evolution of the shape of the ablated region. Importantly, both methods provide best-fit physical parameters that are in close agreement with each other and that are similar to previous estimates in the two systems. Our method provides an accurate and robust means for measuring physical parameters of the actomyosin cortical layer. It can be useful for investigations of actomyosin mechanics at the cellular-scale, but also for providing insights into the active mechanics processes that govern tissue-scale morphogenesis.

Gheorghe Cojoc, Emanuele Roscioli, Lijuan Zhang, Alfonso García-Ulloa, Jagesh V Shah, Michael W Berns, Nenad Pavin, Daniela Cimini, Iva M Tolić, Juraj Gregan
Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore.
J Cell Biol, 212(7) 767-776 (2016)
DOI

Accurate chromosome segregation depends on proper kinetochore-microtubule attachment. Upon microtubule interaction, kinetochores are subjected to forces generated by the microtubules. In this work, we used laser ablation to sever microtubules attached to a merotelic kinetochore, which is laterally stretched by opposing pulling forces exerted by microtubules, and inferred the mechanical response of the kinetochore from its length change. In both mammalian PtK1 cells and in the fission yeast Schizosaccharomyces pombe, kinetochores shortened after microtubule severing. Interestingly, the inner kinetochore-centromere relaxed faster than the outer kinetochore. Whereas in fission yeast all kinetochores relaxed to a similar length, in PtK1 cells the more stretched kinetochores remained more stretched. Simple models suggest that these differences arise because the mechanical structure of the mammalian kinetochore is more complex. Our study establishes merotelic kinetochores as an experimental model for studying the mechanical response of the kinetochore in live cells and reveals a viscoelastic behavior of the kinetochore that is conserved in yeast and mammalian cells.

Kirstin Meyer
An in vivo investigation on the mechanosensory function of the mouse liver
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Saoussen Ben Halima, Sabyashachi Mishra, K Muruga Poopathi Raja, Michael Willem, Antonio Baici, Kai Simons, Oliver Brüstle, Philipp Koch, Christian Haass, Amedeo Caflisch, Lawrence Rajendran
Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.
Cell Rep, 14(9) 2127-2141 (2016)
Open Access PDF DOI

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects.

Alexander Kotzsch, Damian Pawolski, Alexander Milentyev, Anna Shevchenko, André Scheffel, Nicole Poulsen, Andrej Shevchenko, Nils Kröger
Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana.
J Biol Chem, 291(10) 4982-4997 (2016)
DOI

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.

Paola Laurino, Ágnes Tóth-Petróczy, Rubén Meana-Pañeda, Wei Lin, Donald G Truhlar, Dan S Tawfik
An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors.
PLoS Biol, 14(3) Art. No. e1002396 (2016)
Open Access DOI

Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose's ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint--geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif.

Ryota Uehara, Tomoko Kamasaki, Shota Hiruma, Ina Poser, Kinya Yoda, Junichiro Yajima, Daniel W Gerlich, Gohta Goshima
Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission.
Mol Biol Cell, 27(5) 812-827 (2016)
DOI

During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.

Sadhna Phanse, Cuihong Wan, Blake Borgeson, Fan Tu, Kevin Drew, Greg Clark, Xuejian Xiong, Olga Kagan, Julian Kwan, Alexandr Bezginov, Kyle Chessman, Swati Pal, Graham Cromar, Ophelia Papoulas, Zuyao Ni, Daniel R Boutz, Snejana Stoilova, Pierre C Havugimana, Xinghua Guo, Ramy H Malty, Mihail Sarov, Jack Greenblatt, Mohan Babu, W Brent Derry, Elisabeth R Tillier, John Wallingford, John Parkinson, Edward M Marcotte, Andrew Emili
Proteome-wide dataset supporting the study of ancient metazoan macromolecular complexes.
Data Brief, 6 715-721 (2016)
Open Access PDF DOI

Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].

Ibrahim Ömer Çiçek, Samir Karaca, Marko Brankatschk, Suzanne Eaton, Henning Urlaub, Halyna R Shcherbata
Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet.
Genetics, 202(3) 1167-1183 (2016)
DOI

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.

Stefania Tavano
Molecular and cellular mechanisms underlying the role of Insm1 in the generation of basal progenitor cells
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2016)

Julia Mahamid, Stefan Pfeffer, Miroslava Schaffer, Elizabeth Villa, Radostin Danev, Luis Kuhn Cuellar, Friedrich Förster, Anthony Hyman, Jürgen M Plitzko, Wolfgang Baumeister
Visualizing the molecular sociology at the HeLa cell nuclear periphery.
Science, 351(6276) 969-972 (2016)
DOI

The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed the native structure and organization of the cytoplasmic translation machinery. Analysis of a large dynamic structure-the nuclear pore complex-revealed variations detectable at the level of individual complexes. Cryo-ET was used to visualize previously elusive structures, such as nucleosome chains and the filaments of the nuclear lamina, in situ. Elucidation of the lamina structure provides insight into its contribution to metazoan nuclear stiffness.

Roberto Villaseñor, Yannis Kalaidzidis, Marino Zerial
Signal processing by the endosomal system.
Curr Opin Cell Biol, 39 53-60 (2016)
Open Access PDF DOI

Cells need to decode chemical or physical signals from their environment in order to make decisions on their fate. In the case of signalling receptors, ligand binding triggers a cascade of chemical reactions but also the internalization of the activated receptors in the endocytic pathway. Here, we highlight recent studies revealing a new role of the endosomal network in signal processing. The diversity of entry pathways and endosomal compartments is exploited to regulate the kinetics of receptor trafficking, and interactions with specific signalling adaptors and effectors. By governing the spatio-temporal distribution of signalling molecules, the endosomal system functions analogously to a digital-analogue computer that regulates the specificity and robustness of the signalling response.

Svend Bertel Dahl-Jensen, Manuel Figueiredo-Larsen, Anne Grapin-Botton, Kim Sneppen
Short-range growth inhibitory signals from the epithelium can drive non-stereotypic branching in the pancreas.
Physical biology, 13(1) 16007-16007 (2016)
DOI

Many organs such as the vasculature, kidney, lungs, pancreas and several other glands form ramified networks of tubes that either maximize exchange surfaces between two compartments or minimize the volume of an organ dedicated to the production and local delivery of a cell-derived product. The structure of these tubular networks can be stereotyped, as in the lungs, or stochastic with large variations between individuals, as in the pancreas. The principles driving stereotyped branching have attracted much attention and several models have been proposed and refined. Here we focus on the pancreas, as a model of non-stereotyped branching. In many ramified tubular organs, an important role of the mesenchyme as a source of branching signals has been proposed, including in the pancreas. However, our previous work has shown that in the absence of mesenchyme, epithelial cells seeded in vitro in Matrigel form heavily branched organoids. Here we experimentally show that pancreatic organoids grow primarily at the tips. Furthermore, in contrast to classical 'depletion of activator' mechanisms, organoids growing in close vicinity seem not to affect each other's growth before they get in contact. We recapitulate these observations in an in silico model of branching assuming a 'local inhibitor' is secreted by the epithelium. Remarkably this simple mechanism is sufficient to generate branched organoids similar to those observed in vitro, including their transition from filled spheres to a tree like structure. Quantifying the similarity between in silico and in vitro development through a normalized surface to volume ratio, our in silico model predicts that inhibition is likely to be cooperative and that the diffusing inhibitor decays within a length scale of 10-20 μm.

Mihail Sarov^#, Christiane Barz, Helena Jambor, Marco Y Hein, Christopher Schmied, Dana Suchold, Bettina Stender, Stephan Janosch, Vinay Vikas Kj, R T Krishnan, Aishwarya Krishnamoorthy, Irene R S Ferreira, Radoslaw K Ejsmont, Katja Finkl, Susanne Hasse, Philipp Kämpfer, Nicole Plewka, Elisabeth Vinis, Siegfried Schloissnig, Elisabeth Knust, Volker Hartenstein, Matthias Mann, Mani Ramaswami, K VijayRaghavan, Pavel Tomancak^#, Frank Schnorrer^#
A genome-wide resource for the analysis of protein localisation in Drosophila.
Elife, 5 Art. No. e12068 (2016)
Open Access PDF DOI

The Drosophila genome contains >13,000 protein coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here we present a genome-wide fosmid library of 10,000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins we created transgenic lines and for a total of 207 lines we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

Miguel Turrero García, Yoon Jeung Chang, Yoko Arai, Wieland B. Huttner
S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.
J Comp Neurol, 524(3) 456-470 (2016)
DOI

The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. J. Comp. Neurol. 524:456-470, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Sophie Ayciriex, Hermeto Gerber, Guillermo M Garcia Osuna, Mohamed Chami, Henning Stahlberg, Andrej Shevchenko, Patrick C Fraering
The lipidome associated with the γ-secretase complex is required for its integrity and activity.
Biochem J, 473(3) 321-334 (2016)
DOI

γ-Secretase is a multi-subunit membrane protease complex that catalyses the final intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of amyloid-β peptides (Aβ), which are implicated as the causative agents of Alzheimer's disease (AD). In the present study, we report the reconstitution of a highly purified, active γ-secretase complex into proteoliposomes without exogenous lipids and provide the first direct evidence for the existence of a microenvironment of 53 molecular species from 11 major lipid classes specifically associated with the γ-secretase complex, including phosphatidylcholine and cholesterol. Importantly, we demonstrate that the pharmacological modulation of certain phospholipids abolishes both the integrity and the enzymatic activity of the intramembrane protease. Together, our findings highlight the importance of a specific lipid microenvironment for the structure and function of γ-secretase.

Michael Kuhn, Ivica Letunic, Lars Juhl Jensen, Peer Bork
The SIDER database of drugs and side effects.
Nucleic Acids Res, 44(D1) 1075-1079 (2016)
Open Access DOI

Unwanted side effects of drugs are a burden on patients and a severe impediment in the development of new drugs. At the same time, adverse drug reactions (ADRs) recorded during clinical trials are an important source of human phenotypic data. It is therefore essential to combine data on drugs, targets and side effects into a more complete picture of the therapeutic mechanism of actions of drugs and the ways in which they cause adverse reactions. To this end, we have created the SIDER ('Side Effect Resource', http://sideeffects.embl.de) database of drugs and ADRs. The current release, SIDER 4, contains data on 1430 drugs, 5880 ADRs and 140 064 drug-ADR pairs, which is an increase of 40% compared to the previous version. For more fine-grained analyses, we extracted the frequency with which side effects occur from the package inserts. This information is available for 39% of drug-ADR pairs, 19% of which can be compared to the frequency under placebo treatment. SIDER furthermore contains a data set of drug indications, extracted from the package inserts using Natural Language Processing. These drug indications are used to reduce the rate of false positives by identifying medical terms that do not correspond to ADRs.

Wiebke Jahr, Benjamin Schmid, Michael Weber, Jan Huisken
eduSPIM: Light Sheet Microscopy in the Museum.
PLoS ONE, 11(8) Art. No. e0161402 (2016)
Open Access DOI

Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology.

Ronald D Vale, Anthony Hyman
Priority of discovery in the life sciences.
Elife, 5 Art. No. e16931 (2016)
Open Access PDF DOI

The job of a scientist is to make a discovery and then communicate this new knowledge to others. For a scientist to be successful, he or she needs to be able to claim credit or priority for discoveries throughout their career. However, despite being fundamental to the reward system of science, the principles for establishing the "priority of discovery" are rarely discussed. Here we break down priority into two steps: disclosure, in which the discovery is released to the world-wide community; and validation, in which other scientists assess the accuracy, quality and importance of the work. Currently, in biology, disclosure and an initial validation are combined in a journal publication. Here, we discuss the advantages of separating these steps into disclosure via a preprint, and validation via a combination of peer review at a journal and additional evaluation by the wider scientific community.

Robert Haase, Michael Andreeff, Nasreddin Abolmaali
On the Reliability of Automatic Volume Delineation in Low-Contrast [(18)F]FMISO-PET Imaging.
Recent Results Cancer Res, 198 175-187 (2016)
DOI

Hypoxia is a marker of poor prognosis in malignant tumors independent from the selected therapeutic method and the therapy should be intensified in such tumors. Hypoxia imaging with positron emission tomography (PET) is limited by low contrast to noise ratios with every available tracer. In radiation oncology appropriate delineation is required to allow therapy and intensification. While manual segmentation results are highly dependent from experience and observers condition (high inter- and intra observer variability), threshold- and gradient-based algorithms for automatic segmentation frequently fail in low contrast data sets. Likewise, calibration of these algorithms using phantoms is not useful. Complex computational models such as swarm intelligence-based algorithms are promising tools for optimized segmentation results and allow observer independent interpretation of multimodal and multidimensional imaging data.

Elena Taverna, Felipe Mora-Bermúdez, Paulina J. Strzyz, Marta Florio, Jaroslav Icha, Christiane Haffner, Caren Norden, Michaela Wilsch-Bräuninger, Wieland B. Huttner
Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells.
Sci Rep, 6 Art. No. 21206 (2016)
Open Access PDF DOI

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change.

Alexis Webb, Iván M Lengyel, David J. Jörg, Guillaume Valentin, Frank Jülicher, Luis G. Morelli, Andrew C. Oates
Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock.
Elife, 5 Art. No. e08438 (2016)
Open Access PDF DOI

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a "segmentation clock". This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics.

Janko Kajtez, Anastasia Solomatina, Maja Novak, Bruno Polak, Kruno Vukušić, Jonas Rüdiger, Gheorghe Cojoc, Ana Milas, Ivana Šumanovac Šestak, Patrik Risteski, Federica Tavano, Anna Klemm, Emanuele Roscioli, Julie Welburn, Daniela Cimini, Matko Glunčić, Nenad Pavin, Iva M Tolić
Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores.
Nat Commun, 7 Art. No. 10298 (2016)
Open Access PDF DOI

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term 'bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

Mark Leaver, Simone Kienle, Maria L Begasse, Ralf J Sommer, Anthony Hyman
A locus in Pristionchus pacificus that is responsible for the ability to give rise to fertile offspring at higher temperatures.
Biol Open, 5(8) 1111-1117 (2016)
Open Access PDF DOI

Temperature is a stress factor that varies temporally and spatially, and can affect the fitness of cold-blooded organisms, leading to a loss of reproductive output; however, little is understood about the genetics behind the long-term response of organisms to temperature. Here, we approach this problem in the model nematode Pristionchus pacificus by utilising a large collection of natural isolates with diverse phenotypes. From this collection we identify two strains, one from California that can give rise to fertile offspring up to 28°C and one from Japan that is fertile up to 30°C. We show that the optimum temperature and the upper temperature limit for fertility is shifted higher in the Japanese strain suggesting that there is a mechanism that controls the temperature response of fertility across a range of temperatures. By crossing the two strains, and using genetic mapping, we identify a region on chromosome V that is responsible for maintaining fertility at higher temperatures. Thus, we conclude that fitness of P. pacificus at high temperature is under genetic control, suggesting that it could be subject to natural selection.

Matthias Munder, Daniel Midtvedt, Titus Franzmann, Elisabeth Nüske, Oliver Otto, Maik Herbig, Elke Ulbricht, Paul Müller, Anna Taubenberger, Shovamayee Maharana, Liliana Malinovska, Doris Richter, Jochen Guck, Vasily Zaburdaev, Simon Alberti
A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy.
Elife, 5 Art. No. e09347 (2016)
Open Access PDF DOI

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Sandrine Passemard, Alain Verloes, Thierry Billette de Villemeur, Odile Boespflug-Tanguy, Karen Hernandez, Marion Laurent, Bertrand Isidor, Corinne Alberti, Nathalie Pouvreau, Séverine Drunat, Bénédicte Gérard, Vincent El Ghouzzi, Jorge Gallego, Monique Elmaleh-Bergès, Wieland B. Huttner, Stephan Eliez, Pierre Gressens, Marie Schaer
Abnormal spindle-like microcephaly-associated (ASPM) mutations strongly disrupt neocortical structure but spare the hippocampus and long-term memory.
Cortex, 74 158-176 (2016)
DOI

Autosomal recessive primary microcephaly results from abnormal brain development linked to proliferation defects in neural progenitors. The most frequent form, caused by ASPM mutations, is usually defined by a reduced brain volume and is associated with intellectual disability. Although many ASPM cases have now been reported, structural brain abnormalities and their link with cognitive disabilities have rarely been investigated. In this study, we used high resolution T1-weighted magnetic resonance imaging in seven patients with ASPM mutations and 39 healthy age-matched controls to quantify regional volumes, thickness, surface area, gyrification index and white matter volumes of 30 cortical regions. We observed a consistent reduction of 50% or more in the volume and surface area of all cortical regions except for the hippocampus and surrounding medial temporal structures, which were significantly less reduced. Neuropsychologic assessment indicated significant impairments of cognitive abilities. However, these impairments were associated with normal mnesic abilities, in keeping with the relative preservation of the hippocampus and medial temporal structures. These results show that, contrary to current opinion, the cortical volume and surface area of patients with ASPM mutations is reduced depending on a regionally specific fashion and their cognitive profile reflects this heterogeneity. The precise characterization of the cortical map and cognitive abilities of patients with ASPM mutations should allow developing more focused reeducative interventions well-suited to their real abilities.

André Nadler, Dmytro A Yushchenko, Rainer Müller, Frank Stein, Suihan Feng, Christophe Mulle, Mario Carta, Carsten Schultz
Exclusive photorelease of signalling lipids at the plasma membrane.
Nat Commun, 6 Art. No. 10056 (2015)
Open Access PDF DOI

Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

Édgar Roldán, Izaak Neri, Meik Dörpinghaus, Heinrich Meyr, Frank Jülicher
Decision Making in the Arrow of Time.
Phys Rev Lett, 115(25) Art. No. 250602 (2015)
DOI

We show that the steady-state entropy production rate of a stochastic process is inversely proportional to the minimal time needed to decide on the direction of the arrow of time. Here we apply Wald's sequential probability ratio test to optimally decide on the direction of time's arrow in stationary Markov processes. Furthermore, the steady-state entropy production rate can be estimated using mean first-passage times of suitable physical variables. We derive a first-passage time fluctuation theorem which implies that the decision time distributions for correct and wrong decisions are equal. Our results are illustrated by numerical simulations of two simple examples of nonequilibrium processes.

Hernán Morales-Navarrete, Fabián Segovia-Miranda, Piotr Klukowski, Kirstin Meyer, Hidenori Nonaka, Giovanni Marsico, Mikhail Chernykh, Alexander Kalaidzidis, Marino Zerial^#, Yannis Kalaidzidis^#
A versatile pipeline for the multi-scale digital reconstruction and quantitative analysis of 3D tissue architecture.
Elife, 4 Art. No. e11214 (2015)
Open Access PDF DOI

A prerequisite for the systems biology analysis of tissues is an accurate digital 3D reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness.

Kathrin Reichwald, Andreas Petzold, Philipp Koch, Bryan R Downie, Nils Hartmann, Stefan Pietsch, Mario Baumgart, Domitille Chalopin, Marius Felder, Martin Bens, Arne Sahm, Karol Szafranski, Stefan Taudien, Marco Groth, Ivan Arisi, Anja Weise, Samarth S Bhatt, Virag Sharma, Johann M Kraus, Florian Schmid, Steffen Priebe, Thomas Liehr, Matthias Görlach, Manuel E Than, Michael Hiller, Hans A Kestler, Jean-Nicolas Volff, Manfred Schartl, Alessandro Cellerino, Christoph Englert, Matthias Platzer
Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.
Cell, 163(6) 1527-1538 (2015)
PDF DOI

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-β family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

Stephanie Spannl
Identification of the protein Ecdysoneless as a regulator of Hedgehog signaling
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Yen Ting Lin, Daniela Frömberg, Wenwen Huang, Petrina Delivani, Mariola R. Chacón, Iva M Tolić, Frank Jülicher, Vasily Zaburdaev
Pulled Polymer Loops as a Model for the Alignment of Meiotic Chromosomes.
Phys Rev Lett, 115(20) Art. No. 208102 (2015)
DOI

During recombination, the DNA of parents exchange their genetic information to give rise to a genetically unique offspring. For recombination to occur, homologous chromosomes need to find each other and align with high precision. Fission yeast solves this problem by folding chromosomes in loops and pulling them through the viscous nucleoplasm. We propose a theory of pulled polymer loops to quantify the effect of drag forces on the alignment of chromosomes. We introduce an external force field to the concept of a Brownian bridge and thus solve for the statistics of loop configurations in space.

Frances H Marshall, Rachael E H Miles, Young-Chul Song, Peter B Ohm, Rory M Power, Jonathan P Reid, Cari S Dutcher
Diffusion and reactivity in ultraviscous aerosol and the correlation with particle viscosity
Chem Sci, 7 1298-1308 (2015)
Open Access PDF DOI

Oksana Lavrynenko, Jonathan Rodenfels, Maria Carvalho, Natalie Dye, Rene Lafont, Suzanne Eaton, Andrej Shevchenko
The ecdysteroidome of Drosophila: influence of diet and development.
Development, 142(21) 3758-3768 (2015)
DOI

Ecdysteroids are the hormones regulating development, physiology and fertility in arthropods, which synthesize them exclusively from dietary sterols. But how dietary sterol diversity influences the ecdysteroid profile, how animals ensure the production of desired hormones and whether there are functional differences between different ecdysteroids produced in vivo remains unknown. This is because currently there is no analytical technology for unbiased, comprehensive and quantitative assessment of the full complement of endogenous ecdysteroids. We developed a new LC-MS/MS method to screen the entire chemical space of ecdysteroid-related structures and to quantify known and newly discovered hormones and their catabolites. We quantified the ecdysteroidome in Drosophila melanogaster and investigated how the ecdysteroid profile varies with diet and development. We show that Drosophila can produce four different classes of ecdysteroids, which are obligatorily derived from four types of dietary sterol precursors. Drosophila makes makisterone A from plant sterols and epi-makisterone A from ergosterol, the major yeast sterol. However, they prefer to selectively utilize scarce ergosterol precursors to make a novel hormone 24,28-dehydromakisterone A and trace cholesterol to synthesize 20-hydroxyecdysone. Interestingly, epi-makisterone A supports only larval development, whereas all other ecdysteroids allow full adult development. We suggest that evolutionary pressure against producing epi-C-24 ecdysteroids might explain selective utilization of ergosterol precursors and the puzzling preference for cholesterol.

Dimitrios Papadopoulos, Aleksandar J Krmpot, Stanko N Nikolić, Robert Krautz, Lars Terenius, Pavel Tomancak, Rudolf Rigler, Walter J Gehring, Vladana Vukojević
Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.
Mech Dev, 138(2) 218-225 (2015)
PDF DOI

Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

Marta Florio
Transcriptional Signatures of Neocortical Expansion - Evolutionary Role of the Human-Specific Gene ARHGAP11B
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Stefan W Hell, Steffen J. Sahl, Mark Bates, Xiaowei Zhuang, Rainer Heintzmann, Martin J. Booth, Joerg Bewersdorf, Gleb Shtengel, Harald Hess, Philip Tinnefeld, Alf Honigmann, Stefan Jakobs, Ilaria Testa, Laurent Cognet, Brahim Lounis, Helge Ewers, Simon J. Davis, Christian Eggeling, David Klenerman, Katrin I. Willig, Giuseppe Vicidomini, Marco Castello, Alberto Diaspro, Thorben Cordes
The 2015 super-resolution microscopy roadmap
J Phys D: Appl Phys, 48(44) Art. No. 443001 (2015)
Open Access PDF

Mattia Brugiolo
The dynamic RNA-binding behavior of SR-proteins
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Eric R Geertsma^#, Yung-Ning Chang, Farooque R Shaik, Yvonne Neldner, Els Pardon, Jan Steyaert, Raimund Dutzler^#
Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family.
Nat Struct Mol Biol, 22(10) 803-808 (2015)
DOI

The SLC26 family of membrane proteins combines a variety of functions within a conserved molecular scaffold. Its members, besides coupled anion transporters and channels, include the motor protein Prestin, which confers electromotility to cochlear outer hair cells. To gain insight into the architecture of this protein family, we characterized the structure and function of SLC26Dg, a facilitator of proton-coupled fumarate symport, from the bacterium Deinococcus geothermalis. Its modular structure combines a transmembrane unit and a cytoplasmic STAS domain. The membrane-inserted domain consists of two intertwined inverted repeats of seven transmembrane segments each and resembles the fold of the unrelated transporter UraA. It shows an inward-facing, ligand-free conformation with a potential substrate-binding site at the interface between two helix termini at the center of the membrane. This structure defines the common framework for the diverse functional behavior of the SLC26 family.

Alexander von Appen^*, Jan Kosinski^*, Lenore Sparks^*, Alessandro Ori, Amanda L DiGuilio, Benjamin Vollmer, Marie-Therese Mackmull, Niccolo Banterle, Luca Parca, Panagiotis Kastritis, Katarzyna Buczak, Shyamal Mosalaganti, Wim Hagen, Amparo Andres-Pons, Edward A Lemke, Peer Bork, Wolfram Antonin, Joseph S Glavy, Khanh Huy Bui, Martin Beck
In situ structural analysis of the human nuclear pore complex.
Nature, 526(7571) 140-143 (2015)
DOI

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Gesa I Albert, Christoph Schell, Karin M Kirschner, Sebastian Schäfer, Ronald Naumann, Alexandra Müller, Oliver Kretz, Benno Kuropka, Mathias Girbig, Norbert Hübner, Eberhard Krause, Holger Scholz, Tobias B Huber, Klaus-Peter Knobeloch, Christian Freund
The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function.
J Mol Cell Biol, 7(5) 402-414 (2015)
DOI

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

David Martin, Yung-Hae Kim, Dror Sever, Chai-An Mao, Jacques-Antoine Haefliger, Anne Grapin-Botton
REST represses a subset of the pancreatic endocrine differentiation program.
Dev Biol, 405(2) 316-327 (2015)
DOI

To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program.

Marc Bickle, Hakim Djaballah, Lorenz Martin Mayr
The King Is Dead, Long Live the King! JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies.
J Biomol Screen, 20(8) 929-931 (2015)
DOI

Jan Böttger, Katrin Arnold, Carlo Thiel, Christiane Rennert, Susanne Aleithe, Ute Hofmann, Sebastian Vlaic, Susanne Sales, Andrej Shevchenko, Madlen Matz-Soja
RNAi in murine hepatocytes: the agony of choice-a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.
Arch Toxicol, 89(9) 1579-1588 (2015)
DOI

Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4 %), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

Eric Cornes, Montserrat Porta-De-La-Riva, David Aristizábal-Corrales, Ana María Brokate-Llanos, Francisco Javier García-Rodríguez, Iris Ertl, Mònica Díaz, Laura Fontrodona, Kadri Reis, Robert Johnsen, David Baillie, Manuel J Muñoz, Mihail Sarov, Denis Dupuy, Julián Cerón
Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans.
RNA, 21(9) 1544-1553 (2015)
DOI

Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance.

Avinash Patel, Hyun-Ok Kate Lee, Louise Jawerth, Shovamayee Maharana, Marcus Jahnel, Marco Y Hein, Stoyno Stoynov, Julia Mahamid, Shambaditya Saha, Titus Franzmann, Andrei Pozniakovski, Ina Poser, Nicola Maghelli, Loic Royer, Martin Weigert, Eugene W Myers, Stephan W. Grill, David N. Drechsel, Anthony Hyman, Simon Alberti
A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.
Cell, 162(5) 1066-1077 (2015)
DOI

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases. VIDEO ABSTRACT.

Simon Alberti
Don't Go with the Cytoplasmic Flow.
Dev Cell, 34(4) 381-382 (2015)
DOI

Cytoplasmic streaming occurs in a variety of organisms, but its biological function is often unclear. In this issue of Developmental Cell, Pieuchot et al. (2015) report that streaming creates subcellular compartments in the filamentous fungus Neurospora crassa. Nuclei immobilized within these compartments differentiate and may drive a compartment-specific genetic program.

Liat Rockah-Shmuel, Ágnes Tóth-Petróczy, Dan S Tawfik
Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations.
PLoS Comput Biol, 11(8) Art. No. e1004421 (2015)
Open Access DOI

Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII's natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.

Nico Scherf, Jan Huisken
The smart and gentle microscope.
Nat Biotechnol, 33(8) 815-818 (2015)
DOI

Wilhelm J. Walter, Isabel Machens, Fereshteh Rafieian, Stefan Diez
The non-processive rice kinesin-14 OsKCH1 transports actin filaments along microtubules with two distinct velocities.
Nat Plants, 1 Art. No. 15111 (2015)
DOI

Renee W Chow, Alexandra D Almeida, Owen Randlett, Caren Norden^#, William A Harris^#
Inhibitory neuron migration and IPL formation in the developing zebrafish retina.
Development, 142(15) 2665-2677 (2015)
Open Access PDF DOI

The mature vertebrate retina is a highly ordered neuronal network of cell bodies and synaptic neuropils arranged in distinct layers. Little, however, is known about the emergence of this spatial arrangement. Here, we investigate how the three main types of retinal inhibitory neuron (RIN) - horizontal cells (HCs), inner nuclear layer amacrine cells (iACs) and displaced amacrine cells (dACs) - reach their specific laminar positions during development. Using in vivo time-lapse imaging of zebrafish retinas, we show that RINs undergo distinct phases of migration. The first phase, common to all RINs, is bipolar migration directed towards the apicobasal centre of the retina. All RINs then transition to a less directionally persistent multipolar phase of migration. Finally, HCs, iACs and dACs each undergo cell type-specific migration. In contrast to current hypotheses, we find that most dACs send processes into the forming inner plexiform layer (IPL) before migrating through it and inverting their polarity. By imaging and quantifying the dynamics of HCs, iACs and dACs from birth to final position, this study thus provides evidence for distinct and new migration patterns during retinal lamination and insights into the initiation of IPL formation.

Cihan Erkut, Teymuras V. Kurzchalia
The C. elegans dauer larva as a paradigm to study metabolic suppression and desiccation tolerance.
Planta, 242(2) 389-396 (2015)
DOI

The hypometabolic, stress-resistant dauer larva of Caenorhabditis elegans serves as an excellent model to study the molecular mechanisms of desiccation tolerance, such as maintenance of membrane organization, protein folding, xenobiotic and ROS detoxification in the dry state. Many organisms from diverse taxa of life have the remarkable ability to survive extreme desiccation in the nature by entering an ametabolic state known as anhydrobiosis (life without water). The hallmark of the anhydrobiotic state is the achievement and maintenance of an exceedingly low metabolic rate, as well as preservation of the structural integrity of the cell. Although described more than three centuries ago, the biochemical and biophysical mechanisms underlying this phenomenon are still not fully comprehended. This is mainly due to the fact that anhydrobiosis in animals was studied using non-model organisms, which are very difficult, if not impossible, to manipulate at the molecular level. Recently, we introduced the roundworm (nematode) Caenorhabditis elegans as a model for anhydrobiosis. Taking advantage of powerful genetic, biochemical and biophysical tools, we investigated several aspects of anhydrobiosis in a particular developmental stage (the dauer larva) of this organism. First, our studies allowed confirming the previously suggested role of the disaccharide trehalose in the preservation of lipid membranes. Moreover, in addition to known pathways such as reactive oxygen species defense, heat-shock and intrinsically disordered protein expression, evidence for some novel strategies of anhydrobiosis has been obtained. These are increased glyoxalase activity, polyamine and polyunsaturated fatty acid biosynthesis. All these pathways may constitute a generic toolbox of anhydrobiosis, which is possibly conserved between animals and plants.

Mareike Albert, Wieland B. Huttner
Clever space saving-how the cerebral cortex folds.
EMBO J, 34(14) 1845-1847 (2015)
DOI

Katharina Nakel, Fabien Bonneau, Christian R. Eckmann, Elena Conti
Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3.
Proc Natl Acad Sci U.S.A., 112(28) 8614-8619 (2015)
DOI

The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.

S O Dodonova, P Diestelkoetter-Bachert, Alexander von Appen, W J H Hagen, R Beck, M Beck, Felix T. Wieland, John A G Briggs
A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly.
Science, 349(6244) 195-198 (2015)
DOI

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

Jovan Tanevski, Ljupčo Todorovski, Yannis Kalaidzidis, Sašo Džeroski
Domain-specific model selection for structural identification of the Rab5-Rab7 dynamics in endocytosis.
BMC Syst Biol, 9 Art. No. 31 (2015)
Open Access DOI

Given its recent rapid development and the central role that modeling plays in the discipline, systems biology clearly needs methods for automated modeling of dynamical systems. Process-based modeling focuses on explanatory models of dynamical systems; it constructs such models from measured time-course data and formalized modeling knowledge. In this paper, we apply process-based modeling to the practically relevant task of modeling the Rab5-Rab7 conversion switch in endocytosis. The task is difficult due to the limited observability of the system variables and the noisy measurements, which pose serious challenges to the process of model selection. To address these issues, we propose a domain-specific model selection criteria that take into account knowledge about the necessary properties of the simulated model behavior.

Vladimir Despic
Deciphering the post-transcriptional regulatory network during maternal to zygotic transition in zebrafish
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Ya-Huei Lin
The AP2 complex regulates epithelial polarity via regulating Crumbs endocytosis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Shana Elbaum-Garfinkle, Younghoon Kim, Krzysztof Szczepaniak, Carlos Chih-Hsiung Chen, Christian R. Eckmann, Sua Myong, Clifford Brangwynne
The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
Proc Natl Acad Sci U.S.A., 112(23) 7189-7194 (2015)
DOI

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

David Flores-Benitez, Elisabeth Knust
Ménage a Trois to Form the Tricellular Junction.
Dev Cell, 33(5) 501-503 (2015)
DOI

Tricellular junctions tightly seal epithelia at the corners of three cells. In this issue of Developmental Cell, Byri et al. (2015) show that Anakonda, a novel Drosophila transmembrane protein, contains an unusual tripartite extracellular domain organization, which explains the tripartite septum filling the tricellular junction, previously revealed by ultrastructure analysis.

Magdalena A Bukowska, Michael Hohl, Eric R Geertsma, Lea M Hürlimann, Markus G Grütter, Markus A Seeger
A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter.
Biochemistry, 54(19) 3086-3099 (2015)
DOI

ABC exporters are ubiquitous multidomain transport proteins that couple ATP hydrolysis at a pair of nucleotide binding domains to substrate transport across the lipid bilayer mediated by two transmembrane domains. Recently, the crystal structure of the heterodimeric ABC exporter TM287/288 was determined. One of its asymmetric ATP binding sites is called the degenerate site; it binds nucleotides tightly but is impaired in terms of ATP hydrolysis. Here we report the crystal structures of both isolated motor domains of TM287/288. Unexpectedly, structural elements constituting the degenerate ATP binding site are disordered in these crystals and become structured only in the context of the full-length transporter. In addition, hydrogen bonding patterns of key residues, including those of the catalytically important Walker B and the switch loop motifs, are fundamentally different in the solitary NBDs compared to those in the intact transport protein. The structures reveal crucial interdomain contacts that need to be established for the proper assembly of the functional transporter complex.

Anja Zeigerer, Roman L Bogorad, Kirti Sharma, Jerome Gilleron, Sarah Seifert, Susanne Sales, Nikolaus Berndt, Sascha Bulik, Giovanni Marsico, Rochelle C J D'Souza, Naharajan Lakshmanaperumal, Kesavan Meganathan, Karthick Natarajan, Agapios Sachinidis, Andreas Dahl, Hermann-Georg Holzhütter, Andrej Shevchenko, Matthias Mann, Victor Koteliansky, Marino Zerial
Regulation of Liver Metabolism by the Endosomal GTPase Rab5.
Cell Rep, 11(6) 884-892 (2015)
Open Access DOI

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

Wieland B. Huttner
Stem cells: slow and steady wins the race.
Nat Neurosci, 18(5) 613-614 (2015)
DOI

Stefan Kluth, Maria Grazia Pia, Thomas Schoerner-Sadenius, Peter Steinbach
How do particle physicists learn the programming concepts they need?
In: 21ST INTERNATIONAL CONFERENCE ON COMPUTING IN HIGH ENERGY AND NUCLEAR PHYSICS (CHEP2015), PARTS 1-9 (2015) Ch. 62048 (Journal of Physics Conference Series ; 664,6), Bristol, UK, Institute of Physics Publishing (IOP) (2015)
PDF DOI

Julieta Aprea, Mathias Lesche, Simone Massalini, Silvia Prenninger, Dimitra Alexopoulou, Andreas Dahl, Michael Hiller, Federico Calegari
Identification and expression patterns of novel long non-coding RNAs in neural progenitors of the developing mammalian cortex
Neurogenesis, 2(1) Art. No. e995524 (2015)
DOI

Matt Kanke, Helena Jambor, John Reich, Brittany Marches, Ronald Gstir, Young Hee Ryu, Anne Ephrussi, Paul M Macdonald
oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction.
RNA, 21(6) 1096-1109 (2015)
DOI

The Drosophila oskar (osk) mRNA is unusual in that it has both coding and noncoding functions. As an mRNA, osk encodes a protein required for embryonic patterning and germ cell formation. Independent of that function, the absence of osk mRNA disrupts formation of the karyosome and blocks progression through oogenesis. Here we show that loss of osk mRNA also affects the distribution of regulatory proteins, relaxing their association with large RNPs within the germline, and allowing them to accumulate in the somatic follicle cells. This and other noncoding functions of the osk mRNA are mediated by multiple sequence elements with distinct roles. One role, provided by numerous binding sites in two distinct regions of the osk 3' UTR, is to sequester the translational regulator Bruno (Bru), which itself controls translation of osk mRNA. This defines a novel regulatory circuit, with Bru restricting the activity of osk, and osk in turn restricting the activity of Bru. Other functional elements, which do not bind Bru and are positioned close to the 3' end of the RNA, act in the oocyte and are essential. Despite the different roles played by the different types of elements contributing to RNA function, mutation of any leads to accumulation of the germline regulatory factors in the follicle cells.

Marisa P. McShane, Tim Friedrichson, Angelika Giner, Felix Meyenhofer, Rico Barsacchi, Marc Bickle, Marino Zerial
A Combination of Screening and Computational Approaches for the Identification of Novel Compounds That Decrease Mast Cell Degranulation.
J Biomol Screen, 20(6) 720-728 (2015)
PDF DOI

High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.

Helena Khaliullina, Mesut Bilgin, Julio Sampaio, Andrej Shevchenko, Suzanne Eaton
Endocannabinoids are conserved inhibitors of the Hedgehog pathway.
Proc Natl Acad Sci U.S.A., 112(11) 3415-3420 (2015)
DOI

Hedgehog ligands control tissue development and homeostasis by alleviating repression of Smoothened, a seven-pass transmembrane protein. The Hedgehog receptor, Patched, is thought to regulate the availability of small lipophilic Smoothened repressors whose identity is unknown. Lipoproteins contain lipids required to repress Smoothened signaling in vivo. Here, using biochemical fractionation and lipid mass spectrometry, we identify these repressors as endocannabinoids. Endocannabinoids circulate in human and Drosophila lipoproteins and act directly on Smoothened at physiological concentrations to repress signaling in Drosophila and mammalian assays. Phytocannabinoids are also potent Smo inhibitors. These findings link organismal metabolism to local Hedgehog signaling and suggest previously unsuspected mechanisms for the physiological activities of cannabinoids.

Anne Eugster, Aaron Lindner, Mara Catani, Anne-Kristin Heninger, Andreas Dahl, Sylvia Klemroth, Denise Kühn, Susanne M Dietz, Marc Bickle, Anette-Gabrielle Ziegler, Enzio Bonifacio
High Diversity in the TCR Repertoire of GAD65 Autoantigen-Specific Human CD4+ T Cells.
J Immunol, 194(6) 2531-2538 (2015)
DOI

Autoreactive CD4(+) T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4(+) T cells, and TCR α-chain next-generation sequencing to bulk memory CD4(+) T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4(+) T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer(+) cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer(+) CD4(+) T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001-1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies.

Juha M. Torkko, María E Primo, Ronald Dirkx, Anne Friedrich, Antje Viehrig, Elisa Vergari, Barbara Borgonovo, Anke Sönmez, Carolin Wegbrod, Martina Lachnit, Carla Münster, Mauricio P Sica, Mario R Ermácora, Michele Solimena
Stability of proICA512/IA-2 and Its Targeting to Insulin Secretory Granules Require β4-Sheet-Mediated Dimerization of Its Ectodomain in the Endoplasmic Reticulum.
Mol Cell Biol, 35(6) 914-927 (2015)
DOI

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that β2- or β4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 β2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 β4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 β2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 β4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules.

T-Y Dora Tang, Nicholas J Brooks, Oscar Ces, John M Seddon, Richard H Templer
Structural studies of the lamellar to bicontinuous gyroid cubic (Q(G)(II)) phase transitions under limited hydration conditions.
Soft Matter, 11(10) 1991-1997 (2015)
DOI

Non-equilibrium pathways of lyotropic phase transitions such as the lamellar to inverse bicontinuous cubic phase transition are important dynamical processes resembling cellular fusion and fission processes which can be exploited in biotechnological processes such as drug delivery. However, utilising and optimising these structural transformations for applications require a detailed understanding of the energetic pathways which drive the phase transition. We have used the high pressure X-ray diffraction technique to probe the lamellar to Q(G)(II) phase transition in limited hydration monolinolein on the millisecond time scale. Our results show that the phase transition goes via a structural intermediate and once the Q(G)(II) phase initially forms the elastic energy in the bilayer drives this structure to its equilibrium lattice parameter.

Yung Hae Kim, Hjalte List Larsen, Pau Rué, Laurence A Lemaire, Jorge Ferrer, Anne Grapin-Botton
Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas.
PLoS Biol, 13(3) Art. No. e1002111 (2015)
Open Access DOI

Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle-dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro.

Moritz Kreysing, Lorenz Keil, Simon Lanzmich, Dieter Braun
Heat flux across an open pore enables the continuous replication and selection of oligonucleotides towards increasing length.
Nat Chem, 7(3) 203-208 (2015)
PDF DOI

The replication of nucleic acids is central to the origin of life. On the early Earth, suitable non-equilibrium boundary conditions would have been required to surmount the effects of thermodynamic equilibrium such as the dilution and degradation of oligonucleotides. One particularly intractable experimental finding is that short genetic polymers replicate faster and outcompete longer ones, which leads to ever shorter sequences and the loss of genetic information. Here we show that a heat flux across an open pore in submerged rock concentrates replicating oligonucleotides from a constant feeding flow and selects for longer strands. Our experiments utilize the interplay of molecular thermophoresis and laminar convection, the latter driving strand separation and exponential replication. Strands of 75 nucleotides survive whereas strands half as long die out, which inverts the above dilemma of the survival of the shortest. The combined feeding, thermal cycling and positive length selection opens the door for a stable molecular evolution in the long-term microhabitat of heated porous rock.

Cai Guo, Akiko Kumagai, Katharina Schlacher, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
Interaction of Chk1 with Treslin negatively regulates the initiation of chromosomal DNA replication.
Mol Cell, 57(3) 492-505 (2015)
DOI

Treslin helps to trigger the initiation of DNA replication by promoting integration of Cdc45 into the replicative helicase. Treslin is a key positive-regulatory target of cell-cycle control mechanisms; activation of Treslin by cyclin-dependent kinase is essential for the initiation of replication. Here we demonstrate that Treslin is also a critical locus for negative regulatory mechanisms that suppress initiation. We found that the checkpoint-regulatory kinase Chk1 associates specifically with a C-terminal domain of Treslin (designated TRCT). Mutations in the TRCT domain abolish binding of Chk1 to Treslin and thereby eliminate Chk1-catalyzed phosphorylation of Treslin. Significantly, abolition of the Treslin-Chk1 interaction results in elevated initiation of chromosomal DNA replication during an unperturbed cell cycle, which reveals a function for Chk1 during a normal S phase. This increase is due to enhanced loading of Cdc45 onto potential replication origins. These studies provide important insights into how vertebrate cells orchestrate proper initiation of replication.

Maria L Begasse, Mark Leaver, Federico Vazquez, Stephan W. Grill, Anthony Hyman
Temperature Dependence of Cell Division Timing Accounts for a Shift in the Thermal Limits of C. elegans and C. briggsae.
Cell Rep, 10(5) 647-653 (2015)
Open Access DOI

Cold-blooded animals, which cannot directly control their body temperatures, have adapted to function within specific temperature ranges that vary between species. However, little is known about what sets the limits of the viable temperature range. Here we show that the speed of the first cell division in C. elegans N2 varies with temperature according to the Arrhenius equation. However, it does so only within certain limits. Outside these limits we observe alterations in the cell cycle. Interestingly, these temperature limits also correspond to the animal's fertile range. In C. briggsae AF16, isolated from a warmer climatic region, both the fertile range and the temperature range over which the speed of cell division follows the Arrhenius equation, are shifted toward higher temperatures. Our findings suggest that the viable range of an organism can be adapted in part to a different thermal range by adjusting the temperature tolerance of cell division.

Carlos Garzón Coral
The forces that center the mitotic spindle in the C. elegans embryo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Maria L Spletter, Christiane Barz, Assa Yeroslaviz, Cornelia Schönbauer, Irene R S Ferreira, Mihail Sarov, Daniel Gerlach, Alexander Stark, Bianca Habermann, Frank Schnorrer
The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.
EMBO Rep, 16(2) 178-191 (2015)
DOI

In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.

Paulina J. Strzyz
The significance of bringing nuclei to the apical surface for division in the pseudostratified epithelium of the developing zebrafish retina
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2015)

Meritxell Huch
Regenerative biology: The versatile and plastic liver.
Nature, 517(7533) 155-156 (2015)
DOI

Simone Reber, Anthony A. Hyman
Emergent Properties of the Metaphase Spindle
In: Mitosis : a subject collection form Cold Spring Harbor Perspectives in biology. (Eds.) Mitsuhiro Yanagida, Anthony A. Hyman, Jonathon Pines, Cold Spring Harbor, N.Y, Cold Spring Harbor Laboratory Press (2015), 31-52
DOI

Judith Paridaen, Wieland B. Huttner, Michaela Wilsch-Bräuninger
Analysis of primary cilia in the developing mouse brain.
Methods Cell Biol, 127 93-129 (2015)
DOI

Stem and progenitor cells in the developing mammalian brain are highly polarized cells that carry a primary cilium protruding into the brain ventricles. Here, cilia detect signals present in the cerebrospinal fluid that fills the ventricles. Recently, striking observations have been made regarding the dynamics of primary cilia in mitosis and cilium reformation after cell division. In neural progenitors, primary cilia are not completely disassembled during cell division, and some ciliary membrane remnant can be inherited by one daughter cell that tends to maintain a progenitor fate. Furthermore, newborn differentiating cells grow a primary cilium on their basolateral plasma membrane, in spite of them possessing apical membrane and adherens junctions, and thus change the environment to which the primary cilium is exposed. These phenomena are proposed to be involved in cell fate determination and delamination of daughter cells in conjunction with the production of neurons. Here, we describe several methods that can be used to study the structure, localization, and dynamics of primary cilia in the developing mouse brain; these include time-lapse imaging of live mouse embryonic brain tissues, and analysis of primary cilia structure and localization using correlative light- and electron- and serial-block-face scanning electron microscopy.

Eva Sevcsik, Mario Brameshuber, Martin Fölser, Julian Weghuber, Alf Honigmann, Gerhard J Schütz
GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.
Nat Commun, 6 6969-6969 (2015)
Open Access DOI

The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.

Arne Schwelm, Johan Fogelqvist, Andrea Knaust, Sabine Jülke, Tua Lilja, German Bonilla-Rosso, Magnus Karlsson, Andrej Shevchenko, Vignesh Dhandapani, Su Ryun Choi, Hong Gi Kim, Ju Young Park, Yong Pyo Lim, Jutta Ludwig-Müller, Christina Dixelius
The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases.
Sci Rep, 5 Art. No. 11153 (2015)
Open Access DOI

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

Damian Szklarczyk, Andrea Franceschini, Stefan Wyder, Kristoffer Forslund, Davide Heller, Jaime Huerta-Cepas, Milan Simonovic, Alexander Roth, Alberto Santos, Kalliopi P Tsafou, Michael Kuhn, Peer Bork, Lars Juhl Jensen, Christian von Mering
STRING v10: protein-protein interaction networks, integrated over the tree of life.
Nucleic Acids Res, 43(D1) 447-452 (2015)
Open Access DOI

The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

Sonja Kroschwald, Shovamayee Maharana, Daniel Mateju, Liliana Malinovska, Elisabeth Nüske, Ina Poser, Doris Richter, Simon Alberti
Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.
Elife, 4 Art. No. e06807 (2015)
Open Access PDF DOI

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Raphael Etournay, Marko Popović, Matthias Merkel, Amitabha Nandi, Corinna Blasse, Benoit Aigouy, Holger Brandl, Gene Myers, Guillaume Salbreux, Frank Jülicher, Suzanne Eaton
Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing.
Elife, 4 Art. No. e07090 (2015)
Open Access PDF DOI

How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing.

Amulya Priya, Inna Kalaidzidis, Yannis Kalaidzidis, David Lambright, Sunando Datta
Molecular insights into rab7-mediated endosomal recruitment of core retromer: deciphering the role of vps26 and vps35.
Traffic, 16(1) 68-84 (2015)
DOI

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans-Golgi network. It consists of two distinct sub-complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub-complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET-based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N-terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7-mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub-complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7-mediated recruitment of the core retromer complex.

Yannis Kalaidzidis, Inna Kalaidzidis, Marino Zerial
A Probabilistic Method to Quantify the Colocalization of Markers on Intracellular Vesicular Structures Visualized by Light Microscopy.
In: BAYESIAN INFERENCE AND MAXIMUM ENTROPY METHODS IN SCIENCE AND ENGINEERING (MAXENT 2014) (2015)(Eds.) A. Mohammad-Djafari, F. Barbaresco AIP Conference Proceedings ; 1641, Melville, N.Y., AIP (2015), 580-587
DOI

Florian Ehehalt, Dorothée Sturm, Manuela Rösler, Marius Distler, Jürgen Weitz, Stephan Kersting, Barbara Ludwig, Uta Schwanebeck, Hans-Detlev Saeger, Michele Solimena, Robert Grützmann
Blood Glucose Homeostasis in the Course of Partial Pancreatectomy - Evidence for Surgically Reversible Diabetes Induced by Cholestasis.
PLoS ONE, 10(8) Art. No. e0134140 (2015)
Open Access PDF DOI

Partial pancreatic resection is accompanied not only by a reduction in the islet cell mass but also by a variety of other factors that are likely to interfere with glucose metabolism. The aim of this work was to characterize the patient dynamics of blood glucose homeostasis during the course of partial pancreatic resection and to specify the associated clinico-pathological variables.

Sylke Winkler, K. Linke, Nicola Gscheidel, M. Meyer, D. Krabel
PCR-based detection of single sequence variants from a natural collection of the non-model tree species European Aspen Populus tremula (L.)
Silvae Genetica, 64(5-6) 259-269 (2015)
Open Access

In the present study we present and discuss the identification of species-specific SNPs to rule out any experimental influence of species specific primer design (Populus tremula vs. the closely related model-species Populus trichocarpa) on the detectability of SNPs. Applying a species-optimized method, partial sequences of 14 genes involved in xylem cell development, xylogenesis, pectin formation, and drought stress reaction were analyzed at the genomic level. About 3 Mb of sequence information were generated by Sanger sequencing technology and 258 sequence variants were identified. 15 out of these represent insertions/deletions located exclusively in non coding regions and the remaining 243 are SNPs found in coding and non-coding regions of candidate genes. The introduction of a species-specific SNP detection pipeline will help to detect nucleotide variants in P. tremula and to conduct association mapping in natural P tremula populations.

Joshua Alper, Franziska Decker, Bernice Agana, Jonathon Howard
The Motility of Axonemal Dynein Is Regulated by the Tubulin Code
Biophys J, 107(12) 2872-2880 (2014)

Dirk Drasdo, Johannes Bode, Uta Dahmen, Olaf Dirsch, Steven Dooley, Rolf Gebhardt, Ahmed Ghallab, Patricio Godoy, Dieter Häussinger, Seddik Hammad, Stefan Hoehme, Hermann-Georg Holzhütter, Ursula Klingmüller, Lars Kuepfer, Jens Timmer, Marino Zerial, Jan G Hengstler
The virtual liver: state of the art and future perspectives.
Arch Toxicol, 88(12) 2071-2075 (2014)
DOI

Jacqueline Tabler, Trióna G Bolger, John Wallingford, Karen J Liu
Hedgehog activity controls opening of the primary mouth.
Dev Biol, 396(1) 1-7 (2014)
DOI

To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes.

Christopher J Hindley^*, Gianmarco Mastrogiovanni^*, Meritxell Huch
The plastic liver: differentiated cells, stem cells, every cell?
J Clin Invest, 124(12) 5099-5102 (2014)
DOI

The liver is capable of full regeneration following several types and rounds of injury, ranging from hepatectomy to toxin-mediated damage. The source of this regenerative capacity has long been a hotly debated topic. The damage response that occurs when hepatocyte proliferation is impaired is thought to be mediated by oval/dedifferentiated progenitor cells, which replenish the hepatocyte and ductal compartments of the liver. Recently, reports have questioned whether these oval/progenitor cells truly serve as the facultative stem cell of the liver following toxin-mediated damage. In this issue of the JCI, Kordes and colleagues use lineage tracing to follow transplanted rat hepatic stellate cells, a resident liver mesenchymal cell population, in hosts that have suffered liver damage. Transplanted stellate cells repopulated the damaged rat liver by contributing to the oval cell response. These data establish yet another cell type of mesenchymal origin as the progenitor for the oval/ductular response in the rat. The lack of uniformity between different damage models, the extent of the injury to the liver parenchyma, and potential species-specific differences might be at the core of the discrepancy between different studies. Taken together, these data imply a considerable degree of plasticity in the liver, whereby several cell types can contribute to regeneration.

Christine Moessinger, Kristina Klizaite, Almut Steinhagen, Julia Philippou-Massier, Andrej Shevchenko, Michael Hoch, Christer S. Ejsing, Christoph Thiele
Two different pathways of phosphatidylcholine synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate cellular triacylglycerol storage.
BMC Cell Biol, 15(1) Art. No. 43 (2014)
DOI

BackgroundLipids are stored within cells in lipid droplets (LDs). They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and can rapidly change in size upon lipid uptake or release. These dynamics require a fast adaptation of LD surface. We have recently shown that two Lands cycle PC synthesizing enyzmes, LPCAT1 and LPCAT2 can localize to the LD surface.ResultsHere, we show that knock-down of both enzymes leads to an increase in LD size without changes in the total amount of neutral lipids, while interference with the de-novo Kennedy pathway PC biosynthesis is associated with changes in triacylglyceride synthesis. We show that function of LPCAT1 and 2 is conserved in Drosophila melanogaster by the ortholog CG32699. Furthermore we demonstrate that modulation of the LD pool by LPCAT1 influences the release of lipoprotein from liver cells.ConclusionActivity of the Kennedy pathway regulates the balance between phospholipids and neutral lipids, while the Lands cycle regulates lipid droplet size by regulating surface availability and influencing surface to volume ratio. Differences in lipid droplet size may account for differences in lipid dynamics and be relevant to understand lipid overload diseases.

Xin Liang, Johnson Madrid, Jonathon Howard
The microtubule-based cytoskeleton is a component of a mechanical signaling pathway in fly campaniform receptors.
Biophys J, 107(12) 2767-2774 (2014)
DOI

In mechanoreceptors, mechanical stimulation by external forces leads to the rapid opening of transduction channels followed by an electrical response. Despite intensive studies in various model systems, the molecular pathway by which forces are transmitted to the transduction channels remains elusive. In fly campaniform mechanoreceptors, the mechanotransduction channels are gated by compressive forces conveyed via two rows of microtubules that are hypothesized to be mechanically reinforced by an intervening electron-dense material (EDM). In this study, we tested this hypothesis by studying a mutant fly in which the EDM was nearly absent, whereas the other ultrastructural elements in the mechanosensitive organelle were still present at 50% (or greater) of normal levels. We found that the mechanosensory response in this mutant was reduced by 90% and the sensitivity by at least 80%. To test whether loss of the EDM could lead to such a reduction in response, we performed a mechanical analysis and estimated that the loss of the EDM is expected to greatly decrease the overall rigidity, leading to a marked reduction in the gating force conveyed to the channel. We argue that this reduction in force, rather than the reduction in the number of transduction channels, is primarily responsible for the nearly complete loss of mechanosensory response observed in the mutant fly. Based on these experiments and analysis, we conclude that the microtubule-based cytoskeleton (i.e., microtubules and EDM) is an essential component of the mechanical signaling pathway in fly campaniform mechanoreceptor.

Andrea Meinhardt, Dominic Eberle, Akira Tazaki, Adrian Ranga, Marco Niesche, Michaela Wilsch-Bräuninger, Agnieszka Stec, Gabriele Schackert, Matthias Lutolf, Elly M. Tanaka
3D Reconstitution of the Patterned Neural Tube from Embryonic Stem Cells.
Stem Cell Rep, 3(6) 987-999 (2014)
Open Access DOI

Inducing organogenesis in 3D culture is an important aspect of stem cell research. Anterior neural structures have been produced from large embryonic stem cell (ESC) aggregates, but the steps involved in patterning such complex structures have been ill defined, as embryoid bodies typically contained many cell types. Here we show that single mouse ESCs directly embedded in Matrigel or defined synthetic matrices under neural induction conditions can clonally form neuroepithelial cysts containing a single lumen in 3D. Untreated cysts were uniformly dorsal and could be ventralized to floor plate (FP). Retinoic acid posteriorized cysts to cervical levels and induced localize FP formation yielding full patterning along the dorsal/ventral (DV) axis. Correct spatial organization of motor neurons, interneurons, and dorsal interneurons along the DV axis was observed. This system serves as a valuable tool for studying morphogen action in 3D and as a source of patterned spinal cord tissue.

Jonathan Rodenfels, Oksana Lavrynenko, Sophie Ayciriex, Julio Sampaio, Maria Carvalho, Andrej Shevchenko, Suzanne Eaton
Production of systemically circulating Hedgehog by the intestine couples nutrition to growth and development.
Genes Dev, 28(23) 2636-2651 (2014)
DOI

In Drosophila larvae, growth and developmental timing are regulated by nutrition in a tightly coordinated fashion. The networks that couple these processes are far from understood. Here, we show that the intestine responds to nutrient availability by regulating production of a circulating lipoprotein-associated form of the signaling protein Hedgehog (Hh). Levels of circulating Hh tune the rates of growth and developmental timing in a coordinated fashion. Circulating Hh signals to the fat body to control larval growth. It regulates developmental timing by controlling ecdysteroid production in the prothoracic gland. Circulating Hh is especially important during starvation, when it is also required for mobilization of fat body triacylglycerol (TAG) stores. Thus, we demonstrate that Hh, previously known only for its local morphogenetic functions, also acts as a lipoprotein-associated endocrine hormone, coordinating the response of multiple tissues to nutrient availability.

Martin Machyna, Stephanie Kehr, Korinna Straube, Dennis Kappei, Frank Buchholz, Falk Butter, Jernej Ule, Jana Hertel, Peter F Stadler, Karla M. Neugebauer
The Coilin Interactome Identifies Hundreds of Small Noncoding RNAs that Traffic through Cajal Bodies
Mol Cell, 56(3) 389-399 (2014)

Angela Wandinger-Ness, Marino Zerial
Rab proteins and the compartmentalization of the endosomal system.
Cold Spring Harb Perspect Biol, 6(11) Art. No. a022616 (2014)
DOI

Of the approximately 70 human Rab GTPases, nearly three-quarters are involved in endocytic trafficking. Significant plasticity in endosomal membrane transport pathways is closely coupled to receptor signaling and Rab GTPase-regulated scaffolds. Here we review current literature pertaining to endocytic Rab GTPase localizations, functions, and coordination with regulatory proteins and effectors. The roles of Rab GTPases in (1) compartmentalization of the endocytic pathway into early, recycling, late, and lysosomal routes; (2) coordination of individual transport steps from vesicle budding to fusion; (3) effector interactomes; and (4) integration of GTPase and signaling cascades are discussed.

Fabian Rost, Christina Eugster, Christian Schröter, Andrew C. Oates, Lutz Brusch
Chevron formation of the zebrafish muscle segments.
J Exp Biol, 217(21) 3870-3882 (2014)
DOI

The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation.

Sawsan E Abusharkh, Cihan Erkut, Jana Oertel, Teymuras V. Kurzchalia, Karim Fahmy
The Role of Phospholipid Headgroup Composition and Trehalose in the Desiccation Tolerance of Caenorhabditis elegans.
Langmuir, 30(43) 12897-12906 (2014)
DOI

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic state. Distributed to various taxa of life, these organisms have developed strategies to efficiently protect their cell membranes and proteins against extreme water loss. Recently, we showed that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation to harsh desiccation (preconditioning). Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, dauer larvae dramatically reduce their phosphatidylcholine (PC) content. The chemical composition of the phospholipids (PLs) has key consequences not only for their interaction with trehalose, as we demonstrate with Langmuir-Blodgett monolayers, but also, the kinetic response of PLs to hydration transients is strongly influenced as evidenced by time-resolved FTIR spectroscopy. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates of their lyotropic transitions and sub-headgroup H-bond interactions. The different hydration properties of PC and phosphatidylethanolamine (PE) headgroups are crucial for the hydration-dependent rearrangement of the trehalose-mediated H-bond network. As a consequence, the compressibility modulus of PLs from preconditioned larvae is about 2.6-fold smaller than that from non-preconditioned ones. Thus, the biological relevance of reducing the PC:PE ratio by PL headgroup adaptation should be the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose-containing cells during fast rehydration.

Florian Jug, Tobias Pietzsch, Dagmar Kainmueller, Jan Funke, Matthias Kaiser, Erik van Nimwegen, Carsten Rother, Gene Myers
Optimal Joint Segmentation and Tracking of Escherichia Coli in the Mother Machine
In: Bayesian and grAphical models for biomedical imaging first international workshop, BAMBI 2014, Cambridge, MA, USA, September 18, 2014 ; revised selected papers (2014) Lecture Notes in Computer Science ; 8677, New York, Springer (2014), 25-36
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Ines A Ehrnstorfer, Eric R Geertsma, Els Pardon, Jan Steyaert, Raimund Dutzler
Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport.
Nat Struct Mol Biol, 21(11) 990-996 (2014)
DOI

Members of the SLC11 (NRAMP) family transport iron and other transition-metal ions across cellular membranes. These membrane proteins are present in all kingdoms of life with a high degree of sequence conservation. To gain insight into the determinants of ion selectivity, we have determined the crystal structure of Staphylococcus capitis DMT (ScaDMT), a close prokaryotic homolog of the family. ScaDMT shows a familiar architecture that was previously identified in the amino acid permease LeuT. The protein adopts an inward-facing conformation with a substrate-binding site located in the center of the transporter. This site is composed of conserved residues, which coordinate Mn2+, Fe2+ and Cd2+ but not Ca2+. Mutations of interacting residues affect ion binding and transport in both ScaDMT and human DMT1. Our study thus reveals a conserved mechanism for transition-metal ion selectivity within the SLC11 family.

Marnix Wieffer, Marisa P. McShane, Marino Zerial
Rab Proteins and the Organization of Organelle Membrane Domains.
In: Ras Superfamily Small G Proteins: Biology and Mechanisms : 2. (Eds.) Alfred Wittinghofer, Cham, Springer International Publishing (2014), 17-38 Ch. 2
DOI

Elena Taverna, Magdalena Götz, Wieland B. Huttner
The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex.
Annu Rev Cell Dev Biol, 30 465-502 (2014)
DOI

Neural stem and progenitor cells have a central role in the development and evolution of the mammalian neocortex. In this review, we first provide a set of criteria to classify the various types of cortical stem and progenitor cells. We then discuss the issue of cell polarity, as well as specific subcellular features of these cells that are relevant for their modes of division and daughter cell fate. In addition, cortical stem and progenitor cell behavior is placed into a tissue context, with consideration of extracellular signals and cell-cell interactions. Finally, the differences across species regarding cortical stem and progenitor cells are dissected to gain insight into key developmental and evolutionary mechanisms underlying neocortex expansion.

Oliver Wueseke, Jakob Bunkenborg, Marco Y Hein, Andrea Zinke, Valeria Viscardi, Jeffrey Woodruff, Karen Oegema, Matthias Mann, Jens S Andersen, Anthony Hyman
The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix.
Mol Biol Cell, 25(19) 2984-2992 (2014)
DOI

Centrosomes are the main microtubule-organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that assembles from cytoplasmic components. In Caenorhabditis elegans embryos, interactions between the coiled-coil proteins SPD-5 and SPD-2 and the kinase PLK-1 are critical for PCM assembly. However, it is not known whether these interactions promote the formation of cytoplasmic complexes that are added to the PCM or whether the components interact only during incorporation into the PCM matrix. Here we address this problem by using a combination of live-cell fluorescence correlation spectroscopy, mass spectrometry, and hydrodynamic techniques to investigate the native state of PCM components in the cytoplasm. We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1. SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.

Jan Philipp Junker, Emily S. Noël, Victor Guryev, Kevin A Peterson, Gopi Shah, Jan Huisken, Andrew P. McMahon, Eugene Berezikov, Jeroen Bakkers, Alexander van Oudenaarden
Genome-wide RNA Tomography in the Zebrafish Embryo.
Cell, 159(3) 662-675 (2014)
DOI

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Suzanne Eaton, Fernando Martin-Belmonte
Cargo sorting in the endocytic pathway: a key regulator of cell polarity and tissue dynamics.
Cold Spring Harb Perspect Biol, 6(10) Art. No. a016899 (2014)
DOI

The establishment and maintenance of polarized plasma membrane domains is essential for cellular function and proper development of organisms. Epithelial cells polarize along two fundamental axes, the apicobasal and the planar, both depending on finely regulated protein trafficking mechanisms. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans-Golgi network or endosomes. This exocytic trafficking has been identified as essential for proper epithelial polarization. Accumulating evidence now reveals that endocytosis and endocytic recycling play an equally important role in epithelial polarization and the appropriate localization of key polarity proteins. Here, we review recent work in metazoan systems illuminating the connections between endocytosis, postendocytic trafficking, and cell polarity, both apicobasal and planar, in the formation of differentiated epithelial cells, and how these processes regulate tissue dynamics.

Lucie Kalvodova, Kai Simons
Lipidomics is going great guns: Interview with Kai Simons about the power of shotgun lipidomics
Eur J Lipid Sci Technol, 116(10) 1344-1346 (2014)

Christoph Klingner, Anoop V Cherian, Johannes Fels, Philipp M Diesinger, Roland Aufschnaiter, Nicola Maghelli, Thomas Keil, Gisela Beck, Iva M. Tolic-Norrelykke, Mark Bathe, Roland Wedlich-Soldner
Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.
J Cell Biol, 207(1) 107-121 (2014)
PDF DOI

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.

Sophia Millonigg, Ryuji Minasaki, Marco Nousch, Christian R. Eckmann
GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line.
PLoS Genet, 10(9) Art. No. e1004647 (2014)
DOI

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.

Antonella Lauri, Thibaut Brunet, Mette Handberg-Thorsager, Antje H L Fischer, Oleg Simakov, Patrick R H Steinmetz, Raju Tomer, Patrick Keller, Detlev Arendt
Development of the annelid axochord: insights into notochord evolution.
Science, 345(6202) 1365-1368 (2014)
PDF DOI

The origin of chordates has been debated for more than a century, with one key issue being the emergence of the notochord. In vertebrates, the notochord develops by convergence and extension of the chordamesoderm, a population of midline cells of unique molecular identity. We identify a population of mesodermal cells in a developing invertebrate, the marine annelid Platynereis dumerilii, that converges and extends toward the midline and expresses a notochord-specific combination of genes. These cells differentiate into a longitudinal muscle, the axochord, that is positioned between central nervous system and axial blood vessel and secretes a strong collagenous extracellular matrix. Ancestral state reconstruction suggests that contractile mesodermal midline cells existed in bilaterian ancestors. We propose that these cells, via vacuolization and stiffening, gave rise to the chordate notochord.

Matthias Merkel, Andreas Sagner, Franz Gruber, Raphael Etournay, Corinna Blasse, Gene Myers, Suzanne Eaton, Frank Jülicher
The Balance of Prickle/Spiny-Legs Isoforms Controls the Amount of Coupling between Core and Fat PCP Systems.
Curr Biol, 24(18) 2111-2123 (2014)
DOI

The conserved Fat and Core planar cell polarity (PCP) pathways work together to specify tissue-wide orientation of hairs and ridges in the Drosophila wing. Their components form intracellularly polarized complexes at adherens junctions that couple the polarity of adjacent cells and form global patterns. How Fat and Core PCP systems interact is not understood. Some studies suggest that Fat PCP directly orients patterns formed by Core PCP components. Others implicate oriented tissue remodeling in specifying Core PCP patterns.

Marco Nousch, Assa Yeroslaviz, Bianca Habermann, Christian R. Eckmann
The cytoplasmic poly(A) polymerases GLD-2 and GLD-4 promote general gene expression via distinct mechanisms.
Nucleic Acids Res, 42(18) 11622-11633 (2014)
PDF DOI

Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.

Maria Winzi, Maciej Paszkowski-Rogacz, Frank Buchholz
Another Brick in the Wall: RNAi Screens Identify New Barriers in iPSC Reprogramming.
Cell Stem Cell, 15(2) 116-118 (2014)
DOI

Somatic cells can be reprogrammed to induced pluripotent stem cells via exogenous expression of a small set of transcription factors, but the regulatory mechanisms controlling this cell transition are poorly understood. Two recent reports demonstrate the value of RNAi screens as a tool to uncover roadblocks in this inefficient process.

Michaela Mickoleit, Benjamin Schmid, Michael Weber, Florian Fahrbach, Sonja Hombach, Sven Reischauer, Jan Huisken
High-resolution reconstruction of the beating zebrafish heart.
Nat Methods, 11(9) 919-922 (2014)
DOI

The heart's continuous motion makes it difficult to capture high-resolution images of this organ in vivo. We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

Yusuke Toyoda, Cihan Erkut, Francisco Pan-Montojo, Sebastian Boland, Martin P Stewart, Daniel J. Müller, Wolfgang Wurst, Anthony Hyman, Teymuras V. Kurzchalia
Products of the Parkinson's disease-related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival.
Biol Open, 3(8) 777-784 (2014)
PDF DOI

Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

Fong Kuan Wong
Generation of basal radial glia in the embryonic mouse dorsal telencephalon
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2014)

Marta Florio, Wieland B. Huttner
Neural progenitors, neurogenesis and the evolution of the neocortex.
Development, 141(11) 2182-2194 (2014)
DOI

The neocortex is the seat of higher cognitive functions and, in evolutionary terms, is the youngest part of the mammalian brain. Since its origin, the neocortex has expanded in several mammalian lineages, and this is particularly notable in humans. This expansion reflects an increase in the number of neocortical neurons, which is determined during development and primarily reflects the number of neurogenic divisions of distinct classes of neural progenitor cells. Consequently, the evolutionary expansion of the neocortex and the concomitant increase in the numbers of neurons produced during development entail interspecies differences in neural progenitor biology. Here, we review the diversity of neocortical neural progenitors, their interspecies variations and their roles in determining the evolutionary increase in neuron numbers and neocortex size.

Michael Kuhn, Anthony Hyman, Andreas Beyer
Coiled-coil proteins facilitated the functional expansion of the centrosome.
PLoS Comput Biol, 10(6) Art. No. e1003657 (2014)
DOI

Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal-fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions.

Agnes Tóth-Petróczy, Dan S Tawfik
The robustness and innovability of protein folds.
Curr Opin Struct Biol, 26 131-138 (2014)
DOI

Assignment of protein folds to functions indicates that >60% of folds carry out one or two enzymatic functions, while few folds, for example, the TIM-barrel and Rossmann folds, exhibit hundreds. Are there structural features that make a fold amenable to functional innovation (innovability)? Do these features relate to robustness--the ability to readily accumulate sequence changes? We discuss several hypotheses regarding the relationship between the architecture of a protein and its evolutionary potential. We describe how, in a seemingly paradoxical manner, opposite properties, such as high stability and rigidity versus conformational plasticity and structural order versus disorder, promote robustness and/or innovability. We hypothesize that polarity--differentiation and low connectivity between a protein's scaffold and its active-site--is a key prerequisite for innovability.

Anna Shevchenko, Yimin Yang, Andrea Knaust, Henrik Thomas, Hongen Jiang, Enguo Lu, Changsui Wang, Andrej Shevchenko
Proteomics identifies the composition and manufacturing recipe of the 2500-year old sourdough bread from Subeixi cemetery in China.
J Proteomics, 105 363-371 (2014)
DOI

We report on the geLC-MS/MS proteomics analysis of cereals and cereal food excavated in Subeixi cemetery (500-300BC) in Xinjiang, China. Proteomics provided direct evidence that at the Subexi sourdough bread was made from barley and broomcorn millet by leavening with a renewable starter comprising baker's yeast and lactic acid bacteria. The baking recipe and flour composition indicated that barley and millet bread belonged to the staple food already in the first millennium BC and suggested the role of Turpan basin as a major route for cultural communication between Western and Eastern Eurasia in antiquity. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Stephanie Schonegg, Anthony A. Hyman, William B. Wood
Timing and Mechanism of the Initial Cue Establishing Handed Left-Right Asymmetry in Caenorhabditis elegans Embryos
Genesis, 52(6) 572-580 (2014)
DOI

Ellie S Heckscher, Fuhui Long, Michael J Layden, Chein-Hui Chuang, Laurina Manning, Jourdain Richart, Joseph C Pearson, Stephen T Crews, Hanchuan Peng, Gene Myers, Chris Q Doe
Atlas-builder software and the eNeuro atlas: resources for developmental biology and neuroscience.
Development, 141(12) 2524-2532 (2014)
PDF DOI

A major limitation in understanding embryonic development is the lack of cell type-specific markers. Existing gene expression and marker atlases provide valuable tools, but they typically have one or more limitations: a lack of single-cell resolution; an inability to register multiple expression patterns to determine their precise relationship; an inability to be upgraded by users; an inability to compare novel patterns with the database patterns; and a lack of three-dimensional images. Here, we develop new 'atlas-builder' software that overcomes each of these limitations. A newly generated atlas is three-dimensional, allows the precise registration of an infinite number of cell type-specific markers, is searchable and is open-ended. Our software can be used to create an atlas of any tissue in any organism that contains stereotyped cell positions. We used the software to generate an 'eNeuro' atlas of the Drosophila embryonic CNS containing eight transcription factors that mark the major CNS cell types (motor neurons, glia, neurosecretory cells and interneurons). We found neuronal, but not glial, nuclei occupied stereotyped locations. We added 75 new Gal4 markers to the atlas to identify over 50% of all interneurons in the ventral CNS, and these lines allowed functional access to those interneurons for the first time. We expect the atlas-builder software to benefit a large proportion of the developmental biology community, and the eNeuro atlas to serve as a publicly accessible hub for integrating neuronal attributes - cell lineage, gene expression patterns, axon/dendrite projections, neurotransmitters--and linking them to individual neurons.

T-Y Dora Tang, Annela M Seddon, Christoph Jeworrek, Roland Winter, Oscar Ces, John M Seddon, Richard H Templer
The effects of pressure and temperature on the energetics and pivotal surface in a monoacylglycerol/water gyroid inverse bicontinuous cubic phase.
Soft Matter, 10(17) 3009-3015 (2014)
DOI

We have studied the effect of pressure and temperature on the location of the pivotal surface in a lipid inverse bicontinuous gyroid cubic phase (Q(G)(II)), described by the area at the pivotal surface (An), the volume between the pivotal surface and the bilayer midplane (Vn), and the molecular volume of the lipid (V). Small angle X-ray scattering (SAXS) was used to measure the swelling behaviour of the lipid, monolinolein, as a function of pressure and temperature, and the data were fitted to two different geometric models: the parallel interface model (PIM), and the constant mean curvature model (CMCM). The results show that an increase in temperature leads to a shift in the location of the pivotal surface towards the bilayer midplane, whilst an increase in pressure causes the pivotal surface to move towards the interfacial region. In addition, we describe the relevance of An, Vn and V for modeling the energetics of curved mesophases with specific reference to the mean curvature at the pivotal surface and discuss the significance of this parameter for modelling the energetics of curved mesophases.

Marc Bickle
The Technology Development Studio of the MPI-CBG: An Open Access Cell-Based Screening Facility.
Comb Chem High Throughput Screen, 17(4) 322-327 (2014)

In the past decade, academic screening centers have been created in many universities worldwide. Most of these screening centers are organized as core facilities that accept projects from both within their organization and from external users in order to maximize staff and instrument usage. The Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, also created such a screening facility named the Technology Development Studio (TDS). The mission of the facility is to provide cell based screening services focused on high resolution confocal imaging. The acquisition of high resolution images allows the mathematical description of cells with image analysis to a high degree of precision. This precision in turn allows classifying phenotypes accurately and compare different cellular treatments to discover underlying mechanisms.

Maja Petkovic, Aymen Jemaiel, Frédéric Daste, Christian G Specht, Ignacio Izeddin, Daniela Vorkel, Jean-Marc Verbavatz, Xavier Darzacq, Antoine Triller, Karl H Pfenninger, David Tareste, Catherine L Jackson, Thierry Galli
The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion.
Nat Cell Biol, 16(5) 434-444 (2014)
PDF DOI

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.

Liliana Malinovska
Specific adaptations in the proteostasis network of the social amoebae Dictyostelium discoideum lead to an unusual resilience to protein aggregation
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2014)

Isabell Weber, Ana P. Ramos, Paulina J. Strzyz, Louis Leung, Stephen Young, Caren Norden
Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.
Cell Rep, 7(2) 386-397 (2014)
PDF DOI

The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position.

Reinhard Roessler, Sebastien A Smallwood, Jesse V Veenvliet, Petros Pechlivanoglou, Su-Ping Peng, Koushik Chakrabarty, Marian J A Groot-Koerkamp, R Jeroen Pasterkamp, Evelyn Wesseling, Gavin Kelsey, Erik Boddeke, Marten P Smidt, Sjef Copray
Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.
Stem Cell Rep, 2(4) 520-533 (2014)
Open Access DOI

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

Judith Paridaen, Wieland B. Huttner
Neurogenesis during development of the vertebrate central nervous system.
EMBO Rep, 15(4) 351-364 (2014)
DOI

During vertebrate development, a wide variety of cell types and tissues emerge from a single fertilized oocyte. One of these tissues, the central nervous system, contains many types of neurons and glial cells that were born during the period of embryonic and post-natal neuro- and gliogenesis. As to neurogenesis, neural progenitors initially divide symmetrically to expand their pool and switch to asymmetric neurogenic divisions at the onset of neurogenesis. This process involves various mechanisms involving intrinsic as well as extrinsic factors. Here, we discuss the recent advances and insights into regulation of neurogenesis in the developing vertebrate central nervous system. Topics include mechanisms of (a)symmetric cell division, transcriptional and epigenetic regulation, and signaling pathways, using mostly examples from the developing mammalian neocortex.

Florian Jug^*, Tobias Pietzsch^*, Stephan Preibisch, Pavel Tomancak
Bioimage Informatics in the context of Drosophila research.
Methods, 68(1) 60-73 (2014)
PDF DOI

Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of Drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in Drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage Informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the Drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the Drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of Drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development.

Sider Penkov, Akira Ogawa, Ulrike Schmidt, Dhananjay Tate, Vyacheslav Zagoriy, Sebastian Boland, Margit Gruner, Daniela Vorkel, Jean-Marc Verbavatz, Ralf J Sommer, Hans-Joachim Knölker, Teymuras V. Kurzchalia
A wax ester promotes collective host finding in the nematode Pristionchus pacificus.
Nat Chem Biol, 10(4) 281-285 (2014)
DOI

Survival of nematode species depends on how successfully they disperse in the habitat and find a new host. As a new strategy for collective host finding in the nematode Pristionchus pacificus, dauer larvae synthesize an extremely long-chain polyunsaturated wax ester (nematoil) that covers the surface of the animal. The oily coat promotes congregation of up to one thousand individuals into stable 'dauer towers' that can reach a beetle host more easily.

Carsten Wenzel, Björn Riefke, Stephan Gründemann, Alice Krebs, Sven Christian, Florian Prinz, Marc Osterland, Sven Golfier, Sebastian Räse, Nariman Ansari, Milan Esner, Marc Bickle, Francesco Pampaloni, Christian Mattheyer, Ernst H K Stelzer, Karsten Parczyk, Stefan Prechtl, Patrick Steigemann
3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.
Exp Cell Res, 323(1) 131-143 (2014)
DOI

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Nagananda Gurudev, Michaela Yuan, Elisabeth Knust
chaoptin, prominin, eyes shut and crumbs form a genetic network controlling the apical compartment of Drosophila photoreceptor cells.
Biol Open, 3(5) 332-341 (2014)
PDF DOI

The apical surface of epithelial cells is often highly specialised to fulfil cell type-specific functions. Many epithelial cells expand their apical surface by forming microvilli, actin-based, finger-like membrane protrusions. The apical surface of Drosophila photoreceptor cells (PRCs) forms tightly packed microvilli, which are organised into the photosensitive rhabdomeres. As previously shown, the GPI-anchored adhesion protein Chaoptin is required for the stability of the microvilli, whereas the transmembrane protein Crumbs is essential for proper rhabdomere morphogenesis. Here we show that chaoptin synergises with crumbs to ensure optimal rhabdomere width. In addition, reduction of crumbs ameliorates morphogenetic defects observed in PRCs mutant for prominin and eyes shut, known antagonists of chaoptin. These results suggest that these four genes provide a balance of adhesion and anti-adhesion to maintain microvilli development and maintenance. Similar to crumbs mutant PRCs, PRCs devoid of prominin or eyes shut undergo light-dependent retinal degeneration. Given the observation that human orthologues of crumbs, prominin and eyes shut result in progressive retinal degeneration and blindness, the Drosophila eye is ideally suited to unravel the genetic and cellular mechanisms that ensure morphogenesis of PRCs and their maintenance under light-mediated stress.

Ievgeniia Zagoriy
The Role of Polo-like Kinase in Regulating the Prophase-to-Metaphase Transition of Meiosis I
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2014)

Derek Spieler, Maria Kaffe, Franziska Knauf, José Bessa, Juan J Tena, Florian Giesert, Barbara Schormair, Erik Tilch, Hyun-Ok Kate Lee, Marion Horsch, Darina Czamara, Nazanin Karbalai, Christine von Toerne, Melanie Waldenberger, Christian Gieger, Peter Lichtner, Melina Claussnitzer, Ronald Naumann, Bertram Müller-Myhsok, Miguel Torres, Lillian Garrett, Jan Rozman, Martin Klingenspor, Valérie Gailus-Durner, Helmut Fuchs, Martin Hrabe de Angelis, Johannes Beckers, Sabine M Hölter, Thomas Meitinger, Stefanie M Hauck, Helmut Laumen, Wolfgang Wurst, Fernando Casares, Jose Luis Gómez-Skarmeta, Juliane Winkelmann
Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon.
Genome Res, 24(4) 592-603 (2014)
DOI

Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

Julie Hanotel, Nathalie Bessodes, Aurore Thélie, Marie Hedderich, Karine Parain, Benoit Van Driessche, Karina De Oliveira Brandão, Sadia Kricha, Mette C Jorgensen, Anne Grapin-Botton, Palle Serup, Carine Van Lint, Muriel Perron, Tomas Pieler, Kristine A Henningfeld, Eric J Bellefroid
The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.
Dev Biol, 386(2) 340-357 (2014)
DOI

The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

Ashley L Alvers, Sean Ryan, Paul J Scherz, Jan Huisken, Michel Bagnat
Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling.
Development, 141(5) 1110-1119 (2014)
DOI

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.

Varadharajan Sundaramurthy, Rico Barsacchi, Mikhail Chernykh, Martin Stöter, Nadine Tomschke, Marc Bickle, Yannis Kalaidzidis, Marino Zerial
Deducing the mechanism of action of compounds identified in phenotypic screens by integrating their multiparametric profiles with a reference genetic screen.
Nat Protoc, 9(2) 474-490 (2014)
PDF DOI

Cell-based high-content screens are increasingly used to discover bioactive small molecules. However, identifying the mechanism of action of the selected compounds is a major bottleneck. Here we describe a protocol consisting of experimental and computational steps to identify the cellular pathways modulated by chemicals, and their mechanism of action. The multiparametric profiles from a 'query' chemical screen are used as constraints to select genes with similar profiles from a 'reference' genetic screen. In our case, the query screen is the intracellular survival of mycobacteria and the reference is a genome-wide RNAi screen of endocytosis. The two disparate screens are bridged by an 'intermediate' chemical screen of endocytosis, so that the similarity in the multiparametric profiles between the chemical and the genetic perturbations can generate a testable hypothesis of the cellular pathways modulated by the chemicals. This approach is not assay specific, but it can be broadly applied to various quantitative, multiparametric data sets. Generation of the query system takes 3-6 weeks, and data analysis and integration with the reference data set takes an 3 additional weeks.

Patricia Heyn, Martin Kircher, Andreas Dahl, Janet Kelso, Pavel Tomancak^#, Alex T. Kalinka, Karla M. Neugebauer^#
The Earliest Transcribed Zygotic Genes Are Short, Newly Evolved, and Different across Species.
Cell Rep, 6(2) 285-292 (2014)
DOI

The transition from maternal to zygotic control is fundamental to the life cycle of all multicellular organisms. It is widely believed that genomes are transcriptionally inactive from fertilization until zygotic genome activation (ZGA). Thus, the earliest genes expressed probably support the rapid cell divisions that precede morphogenesis and, if so, might be evolutionarily conserved. Here, we identify the earliest zygotic transcripts in the zebrafish, Danio rerio, through metabolic labeling and purification of RNA from staged embryos. Surprisingly, the mitochondrial genome was highly active from the one-cell stage onwards, showing that significant transcriptional activity exists at fertilization. We show that 592 nuclear genes become active when cell cycles are still only 15 min long, confining expression to relatively short genes. Furthermore, these zygotic genes are evolutionarily younger than those expressed at other developmental stages. Comparison of fish, fly, and mouse data revealed different sets of genes expressed at ZGA. This species specificity uncovers an evolutionary plasticity in early embryogenesis that probably confers substantial adaptive potential.

Moritz Kreysing, Dino Ott, Michael J Schmidberger, Oliver Otto, Markus Schürmann, Estela Martín-Badosa, Graeme Whyte, Jochen Guck
Dynamic operation of optical fibres beyond the single-mode regime facilitates the orientation of biological cells.
Nat Commun, 5 Art. No. 5481 (2014)
PDF DOI

The classical purpose of optical fibres is delivery of either optical power, as for welding, or temporal information, as for telecommunication. Maximum performance in both cases is provided by the use of single-mode optical fibres. However, transmitting spatial information, which necessitates higher-order modes, is difficult because their dispersion relation leads to dephasing and a deterioration of the intensity distribution with propagation distance. Here we consciously exploit the fundamental cause of the beam deterioration-the dispersion relation of the underlying vectorial electromagnetic modes-by their selective excitation using adaptive optics. This allows us to produce output beams of high modal purity, which are well defined in three dimensions. The output beam distribution is even robust against significant bending of the fibre. The utility of this approach is exemplified by the controlled rotational manipulation of live cells in a dual-beam fibre-optical trap integrated into a modular lab-on-chip system.

Fabian Multrus
Calculation of the Electric Potential for a Neuronal Activity Model in the Brain
Diploma Thesis,Technische Universität Dresden, Dresden, Germany (2014)
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Sinem K Saka, Alf Honigmann, Christian Eggeling, Stefan W Hell, Thorsten Lang, Silvio O Rizzoli
Multi-protein assemblies underlie the mesoscale organization of the plasma membrane.
Nat Commun, 5 4509-4509 (2014)
DOI

Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity.

Chiara Gabella, Elena Bertseva, Céline Bottier, Niccolo Piacentini, Alicia Bornert, Sylvia Jeney, Laszlo Forro, Ivo F. Sbalzarini, J-J Meister, A B Verkhovsky
Contact Angle at the Leading Edge Controls Cell Protrusion Rate
Curr Biol, 24(10) 1126-1132 (2014)
PDF DOI

Matthew A Benton, Anastasios Pavlopoulos
Tribolium embryo morphogenesis: May the force be with you.
Bioarchitecture, 4(1) 16-21 (2014)
PDF

Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis.

Kimberley Gibson, Daniela Vorkel, Jana Meissner, Jean-Marc Verbavatz
Fluorescing the electron: strategies in correlative experimental design.
Methods Cell Biol, 124 23-54 (2014)
PDF DOI

Correlative light and electron microscopy (CLEM) encompasses a growing number of imaging techniques aiming to combine the benefits of light microscopy, which allows routine labeling of molecules and live-cell imaging of fluorescently tagged proteins with the resolution and ultrastructural detail provided by electron microscopy (EM). Here we review three different strategies that are commonly used in CLEM and we illustrate each approach with one detailed example of their application. The focus is on different options for sample preparation with their respective benefits as well as on the imaging workflows that can be used. The three strategies cover: (1) the combination of live-cell imaging with the high resolution of EM (time-resolved CLEM), (2) the need to identify a fluorescent cell of interest for further exploration by EM (cell sorting), and (3) the subcellular correlation of a fluorescent feature in a cell with its associated ultrastructural features (spatial CLEM). Finally, we discuss future directions for CLEM exploring the possibilities for combining super-resolution microscopy with EM.

Stefanie Redemann, Britta Weber, Marit Möller, Jean-Marc Verbavatz, Anthony Hyman, Daniel Baum, Steffen Prohaska, Thomas Müller-Reichert
The segmentation of microtubules in electron tomograms using Amira.
Methods Mol Biol, 1136 261-278 (2014)
PDF DOI

The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129-138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model systems.

Christian Bläsche
Setting Young’s modulus in the subcellular element model
Diploma Thesis,Technische Universität Dresden, Dresden, Germany (2014)
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Claudia Lengerke, Oscar Fernandez-Capetillo, Iva M. Tolic-Norrelykke
When the Going Gets Tough: Scientists’ Personal Challenges
Cell, 159(2) 225-226 (2014)
PDF DOI

Vibor Laketa, Sirus Zarbakhsh, Alexis Traynor-Kaplan, Aidan Macnamara, Devaraj Subramanian, Mateusz Putyrski, Rainer Mueller, André Nadler, Matthias Mentel, Julio Saez-Rodriguez, Rainer Pepperkok, Carsten Schultz
PIP3 induces the recycling of receptor tyrosine kinases.
Sci Signal, 7(308) Art. No. ra5 (2014)
DOI

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Alexandra Kumichel, Elisabeth Knust
Apical localisation of crumbs in the boundary cells of the Drosophila hindgut is independent of its canonical interaction partner stardust.
PLoS ONE, 9(4) Art. No. e94038 (2014)
PDF DOI

The transmembrane protein Crumbs/Crb is a key regulator of apico-basal epithelial cell polarity, both in Drosophila and in vertebrates. In most cases studied so far, the apical localisation of Drosophila Crumbs depends on the interaction of its C-terminal amino acids with the scaffolding protein Stardust. Consequently, embryos lacking either Crumbs or Stardust develop a very similar phenotype, characterised by the loss of epithelial tissue integrity and cell polarity in many epithelia. An exception is the hindgut, which is not affected by the loss of either gene. The hindgut is a single layered epithelial tube composed of two cell populations, the boundary cells and the principal cells. Here we show that Crumbs localisation in the principal cells depends on Stardust, similarly to other embryonic epithelia. In contrast, localisation of Crumbs in the boundary cells does not require Stardust and is independent of its PDZ domain- and FERM-domain binding motifs. In line with this, the considerable upregulation of Crumbs in boundary cells is not followed by a corresponding upregulation of its canonical binding partners. Our data are the first to suggest a mechanism controlling apical Crumbs localisation, which is independent of its conserved FERM- and PDZ-domain binding motifs.

Daiki Umetsu, Sebastian Dunst, Christian Dahmann
An RNA interference screen for genes required to shape the anteroposterior compartment boundary in Drosophila identifies the eph receptor.
PLoS ONE, 9(12) Art. No. e114340 (2014)
Open Access PDF DOI

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.

Katharina Seitz, Verena Dürsch, Jakub Harnoš, Vitezslav Bryja, Marc Gentzel, Alexandra Schambony
β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+) pathway in xenopus gastrulation.
PLoS ONE, 9(1) Art. No. e87132 (2014)
DOI

β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+) cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+) pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+) signaling cascade upstream of Protein Kinase C (PKC) and Ca(2+)/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+) signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

Yuri K. Shestopaloff, Ivo F. Sbalzarini
A method for modeling growth of organs and transplants based on the general growth law: application to the liver in dogs and humans.
PLoS ONE, 9(6) Art. No. e99275 (2014)
Open Access DOI

Understanding biological phenomena requires a systemic approach that incorporates different mechanisms acting on different spatial and temporal scales, since in organisms the workings of all components, such as organelles, cells, and organs interrelate. This inherent interdependency between diverse biological mechanisms, both on the same and on different scales, provides the functioning of an organism capable of maintaining homeostasis and physiological stability through numerous feedback loops. Thus, developing models of organisms and their constituents should be done within the overall systemic context of the studied phenomena. We introduce such a method for modeling growth and regeneration of livers at the organ scale, considering it a part of the overall multi-scale biochemical and biophysical processes of an organism. Our method is based on the earlier discovered general growth law, postulating that any biological growth process comprises a uniquely defined distribution of nutritional resources between maintenance needs and biomass production. Based on this law, we introduce a liver growth model that allows to accurately predicting the growth of liver transplants in dogs and liver grafts in humans. Using this model, we find quantitative growth characteristics, such as the time point when the transition period after surgery is over and the liver resumes normal growth, rates at which hepatocytes are involved in proliferation, etc. We then use the model to determine and quantify otherwise unobservable metabolic properties of livers.

Michaela Müller-McNicoll, Karla M. Neugebauer
Good cap/bad cap: how the cap-binding complex determines RNA fate
Nat Struct Mol Biol, 21(1) 9-12 (2014)
DOI

Sundar Naganathan, Sebastian Fürthauer, Masatoshi Nishikawa, Frank Jülicher, Stephan W. Grill
Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking.
Elife, 3 Art. No. e04165 (2014)
Open Access PDF DOI

Many developmental processes break left-right (LR) symmetry with a consistent handedness. LR asymmetry emerges early in development, and in many species the primary determinant of this asymmetry has been linked to the cytoskeleton. However, the nature of the underlying chirally asymmetric cytoskeletal processes has remained elusive. In this study, we combine thin-film active chiral fluid theory with experimental analysis of the C. elegans embryo to show that the actomyosin cortex generates active chiral torques to facilitate chiral symmetry breaking. Active torques drive chiral counter-rotating cortical flow in the zygote, depend on myosin activity, and can be altered through mild changes in Rho signaling. Notably, they also execute the chiral skew event at the 4-cell stage to establish the C. elegans LR body axis. Taken together, our results uncover a novel, large-scale physical activity of the actomyosin cytoskeleton that provides a fundamental mechanism for chiral morphogenesis in development.

Alexander Krull, André Steinborn, Vaishnavi Ananthanarayanan, Damien Ramunno-Johnson, Uwe Petersohn, Iva M. Tolic-Norrelykke
A divide and conquer strategy for the maximum likelihood localization of low intensity objects.
Opt express, 22(1) 210-228 (2014)
PDF DOI

In cell biology and other fields the automatic accurate localization of sub-resolution objects in images is an important tool. The signal is often corrupted by multiple forms of noise, including excess noise resulting from the amplification by an electron multiplying charge-coupled device (EMCCD). Here we present our novel Nested Maximum Likelihood Algorithm (NMLA), which solves the problem of localizing multiple overlapping emitters in a setting affected by excess noise, by repeatedly solving the task of independent localization for single emitters in an excess noise-free system. NMLA dramatically improves scalability and robustness, when compared to a general purpose optimization technique. Our method was successfully applied for in vivo localization of fluorescent proteins.

Marko Brankatschk, Sebastian Dunst, Linda Nemetschke, Suzanne Eaton
Delivery of circulating lipoproteins to specific neurons in the Drosophila brain regulates systemic insulin signaling.
Elife, 3 Art. No. e02862 (2014)
Open Access DOI

The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. Here, we demonstrate a function for the Drosophila lipoprotein LTP in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in Blood Brain Barrier glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content.

Alf Honigmann, Veronika Mueller, Haisen Ta, Andreas Schoenle, Erdinc Sezgin, Stefan W Hell, Christian Eggeling
Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.
Nat Commun, 5 5412-5412 (2014)
DOI

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.

P. Rué, Y.H. Kim, H. List Larsen, A. Grapin-Botton, A. Martinez Arias
A framework for the analysis of symmetric and asymmetric divisions in developmental processes.
bioRxiv, Art. No. https://doi.org/10.1101/010835 (2014)
Open Access DOI

Marie-Theres Schmid, Franziska Weinandy, Michaela Wilsch-Bräuninger, Wieland B. Huttner, Silvia Cappello, Magdalena Götz
The role of α-E-catenin in cerebral cortex development: radial glia specific effect on neuronal migration.
Front Cell Neurosci, 8 Art. No. 215 (2014)
Open Access DOI

During brain development, radial glial cells possess an apico-basal polarity and are coupled by adherens junctions (AJs) to an F-actin belt. To elucidate the role of the actin, we conditionally deleted the key component α-E-catenin in the developing cerebral cortex. Deletion at early stages resulted in severe disruption of tissue polarity due to uncoupling of AJs with the intracellular actin fibers leading to the formation of subcortical band heterotopia. Interestingly, this phenotype closely resembled the phenotype obtained by conditional RhoA deletion, both in regard to the macroscopic subcortical band heterotopia and the subcellular increase in G-actin/F-actin ratio. These data therefore together corroborate the role of the actin cytoskeleton and its anchoring to the AJs for neuronal migration disorders.

Alexis Webb, Daniele Soroldoni, Annelie Oswald, Johannes Schindelin, Andrew C. Oates
Generation of dispersed presomitic mesoderm cell cultures for imaging of the zebrafish segmentation clock in single cells.
J Vis Exp, (89) Art. No. e50307 (2014)
DOI

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Marija Podolski
Characterization of the budding yeast microtubule polymerase Stu2
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2013)

Khanh Huy Bui^*, Alexander von Appen^*, Amanda L DiGuilio, Alessandro Ori, Lenore Sparks, Marie-Therese Mackmull, Thomas Bock, Wim Hagen, Amparo Andrés-Pons, Joseph S Glavy^#, Martin Beck^#
Integrated structural analysis of the human nuclear pore complex scaffold.
Cell, 155(6) 1233-1243 (2013)
DOI

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

Cihan Erkut, Andrej Vasilj, Sebastian Boland, Bianca Habermann, Andrej Shevchenko, Teymuras V. Kurzchalia
Molecular Strategies of the Caenorhabditis elegans Dauer Larva to Survive Extreme Desiccation.
PLoS ONE, 8(12) Art. No. e82473 (2013)
DOI

Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for anhydrobiosis in plants and animals.

Peter Refsing Andersen, Michal Domanski, Maiken S Kristiansen, Helena Storvall, Evgenia Ntini, Celine Verheggen, Aleks Schein, Jakob Bunkenborg, Ina Poser, Marie Hallais, Rickard Sandberg, Anthony Hyman, John Lacava, Michael P Rout, Jens S Andersen, Edouard Bertrand, Torben Heick Jensen
The human cap-binding complex is functionally connected to the nuclear RNA exosome.
Nat Struct Mol Biol, 20(12) 1367-1376 (2013)
DOI

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.

Norio Takeshita, Daniel Mania, Saturnino Herrero, Yuji Ishitsuka, G Ulrich Nienhaus, Marija Podolski, Jonathon Howard, Reinhard Fischer
The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.
J Cell Sci, 126(Pt 23) 5400-5411 (2013)
DOI

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

Marta Maria Swierczynska, Valéria Lamounier-Zepter, Stefan R. Bornstein, Suzanne Eaton
Lipoproteins and Hedgehog signalling - possible implications for the adrenal gland function.
Eur J Clin Invest, 43(11) 1178-1183 (2013)
DOI

Metabolic syndrome is a common metabolic disorder that is associated with an increased risk of type 2 diabetes and cardiovascular diseases. Disturbances in adrenal steroid hormone production significantly contribute to the development of this disorder. Therefore, it is extremely important to fully understand the mechanisms governing adrenal gland function, both in physiological and pathological conditions.

Jarmila Hnilicová, Samira Hozeifi, Eva Stejskalová, Eva Dušková, Ina Poser, Jana Humpolíčková, Martin Hof, David Staněk
The C-terminal domain of Brd2 is important for chromatin interaction and regulation of transcription and alternative splicing.
Mol Biol Cell, 24(22) 3557-3568 (2013)
DOI

Brd2 is a member of the bromodomain extra terminal (BET) protein family, which consists of four chromatin-interacting proteins that regulate gene expression. Each BET protein contains two N-terminal bromodomains, which recognize acetylated histones, and the C-terminal protein-protein interaction domain. Using a genome-wide screen, we identify 1450 genes whose transcription is regulated by Brd2. In addition, almost 290 genes change their alternative splicing pattern upon Brd2 depletion. Brd2 is specifically localized at promoters of target genes, and our data show that Brd2 interaction with chromatin cannot be explained solely by histone acetylation. Using coimmunoprecipitation and live-cell imaging, we show that the C-terminal part is crucial for Brd2 association with chromatin. Live-cell microscopy also allows us to map the average binding time of Brd2 to chromatin and quantify the contributions of individual Brd2 domains to the interaction with chromatin. Finally, we show that bromodomains and the C-terminal domain are equally important for transcription and splicing regulation, which correlates with the role of these domains in Brd2 binding to chromatin.

Meritxell Huch^*, Paola Bonfanti^*, Sylvia F Boj^*, Toshiro Sato^*, Cindy J M Loomans, Marc van de Wetering, Mozhdeh Sojoodi, Vivian S W Li, Jurian Schuijers, Ana Gracanin, Femke Ringnalda, Harry Begthel, Karien Hamer, Joyce Mulder, Johan H van Es, Eelco de Koning, Robert R G Vries, Harry Heimberg^*^#, Hans Clevers^*^#
Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.
EMBO J, 32(20) 2708-2721 (2013)
Open Access DOI

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Wieland B. Huttner, Iva Kelava, Eric Lewitus
Progenitor networking in the fetal primate neocortex.
Neuron, 80(2) 259-262 (2013)
DOI

Basal radial glia (bRG) is a recently identified major type of neural stem cell in fetal primate, notably human, neocortex. In this issue of Neuron, Betizeau et al. (2013) now demonstrate that four morphologically distinct bRG subtypes exist in the outer subventricular zone of fetal macaque neocortex, and reveal an unexpected complexity of lineages generating neurons.

Martin Depken, Juan Parrondo, Stephan W. Grill
Intermittent transcription dynamics for the rapid production of long transcripts of high fidelity.
Cell Rep, 5(2) 521-530 (2013)
Open Access DOI

Normal cellular function relies on the efficient and accurate readout of the genetic code. Single-molecule experiments show that transcription and replication are highly intermittent processes that are frequently interrupted by polymerases pausing and reversing directions. Although intermittent dynamics in replication are known to result from proofreading, their origin and significance during transcription remain controversial. Here, we theoretically investigate transcriptional fidelity and show that the kinetic scheme provided by the RNA-polymerase backtracking and transcript-cleavage pathway can account for measured error rates. Importantly, we find that intermittent dynamics provide an enormous increase in the rate of producing long transcripts of high fidelity. Our results imply that intermittent dynamics during transcription may have evolved as a way to mitigate the competing demands of speed and fidelity in the transcription of extended sequences.

Julia Phieler, Kj Chung, Antonios Chatzigeorgiou, Anne Klotzsche-von Ameln, Ruben Garcia-Martin, David Sprott, Maria Moisidou, Theodora Tzanavari, Barbara Ludwig, Elena Baraban, Monika Ehrhart-Bornstein, Stefan Bornstein, Hassan Mziaut, Michele Solimena, Katia P Karalis, Matina Economopoulou, John D Lambris, Trian Chavakis
The complement anaphylatoxin C5a receptor contributes to obese adipose tissue inflammation and insulin resistance.
J Immunol, 191(8) 4367-4374 (2013)
DOI

Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR(-/-) mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR(-/-) mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in β cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.

Marcus Braun, Zdenek Lansky, Seweryn Bajer, Gero Fink, Andrzej A Kasprzak, Stefan Diez
The human kinesin-14 HSET tracks the tips of growing microtubules in vitro.
Cytoskeleton (Hoboken), 70(9) 515-521 (2013)
PDF DOI

Tip-tracking of kinesin-14 motor proteins is believed to be crucial for the assembly and maintenance of dynamic microtubule arrays. However, in contrast to other members of the kinesin-14 family, H. sapiens kinesin-14 HSET has so far never been observed to be prominently located at microtubule plus ends. Here, using an in vitro microtubule dynamics reconstitution assay we observe tip-tracking of GFP-HSET in the presence of H. sapiens EB1 (hsEB1). Tip-tracking depended on the SxIP-like motif in HSET as well as on the EB homology domain in hsEB1. D. melanogaster Ncd and S. pombe Klp2 tip-tracking reconstitution assays accompanied by kinesin-14 amino acid sequence comparisons suggest that SxIP-like motif mediated tip-tracking dependent on EB family proteins is conserved in the kinesin-14 family of molecular motors.

Marco Nousch, Nora Techritz, Daniel Hampel, Sophia Millonigg, Christian R. Eckmann
The Ccr4-Not deadenylase complex constitutes the main poly(A) removal activity in C. elegans.
J Cell Sci, 126(18) 4274-4285 (2013)
DOI

Post-transcriptional regulatory mechanisms are widely used to control gene expression programs of tissue development and physiology. Controlled 3' poly(A) tail-length changes of mRNAs provide a mechanistic basis of such regulation, affecting mRNA stability and translational competence. Deadenylases are a conserved class of enzymes that facilitate poly(A) tail removal, and their biochemical activities have been mainly studied in the context of single-cell systems. Little is known about the different deadenylases and their biological role in multicellular organisms. In this study, we identify and characterize all known deadenylases of Caenorhabditis elegans, and identify the germ line as tissue that depends strongly on deadenylase activity. Most deadenylases are required for hermaphrodite fertility, albeit to different degrees. Whereas ccr-4 and ccf-1 deadenylases promote germline function under physiological conditions, panl-2 and parn-1 deadenylases are only required under heat-stress conditions. We also show that the Ccr4-Not core complex in nematodes is composed of the two catalytic subunits CCR-4 and CCF-1 and the structural subunit NTL-1, which we find to regulate the stability of CCF-1. Using bulk poly(A) tail measurements with nucleotide resolution, we detect strong deadenylation defects of mRNAs at the global level only in the absence of ccr-4, ccf-1 and ntl-1, but not of panl-2, parn-1 and parn-2. Taken together, this study suggests that the Ccr4-Not complex is the main deadenylase complex in C. elegans germ cells. On the basis of this and as a result of evidence in flies, we propose that the conserved Ccr4-Not complex is an essential component in post-transcriptional regulatory networks promoting animal reproduction.

Simone Reber, Johannes Baumgart, Per Widlund, Andrei I. Pozniakovsky, Jonathon Howard, Anthony Hyman^#, Frank Jülicher^#
XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules
Nat Cell Biol, 15(9) 1116-1122 (2013)
DOI

Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules.

Anastasiya Trushko, Erik Schäffer^#, Jonathon Howard^#
The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force.
Proc Natl Acad Sci U.S.A., 110(36) 14670-14675 (2013)
PDF DOI

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.

Xicotencatly Gracida, Christian R. Eckmann
Mind the gut: Dietary impact on germline stem cells and fertility
Commun Integr Biol, 6(6) Art. No. e26004 (2013)
DOI

Eric Lewitus, Iva Kelava, Wieland B. Huttner
Conical expansion of the outer subventricular zone and the role of neocortical folding in evolution and development
Front Hum Neurosci, 7 Art. No. 424 (2013)
DOI

as it expands, so does it fold. The degree to which it folds, however, cannot strictly be attributed to its expansion. Across species, cortical volume does not keep pace with cortical surface area, but rather folds appear more rapidly than expected. As a result, larger brains quickly become disproportionately more convoluted than smaller brains. Both the absence (lissencephaly) and presence (gyrencephaly) of cortical folds is observed in all mammalian orders and, while there is likely some phylogenetic signature to the evolutionary appearance of gyri and sulci, there are undoubtedly universal trends to the acquisition of folds in an expanding neocortex. Whether these trends are governed by conical expansion of neocortical germinal zones, the distribution of cortical connectivity, or a combination of growth- and connectivity-driven forces remains an open question. But the importance of cortical folding for evolution of the uniquely mammalian neocortex, as well as for the incidence of neuropathologies in humans, is undisputed. In this hypothesis and theory article, we will summarize the development of cortical folds in the neocortex, consider the relative influence of growth- vs. connectivity-driven forces for the acquisition of cortical folds between and within species, assess the genetic, cell-biological, and mechanistic implications for neocortical expansion, and discuss the significance of these implications for human evolution, development, and disease. We will argue that evolutionary increases in the density of neuron production, achieved via maintenance of a basal proliferative niche in the neocortical germinal zones, drive the conical migration of neurons toward the cortical surface and ultimately lead to the establishment of cortical folds in large-brained mammal species.

Marta Pabis, Noa Neufeld, Michaela Steiner, Teodora Bojic, Yaron Shav-Tal, Karla M. Neugebauer
The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells
RNA, 19(8) 1054-1063 (2013)
DOI

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

Chiu Fan Lee, Clifford Brangwynne, Joebin Gharakhani, Anthony Hyman, Frank Jülicher
Spatial organization of the cell cytoplasm by position-dependent phase separation.
Phys Rev Lett, 111(8) Art. No. 088101 (2013)
DOI

During asymmetric cell division, cytoplasmic components are segregated to opposite sides of the cell. We discuss how the observed segregation can be achieved by a position-dependent phase separation mechanism controlled by a protein concentration gradient. We show that effects of even a weak gradient can be amplified by the phase transition to achieve strong segregation. We compare our theory to the segregation of germ granules observed during the divisions in the C. elegans embryo. Our study demonstrates how liquid-liquid phase separation can play a key role in the organization of the cytoplasm.

Suzanne Eaton
Suzanne Eaton: the beautiful logic of development.
J Cell Biol, 202(2) 184-185 (2013)
DOI

Denise Stenzel, Wieland B. Huttner
Role of maternal thyroid hormones in the developing neocortex and during human evolution
Front Neuroanat, 7 Art. No. 19 (2013)
DOI

The importance of thyroid hormones during brain development has been appreciated for many decades. In humans, low levels of circulating maternal thyroid hormones, e.g., caused by maternal hypothyroidism or lack of iodine in diet, results in a wide spectrum of severe neurological defects, including neurological cretinism characterized by profound neurologic impairment and mental retardation, underlining the importance of the maternal thyroid hormone contribution. In fact, iodine intake, which is essential for thyroid hormone production in the thyroid gland, has been related to the expansion of the brain, associated with the increased cognitive capacities during human evolution. Because thyroid hormones regulate transcriptional activity of target genes via their nuclear thyroid hormone receptors (THRs), even mild and transient changes in maternal thyroid hormone levels can directly affect and alter the gene expression profile, and thus disturb fetal brain development. Here we summarize how thyroid hormones may have influenced human brain evolution through the adaptation to new habitats, concomitant with changes in diet and, therefore, iodine intake. Further, we review the current picture we gained from experimental studies in rodents on the function of maternal thyroid hormones during developmental neurogenesis. We aim to evaluate the effects of maternal thyroid hormone deficiency as well as lack of THRs and transporters on brain development and function, shedding light on the cellular behavior conducted by thyroid hormones.

Iva Kelava, Eric Lewitus, Wieland B. Huttner
The secondary loss of gyrencephaly as an example of evolutionary phenotypical reversal
Front Neuroanat, 7 Art. No. 16 (2013)
DOI

Gyrencephaly (the folding of the surface of the neocortex) is a mammalian-specific trait present in almost all mammalian orders. Despite the widespread appearance of the trait, little is known about the mechanism of its genesis or its adaptive significance. Still, most of the hypotheses proposed concentrated on the pattern of connectivity of mature neurons as main components of gyri formation. Recent work on embryonic neurogenesis in several species of mammals revealed different progenitor and stem cells and their neurogenic potential as having important roles in the process of gyrification. Studies in the field of comparative neurogenesis revealed that gyrencephaly is an evolutionarily labile trait, and that some species underwent a secondary loss of a convoluted brain surface and thus reverted to a more ancient form, a less folded brain surface (lissencephaly). This phenotypic reversion provides an excellent system for understanding the phenomenon of secondary loss. In this review, we will outline the theory behind secondary loss and, as specific examples, present species that have undergone this transition with respect to neocortical folding. We will also discuss different possible pathways for obtaining (or losing) gyri. Finally, we will explore the potential adaptive consequence of gyrencephaly relative to lissencephaly and vice versa.

Simon Alberti
Aggregating the message to control the cell cycle.
Dev Cell, 25(6) 551-552 (2013)
DOI

Localization of messenger RNAs (mRNAs) enables the precise regulation of protein expression in space and time. In this issue of Developmental Cell, Lee et al. (2013) report that the RNA-binding protein Whi3 spatially constrains a cyclin-encoding mRNA in the cytoplasm of multinucleate cells, thus allowing independent cell-cycle control of individual nuclei.

André Nadler, Gregor Reither, Suihan Feng, Frank Stein, Sabine Reither, Rainer Müller, Carsten Schultz
The fatty acid composition of diacylglycerols determines local signaling patterns.
Angew Chem Int Ed Engl, 52(24) 6330-6334 (2013)
DOI

Andrea J Yool, Johann Morelle, Yvette Cnops, Jean-Marc Verbavatz, Ewan M Campbell, Elizabeth A H Beckett, Grant W Booker, Gary Flynn, Olivier Devuyst
AqF026 is a pharmacologic agonist of the water channel aquaporin-1.
J Am Soc Nephrol, 24(7) 1045-1052 (2013)
PDF DOI

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

József Jászai, Sylvi Graupner, Elly M. Tanaka, Richard Funk, Wieland B. Huttner, Michael Brand, Denis Corbeil
Spatial distribution of prominin-1 (CD133)-positive cells within germinative zones of the vertebrate brain
PLoS ONE, 8(5) Art. No. e63457 (2013)
DOI

In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133), a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS) as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS.

Sundar Naganathan
Regulation of mesoscale physical properties of the C.elegans actomyosin cortex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2013)

Apriliana E R Kartikasari, Josie X Zhou, Murtaza S Kanji, David N Chan, Arjun Sinha, Anne Grapin-Botton, Mark A Magnuson, William E Lowry, Anil Bhushan
The histone demethylase Jmjd3 sequentially associates with the transcription factors Tbx3 and Eomes to drive endoderm differentiation.
EMBO J, 32(10) 1393-1408 (2013)
DOI

Stem cell differentiation depends on transcriptional activation driven by lineage-specific regulators as well as changes in chromatin organization. However, the coordination of these events is poorly understood. Here, we show that T-box proteins team up with chromatin modifying enzymes to drive the expression of the key lineage regulator, Eomes during endodermal differentiation of embryonic stem (ES) cells. The Eomes locus is maintained in a transcriptionally poised configuration in ES cells. During early differentiation steps, the ES cell factor Tbx3 associates with the histone demethylase Jmjd3 at the enhancer element of the Eomes locus to allow enhancer-promoter interactions. This spatial reorganization of the chromatin primes the cells to respond to Activin signalling, which promotes the binding of Jmjd3 and Eomes to its own bivalent promoter region to further stimulate Eomes expression in a positive feedback loop. In addition, Eomes activates a transcriptional network of core regulators of endodermal differentiation. Our results demonstrate that Jmjd3 sequentially associates with two T-box factors, Tbx3 and Eomes to drive stem cell differentiation towards the definitive endoderm lineage.

Athena Kyrkou, M Soufi, R Bahtz, Charles Ferguson, M Bai, Robert G. Parton, Ingrid Hoffmann, Marino Zerial, Theodore Fotsis, Carol Murphy
The RhoD to centrosomal duplication.
Small GTPases, 4(2) 116-122 (2013)
DOI

The main functional roles attributed to the centrosome, the major microtubule organizing center (MTOC) of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. In this commentary, we discuss our recent study, which shows the involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome's structural and functional integrity to vesicular trafficking.

Miguel Coelho, Nicola Maghelli, Iva M. Tolic-Norrelykke
Single-molecule imaging in vivo: The dancing building blocks of the cell.
Integr Biol (Camb), 5(5) 748-758 (2013)
PDF DOI

A cell can be viewed as a dynamic puzzle, where single pieces shuffle in space, change their conformation to fit different partners, and new pieces are generated while old ones are destroyed. Microscopy has become capable of directly observing the pieces of the puzzle, which are single molecules. Single-molecule microscopy in vivo provides new insights into the molecular processes underlying the physiology of a cell, allowing not only for visualizing how molecules distribute with nanometer resolution in the cellular environment, but also for characterizing their movement with high temporal precision. This approach reveals molecular behaviors normally invisible in ensemble measurements. Depending on the molecule, the process, and the cellular region studied, single molecules can be followed by conventional epifluorescence microscopy, or by illuminating only a thin region of the cell, as in Total Internal Reflection Fluorescence (TIRF) and Selective Plane Illumination Microscopy (SPIM), and by limiting the amount of detectable molecules, as in Fluorescence Speckle Microscopy (FSM) and Photo-Activation (PA). High spatial resolution can be obtained by imaging only a fraction of the molecules at a time, as in Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), or by de-exciting molecules in the periphery of the detection region as in Stimulated Emission-Depletion (STED) microscopy. Single-molecule techniques in vivo are becoming widespread; however, it is important to choose the most suited technique for each biological question or sample. Here we review single-molecule microscopy techniques, describe their basic principles, advantages for in vivo application, and discuss the lessons that can be learned from live single-molecule imaging.

Jörg Schönheit, Christiane Kuhl, Marie Luise Gebhardt, Francisco Fernández Klett, Pia Riemke, Marina Scheller, Gang Huang, Ronald Naumann, Achim Leutz, Carol Stocking, Josef Priller, Miguel A Andrade-Navarro, Frank Rosenbauer
PU.1 level-directed chromatin structure remodeling at the Irf8 gene drives dendritic cell commitment.
Cell Rep, 3(5) 1617-1628 (2013)
DOI

Dendritic cells (DCs) are essential regulators of immune responses; however, transcriptional mechanisms that establish DC lineage commitment are poorly defined. Here, we report that the PU.1 transcription factor induces specific remodeling of the higher-order chromatin structure at the interferon regulatory factor 8 (Irf8) gene to initiate DC fate choice. An Irf8 reporter mouse enabled us to pinpoint an initial progenitor stage at which DCs separate from other myeloid lineages in the bone marrow. In the absence of Irf8, this progenitor undergoes DC-to-neutrophil reprogramming, indicating that DC commitment requires an active, Irf8-dependent escape from alternative myeloid lineage potential. Mechanistically, myeloid Irf8 expression depends on high PU.1 levels, resulting in local chromosomal looping and activation of a lineage- and developmental-stage-specific cis-enhancer. These data delineate PU.1 as a concentration-dependent rheostat of myeloid lineage selection by controlling long-distance contacts between regulatory elements and suggest that specific higher-order chromatin remodeling at the Irf8 gene determines DC differentiation.

Xin Liang, Johnson Madrid, Roland Gärtner, Jean-Marc Verbavatz, Christoph Schiklenk, Michaela Wilsch-Bräuninger, Aliona Bogdanova, Florian Stenger, Axel Voigt, Jonathon Howard
A NOMPC-dependent membrane-microtubule connector is a candidate for the gating spring in fly mechanoreceptors.
Curr Biol, 23(9) 755-763 (2013)
PDF DOI

Mechanoreceptors contain compliant elements, termed "gating springs," that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments; they have not been identified molecularly or ultrastructurally.

Andrea Knaust, Jutta Ludwig-Müller
The Ethylene Signaling Pathway is Needed to Restrict Root Gall Growth in Arabidopsis after Infection with the Obligate Biotrophic Protist Plasmodiophora brassicae
J Plant Growth Regul, 32(1) 9-21 (2013)

A Kyrkou, M Soufi, R Bahtz, Charles Ferguson, M Bai, Robert G. Parton, I Hoffmann, Marino Zerial, Theodore Fotsis, C Murphy
RhoD participates in the regulation of cell-cycle progression and centrosome duplication.
Oncogene, 32(14) 1831-1842 (2013)
PDF DOI

We have previously identified a Rho protein, RhoD, which localizes to the plasma membrane and the early endocytic compartment. Here, we show that a GTPase-deficient mutant of RhoD, RhoDG26V, causes hyperplasia and perturbed differentiation of the epidermis, when targeted to the skin of transgenic mice. In vitro, gain-of-function and loss-of-function approaches revealed that RhoD is involved in the regulation of G1/S-phase progression and causes overduplication of centrosomes. Centriole overduplication assays in aphidicolin-arrested p53-deficient U2OS cells, in which the cell and the centrosome cycles are uncoupled, revealed that the effects of RhoD and its mutants on centrosome duplication and cell cycle are independent. Enhancement of G1/S-phase progression was mediated via Diaph1, a novel effector of RhoD, which we have identified using a two-hybrid screen. These results indicate that RhoD participates in the regulation of cell-cycle progression and centrosome duplication.Oncogene advance online publication, 4 June 2012; doi:10.1038/onc.2012.195.

Lorenz Pammer, Daniel T O'Connor, S Andrew Hires, Nathan G Clack, Daniel Huber, Eugene W Myers, Karel Svoboda
The mechanical variables underlying object localization along the axis of the whisker.
J Neurosci, 33(16) 6726-6741 (2013)
PDF DOI

Rodents move their whiskers to locate objects in space. Here we used psychophysical methods to show that head-fixed mice can localize objects along the axis of a single whisker, the radial dimension, with one-millimeter precision. High-speed videography allowed us to estimate the forces and bending moments at the base of the whisker, which underlie radial distance measurement. Mice judged radial object location based on multiple touches. Both the number of touches (1-17) and the forces exerted by the pole on the whisker (up to 573 μN; typical peak amplitude, 100 μN) varied greatly across trials. We manipulated the bending moment and lateral force pressing the whisker against the sides of the follicle and the axial force pushing the whisker into the follicle by varying the compliance of the object during behavior. The behavioral responses suggest that mice use multiple variables (bending moment, axial force, lateral force) to extract radial object localization. Characterization of whisker mechanics revealed that whisker bending stiffness decreases gradually with distance from the face over five orders of magnitude. As a result, the relative amplitudes of different stress variables change dramatically with radial object distance. Our data suggest that mice use distance-dependent whisker mechanics to estimate radial object location using an algorithm that does not rely on precise control of whisking, is robust to variability in whisker forces, and is independent of object compliance and object movement. More generally, our data imply that mice can measure the amplitudes of forces in the sensory follicles for tactile sensation.

Karl F Lechtreck, Jason M Brown, Julio Sampaio, Julie M Craft, Andrej Shevchenko, James E Evans, George B Witman
Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase.
J Cell Biol, 201(2) 249-261 (2013)
DOI

The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched ∼150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency.

Sandra Tenreiro^*, Matthias Munder^*, Simon Alberti^#, Tiago Fleming Outeiro^#
Harnessing the power of yeast to unravel the molecular basis of neurodegeneration.
J Neurochem, 127(4) 438-452 (2013)
DOI

Several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration have been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well-characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders. This article is protected by copyright. All rights reserved.

Hung-Hsiang Yu, Takeshi Awasaki, Michaela Schroeder, Fuhui Long, Jacob S Yang, Yisheng He, Peng Ding, Jui-Chun Kao, Gloria Yueh-Yi Wu, Hanchuan Peng, Gene Myers, Tzumin Lee
Clonal development and organization of the adult Drosophila central brain.
Curr Biol, 23(8) 633-643 (2013)
PDF DOI

The insect brain can be divided into neuropils that are formed by neurites of both local and remote origin. The complexity of the interconnections obscures how these neuropils are established and interconnected through development. The Drosophila central brain develops from a fixed number of neuroblasts (NBs) that deposit neurons in regional clusters.

Zoltan Ungvari, Anna Csiszar, Danuta Sosnowska, Eva E Philipp, Courtney M Campbell, Philip McQuary, Tracy T Chow, Miguel Coelho, Elizabeth S Didier, Sara Gelino, Marissa A Holmbeck, Insil Kim, Erik Levy, William E Sonntag, Paul W Whitby, Steven N Austad, Iain Ridgway
Testing predictions of the oxidative stress hypothesis of aging using a novel invertebrate model of longevity: the giant clam (Tridacna derasa).
J Gerontol A Biol Sci Med Sci, 68(4) 359-367 (2013)
DOI

Bivalve species with exceptional longevity are newly introduced model systems in biogerontology to test evolutionarily conserved mechanisms of aging. Here, we tested predictions based on the oxidative stress hypothesis of aging using one of the tropical long-lived sessile giant clam species, the smooth giant clam (Tridacna derasa; predicted maximum life span: >100 years) and the short-lived Atlantic bay scallop (Argopecten irradians irradians; maximum life span: 2 years). The warm water-dwelling giant clams warrant attention because they challenge the commonly held view that the exceptional longevity of bivalves is a consequence of the cold water they reside in. No significant interspecific differences in production of H2O2 and O2- in the gills, heart, or adductor muscle were observed. Protein carbonyl content in gill and muscle tissues were similar in T derasa and A i irradians. In tissues of T derasa, neither basal antioxidant capacities nor superoxide dismutase and catalase activities were consistently greater than in A i irradians. We observed a positive association between longevity and resistance to mortality induced by exposure to tert-butyl hydroperoxide (TBHP). This finding is consistent with the prediction based on the oxidative stress hypothesis of aging. The findings that in tissues of T derasa, proteasome activities are significantly increased as compared with those in tissues of A i irradians warrant further studies to test the role of enhanced protein recycling activities in longevity of bivalves.

Michaela Müller-McNicoll, Karla M. Neugebauer
How cells get the message: dynamic assembly and function of mRNA-protein complexes.
Nat Rev Genet, 14(4) 275-287 (2013)
PDF DOI

mRNA is packaged into ribonucleoprotein particles called mRNPs. A multitude of RNA-binding proteins as well as a host of associated proteins participate in the fate of mRNA from transcription and processing in the nucleus to translation and decay in the cytoplasm. Methodological innovations in cell biology and genome-wide high-throughput approaches have revealed an unexpected diversity of mRNA-associated proteins and unforeseen interconnections between mRNA-processing steps. Recent insights into mRNP formation in vivo have also highlighted the importance of mRNP packaging, which can sort RNAs on the basis of their length and determine mRNA fate through alternative mRNP assembly, processing and export pathways.

Tomislav Maricic, Viola Günther, Oleg Georgiev, Sabine Gehre, Marija Curlin, Christiane Schreiweis, Ronald Naumann, Hernán A Burbano, Matthias Meyer, Carles Lalueza-Fox, Marco de la Rasilla, Antonio Rosas, Srecko Gajovic, Janet Kelso, Wolfgang Enard, Walter Schaffner, Svante Pääbo
A Recent Evolutionary Change Affects a Regulatory Element in the Human FOXP2 Gene.
Mol Biol Evol, 30(4) 844-852 (2013)
DOI

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Manli Shen, Stanislav Bellaousov, Michael Hiller, Pierre de la Grange, Trevor P Creamer, Orit Malina, Ruth Sperling, David H Mathews, Peter Stoilov, Stefan Stamm
Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure.
Nucleic Acids Res, 41(6) 3819-3832 (2013)
PDF DOI

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2'-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5'-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A-U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch.

Enrico Perini
In vitro reconstitution of the molecular mechanisms of vesicle tethering and membrane fusion
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2013)

Andrew Clark
Thickness, Dynamics and Mechanics of the Actomyosin Cortex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2013)

Chun-Ting Chen, Andreas Ettinger, Wieland B. Huttner, Stephen J Doxsey
Resurrecting remnants: the lives of post-mitotic midbodies
Trends Cell Biol, 23(3) 118-128 (2013)
DOI

Around a century ago, the midbody (MB) was described as a structural assembly within the intercellular bridge during cytokinesis that served to connect the two future daughter cells. The MB has become the focus of intense investigation through the identification of a growing number of diverse cellular and molecular pathways that localize to the MB and contribute to its cytokinetic functions, ranging from selective vesicle trafficking and regulated microtubule (MT), actin, and endosomal sorting complex required for transport (ESCRT) filament assembly and disassembly to post-translational modification, such as ubiquitination. More recent studies have revealed new and unexpected functions of MBs in post-mitotic cells. In this review, we provide a historical perspective, discuss exciting new roles for MBs beyond their cytokinetic function, and speculate on their potential contributions to pluripotency.

Alexi Nott, Justyna Nitarska, Jesse V Veenvliet, Stephan Schacke, Alwin A H A Derijck, Piotr Sirko, Christian Muchardt, R Jeroen Pasterkamp, Marten P Smidt, Antonella Riccio
S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration.
Proc Natl Acad Sci U.S.A., 110(8) 3113-3118 (2013)
DOI

Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.

Paola Picotti, Mathieu Clément-Ziza, Hugo Y K Lam, David S Campbell, Alexander Schmidt, Eric W Deutsch, Hannes Röst, Zhongwei Sun, Oliver Rinner, Lukas Reiter, Qin Shen, Jacob J Michaelson, Andreas Frei, Simon Alberti, Ulrike Kusebauch, Bernd Wollscheid, Robert L Moritz, Andreas Beyer, Ruedi Aebersold
A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis.
Nature, 494(7436) 266-270 (2013)
DOI

Experience from different fields of life sciences suggests that accessible, complete reference maps of the components of the system under study are highly beneficial research tools. Examples of such maps include libraries of the spectroscopic properties of molecules, or databases of drug structures in analytical or forensic chemistry. Such maps, and methods to navigate them, constitute reliable assays to probe any sample for the presence and amount of molecules contained in the map. So far, attempts to generate such maps for any proteome have failed to reach complete proteome coverage. Here we use a strategy based on high-throughput peptide synthesis and mass spectrometry to generate an almost complete reference map (97% of the genome-predicted proteins) of the Saccharomyces cerevisiae proteome. We generated two versions of this mass-spectrometric map, one supporting discovery-driven (shotgun) and the other supporting hypothesis-driven (targeted) proteomic measurements. Together, the two versions of the map constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. To show the utility of the maps, we applied them to a protein quantitative trait locus (QTL) analysis, which requires precise measurement of the same set of peptides over a large number of samples. Protein measurements over 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, influencing the levels of related proteins. Our results suggest that selective pressure favours the acquisition of sets of polymorphisms that adapt protein levels but also maintain the stoichiometry of functionally related pathway members.

Bhavna Rajasekaran
Analysis of Movement of Cellular Oscillators in the Pre-somitic mesoderm of the zebrafish embryo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2013)

André Nadler, Carsten Schultz
The power of fluorogenic probes.
Angew Chem Int Ed Engl, 52(9) 2408-2410 (2013)
DOI

A definite turn-on: Turning on fluorescence only where successful labeling is happening sounds as desirable as delivering a drug only where the drug target resides. New fluorogenic xanthene derivatives from the Bertozzi research group are getting us closer to "magic bullet" dyes.

Eugeniu Nacu, Mareen Glausch, Huy Quang Le, Febriyani Damanik, Maritta Schuez, Dunja Knapp, Shahryar Khattak, Tobias Richter, Elly M. Tanaka
Connective tissue cells, but not muscle cells, are involved in establishing the proximo-distal outcome of limb regeneration in the axolotl.
Development, 140(3) 513-518 (2013)
DOI

During salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as the rule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell types participate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved. We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guide muscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by β-catenin signalling.

Sven Klose, David Flores-Benitez, Falko Riedel, Elisabeth Knust
Fosmid-based structure-function analysis reveals functionally distinct domains in the cytoplasmic domain of Drosophila crumbs.
G3 (Bethesda), 3(2) 153-165 (2013)
PDF DOI

The evolutionarily conserved transmembrane protein Crumbs is required for epithelial polarity and morphogenesis in the embryo, control of tissue size in imaginal discs and morphogenesis of photoreceptor cells, and prevents light-dependent retinal degeneration. The small cytoplasmic domain contains two highly conserved regions, a FERM (i.e., protein 4.1/ezrin/radixin/moesin)-binding and a PDZ (i.e., postsynaptic density/discs large/ZO-1)-binding domain. Using a fosmid-based transgenomic approach, we analyzed the role of the two domains during invagination of the tracheae and the salivary glands in the Drosophila embryo. We provide data to show that the PDZ-binding domain is essential for the maintenance of cell polarity in both tissues. In contrast, in embryos expressing a Crumbs protein with an exchange of a conserved Tyrosine residue in the FERM-binding domain to an Alanine, both tissues are internalized, despite some initial defects in apical constriction, phospho-Moesin recruitment, and coordinated invagination movements. However, at later stages these embryos fail to undergo dorsal closure, germ band retraction, and head involution. In addition, frequent defects in tracheal fusion were observed. These results suggest stage and/or tissue specific binding partners. We discuss the power of this fosmid-based system for detailed structure-function analyses in comparison to the UAS/Gal4 system.

Alex T. Kalinka
The probability of drawing intersections: extending the hypergeometric distribution
arXiv, Art. No. arXiv:1305.0717 (2013)

Nagananda Gurudev, Mareike Florek, Denis Corbeil, Elisabeth Knust
Prominent role of prominin in the retina.
Adv Exp Med Biol, 777 55-71 (2013)
DOI

Prominin molecules represent a new family of pentaspan membrane glycoproteins expressed throughout the animal kingdom. The name originates from its localization on membrane protrusion, such as microvilli, filopodia, lamellipodia, and microspikes. Following the original description in mouse and human, representative prominin members were found in fish (e.g., Danio rerio), amphibian (Ambystoma mexicanum, Xenopus laevis), worm (Caenorhabditis elegans), and flies (Drosophila melanogaster). Mammalian prominin-1 was identified as a marker of somatic and cancer stem cells and plays an essential role in the visual system, which contributed to increased interest of the medical field in this molecule. Here we summarize recent data from various fields, including Drosophila, which will aid to our understanding of its still elusive function.

Marco Nousch, Christian R. Eckmann
Translational Control in the Caenorhabditis elegans Germ Line.
Adv Exp Med Biol, 757 205-247 (2013)
PDF DOI

Translational control is a prevalent form of gene expression regulation in the Caenorhabditis elegans germ line. Linking the amount of protein synthesis to mRNA quantity and translational accessibility in the cell cytoplasm provides unique advantages over DNA-based controls for developing germ cells. This mode of gene expression is especially exploited in germ cell fate decisions and during oogenesis, when the developing oocytes stockpile hundreds of different mRNAs required for early embryogenesis. Consequently, a dense web of RNA regulators, consisting of diverse RNA-binding proteins and RNA-modifying enzymes, control the translatability of entire mRNA expression programs. These RNA regulatory networks are tightly coupled to germ cell developmental progression and are themselves under translational control. The underlying molecular mechanisms and RNA codes embedded in the mRNA molecules are beginning to be understood. Hence, the C. elegans germ line offers fertile grounds for discovering post-transcriptional mRNA regulatory mechanisms and emerges as great model for a systems level understanding of translational control during development.

Julia Mahamid, Anthony Hyman, Wolfgang Baumeister
Toward Exploring the 3D Supramolecular Architecture of Centrosomes In-Situ
In: Advances in Imaging and Electron Physics ; Vol. 179. (Eds.) Peter W. Hawkes, Amsterdam, Netherlands, Academic Press (2013), 147-147

Iana M. Kalinina, Amitabha Nandi, Petrina Delivani, Mariola R. Chacón, Anna Klemm, Damien Ramunno-Johnson, Alexander Krull, Benjamin Lindner, Nenad Pavin, Iva M. Tolic-Norrelykke
Pivoting of microtubules around the spindle pole accelerates kinetochore capture.
Nat Cell Biol, 15(1) 82-87 (2013)
PDF DOI

Vincent Fraisier, Amal Kasri, Stéphanie Miserey-Lenkei, Jean-Baptiste Sibarita, Deepak Nair, Adeline Mayeux, Sabine Bardin, Yusuke Toyoda, Ina Poser, Anton A Poznyakovskiy, Bruno Goud, Anthony Hyman, Ariane Dimitrov
C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.
PLoS ONE, 8(12) Art. No. e82223 (2013)
DOI

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.

Monalisa Mishra, Elisabeth Knust
Analysis of the Drosophila compound eye with light and electron microscopy.
Methods Mol Biol, 935 161-182 (2013)
PDF DOI

The Drosophila compound eye is a regular structure, in which about 750 units, called ommatidia, are arranged in a highly regular pattern. Eye development proceeds in a stereotypical fashion, where epithelial cells of the eye imaginal discs are specified, recruited, and differentiated in a sequential order that leads to the highly precise structure of an adult eye. Even small perturbations, for example in signaling pathways that control proliferation, cell death, or differentiation, can impair the regular structure of the eye, which can be easily detected and analyzed. In addition, the Drosophila eye has proven to be an ideal model for studying the genetic control of neurodegeneration, since the eye is not essential for viability. Several human neurodegeneration diseases have been modeled in the fly, leading to a better understanding of the function/misfunction of the respective gene. In many cases, the genes involved and their function are conserved between flies and human. More strikingly, when ectopically expressed in the fly eye some human genes without a Drosophila counterpart can induce neurodegeneration, detectable by aberrant phototaxis, impaired electrophysiology, or defects in eye morphology. These defects are often rather subtle alteration in shape, size, or arrangement of the cells, and can be easily scored at the ultrastructural level. This chapter aims to provide an overview regarding the analysis of the retina by various means.

Joao Firmino, Jean-Yves Tinevez, Elisabeth Knust
Crumbs affects protein dynamics in anterior regions of the developing Drosophila embryo.
PLoS ONE, 8(3) Art. No. e58839 (2013)
PDF DOI

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

Marta Luz, Elisabeth Knust
Fluorescently tagged Lin7c is a dynamic marker for polarity maturation in the zebrafish retinal epithelium.
Biol Open, 2(9) 867-871 (2013)
DOI

Development of epithelial cell polarity is a highly dynamic process, and often established by the sequential recruitment of conserved protein complexes, such as the Par or the Crumbs (Crb) complex. However, detailed insights into the refinement of polarity and the formation of the complexes are still lacking. Here, we established fluorescently tagged Lin7c, a core member of the Crb complex, as an ideal tool to follow development of polarity in zebrafish epithelia. We find that in gastrula stages, RFP-Lin7c is found in the cytosol of the enveloping layer, while Pard3-GFP is already polarized at this stage. During development of the retinal epithelium, RFP-Lin7c localization is refined from being cytosolic at 14 hours post fertilization (hpf) to almost entirely apical in cells of the eye cup at 28 hpf. This apical Lin7c localization depends on the Crb complex members Oko meduzy and Nagie oko. Thus, fluorescently tagged Lin7c can be used in a broad range of epithelia to follow polarity maturation in vivo and specifically to elucidate the sequence of events determining Crb complex-mediated polarity.

Marina N Nedelcheva-Veleva, Mihail Sarov, Ivan Yanakiev, Eva Mihailovska, Miroslav P Ivanov, Greta C Panova, Stoyno Stoynov
The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron-exon recognition.
Nat Commun, 4 Art. No. 2101 (2013)
DOI

The essential cis- and trans-acting elements required for RNA splicing have been defined, however, the detailed molecular mechanisms underlying intron-exon recognition are still unclear. Here we demonstrate that the ratio between stability of mRNA/DNA and DNA/DNA duplexes near 3'-spice sites is a characteristic feature that can contribute to intron-exon differentiation. Remarkably, throughout all transcripts, the most unstable mRNA/DNA duplexes, compared with the corresponding DNA/DNA duplexes, are situated upstream of the 3'-splice sites and include the polypyrimidine tracts. This characteristic instability is less pronounced in weak alternative splice sites and disease-associated cryptic 3'-splice sites. Our results suggest that this thermodynamic pattern can prevent the re-annealing of mRNA to the DNA template behind the RNA polymerase to ensure access of the splicing machinery to the polypyrimidine tract and the branch point. In support of this mechanism, we demonstrate that RNA/DNA duplex formation at this region prevents pre-spliceosome A complex assembly.

Dunja Knapp, Herbert Schulz, Cynthia Alexander Rascon, Michael Volkmer, Juliane Scholz, Eugeniu Nacu, Mu Le, Sergey Novozhilov, Akira Tazaki, Stephanie Protze, Tina Jacob, Norbert Hubner, Bianca Habermann, Elly M. Tanaka
Comparative transcriptional profiling of the axolotl limb identifies a tripartite regeneration-specific gene program.
PLoS ONE, 8(5) Art. No. e61352 (2013)
DOI

Understanding how the limb blastema is established after the initial wound healing response is an important aspect of regeneration research. Here we performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. This comparison between wound healing and regeneration allowed us to identify amputation-specific genes. By clustering the expression profiles of these samples, we could detect three distinguishable phases of gene expression - early wound healing followed by a transition-phase leading to establishment of the limb development program, which correspond to the three phases of limb regeneration that had been defined by morphological criteria. By focusing on the transition-phase, we identified 93 strictly amputation-associated genes many of which are implicated in oxidative-stress response, chromatin modification, epithelial development or limb development. We further classified the genes based on whether they were or were not significantly expressed in the developing limb bud. The specific localization of 53 selected candidates within the blastema was investigated by in situ hybridization. In summary, we identified a set of genes that are expressed specifically during regeneration and are therefore, likely candidates for the regulation of blastema formation.

A.M. Sykes, Wieland B. Huttner
Prominin-1 (CD133) and the cell biology of neural progenitors and their progeny.
In: Prominin-1 (CD133): New Insights on Stem and Cancer Stem Cell Biology. (Eds.) Denis Corbeil Advances in Experimental Medicine and Biology ; 777., New York, Springer (2013), 89-98 Ch. 6
DOI

Jacopo Marino, Eric R Geertsma, Oliver Zerbe
Topogenesis of heterologously expressed fragments of the human Y4 GPCR.
Biochim Biophys Acta, 1818(12) 3055-3063 (2012)
DOI

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.

David Richmond, Andrew C. Oates
The segmentation clock: inherited trait or universal design principle?
Curr Opin Genet Dev, 22(6) 600-606 (2012)
DOI

Metamerism is a widespread feature of multicellular body plans; however, our understanding of the underlying mechanisms that generate these patterns is currently based on only a few model organisms. In particular, vertebrate embryos use a segmentation clock to rhythmically and sequentially add segments in concert with posterior elongation of their body. Recent evidence of a segmentation clock acting in arthropods indicates that this mechanism may be a widely used strategy for generating serial anatomy in animals. Whether this is due to homology or convergence is not yet known, but the recent discovery of an oscillatory process associated with the production of sequential root primordia in plants suggests that a segmentation clock is a fundamental patterning principle in growing tissues, independent of ancestry. In this review, we consider the principles of the segmentation clock that may be conserved across the animal and plant kingdoms, and discuss opportunities for cross-fertilization between these active fields of research.

Melek Asli Kayserili^*, Dave T. Gerrard^*, Pavel Tomancak^#, Alex T. Kalinka^#
An excess of gene expression divergence on the x chromosome in Drosophila embryos: implications for the faster-x hypothesis.
PLoS Genet, 8(12) Art. No. e1003200 (2012)
DOI

The X chromosome is present as a single copy in the heterogametic sex, and this hemizygosity is expected to drive unusual patterns of evolution on the X relative to the autosomes. For example, the hemizgosity of the X may lead to a lower chromosomal effective population size compared to the autosomes, suggesting that the X might be more strongly affected by genetic drift. However, the X may also experience stronger positive selection than the autosomes, because recessive beneficial mutations will be more visible to selection on the X where they will spend less time being masked by the dominant, less beneficial allele-a proposal known as the faster-X hypothesis. Thus, empirical studies demonstrating increased genetic divergence on the X chromosome could be indicative of either adaptive or non-adaptive evolution. We measured gene expression in Drosophila species and in D. melanogaster inbred strains for both embryos and adults. In the embryos we found that expression divergence is on average more than 20% higher for genes on the X chromosome relative to the autosomes; but in contrast, in the inbred strains, gene expression variation is significantly lower on the X chromosome. Furthermore, expression divergence of genes on Muller's D element is significantly greater along the branch leading to the obscura sub-group, in which this element segregates as a neo-X chromosome. In the adults, divergence is greatest on the X chromosome for males, but not for females, yet in both sexes inbred strains harbour the lowest level of gene expression variation on the X chromosome. We consider different explanations for our results and conclude that they are most consistent within the framework of the faster-X hypothesis.

Robert Opitz, Emilie Maquet, Jan Huisken, Francesco Antonica, Achim Trubiroha, Gaëlle Pottier, Veerle Janssens, Sabine Costagliola
Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development.
Dev Biol, 372(2) 203-216 (2012)
DOI

Among the various organs derived from foregut endoderm, the thyroid gland is unique in that major morphogenic events such as budding from foregut endoderm, descent into subpharyngeal mesenchyme and growth expansion occur in close proximity to cardiovascular tissues. To date, research on thyroid organogenesis was missing one vital tool-a transgenic model that allows to track the dynamic changes in thyroid size, shape and location relative to adjacent cardiovascular tissues in live embryos. In this study, we generated a novel transgenic zebrafish line, tg(tg:mCherry), in which robust and thyroid-specific expression of a membrane version of mCherry enables live imaging of thyroid development in embryos from budding stage throughout formation of functional thyroid follicles. By using various double transgenic models in which EGFP expression additionally labels cardiovascular structures, a high coordination was revealed between thyroid organogenesis and cardiovascular development. Early thyroid development was found to proceed in intimate contact with the distal ventricular myocardium and live imaging confirmed that thyroid budding from the pharyngeal floor is tightly coordinated with the descent of the heart. Four-dimensional imaging of live embryos by selective plane illumination microscopy and 3D-reconstruction of confocal images of stained embryos yielded novel insights into the role of specific pharyngeal vessels, such as the hypobranchial artery (HA), in guiding late thyroid expansion along the pharyngeal midline. An important role of the HA was corroborated by the detailed examination of thyroid development in various zebrafish models showing defective cardiovascular development. In combination, our results from live imaging as well es from 3D-reconstruction of thyroid development in tg(tg:mCherry) embryos provided a first dynamic view of late thyroid organogenesis in zebrafish-a critical resource for the design of future studies addressing the molecular mechanisms of these thyroid-vasculature interactions.

Silvia Brambillasca, Anke Altkrueger, Sara Francesca Colombo, Anne Friederich, Peter Eickelmann, Michael Mark, Nica Borgese, Michele Solimena
CDK5 regulatory subunit-associated protein 1-like 1 (CDKAL1) is a tail-anchored protein in the endoplasmic reticulum (ER) of insulinoma cells.
J Biol Chem, 287(50) 41808-41819 (2012)
DOI

Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced β-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNA(Lys) to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-β-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.

Clifford Brangwynne, Anthony A. Hyman
The Origin of Life
Nature, 491(7425) 524-525 (2012)

Iva Kelava, Wieland B. Huttner
Neurogenesis in the developing mammalian neocortex.
In: Encyclopedia of Life Sciences (eLS)., Chichester, John Wiley & Sons Ltd. (2012)
DOI

Senthil Arumugam, Grzegorz Chwastek, Elisabeth Fischer-Friedrich, Carina Ehrig, Ingolf Mönch, Petra Schwille
Surface topology engineering of membranes for the mechanical investigation of the tubulin homologue FtsZ.
Angew Chem Int Ed Engl, 51(47) 11858-11862 (2012)
DOI

Andrew Dauber, Stephen H Lafranchi, Zoltan Maliga, Julian C Lui, Jennifer E Moon, Cailin McDeed, Katrin Henke, Jonathan Zonana, Garrett A Kingman, Tune H Pers, Jeffrey Baron, Ron G Rosenfeld, Joel N Hirschhorn, Matthew P Harris, Vivian Hwa
Novel microcephalic primordial dwarfism disorder associated with variants in the centrosomal protein ninein.
J Clin Endocrinol Metab, 97(11) 2140-2151 (2012)
DOI

Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions.

Kristina Uzunova, Billy T Dye, Hannelore Schutz, Rene Ladurner, Georg Petzold, Yusuke Toyoda, Marc A Jarvis, Nicholas G Brown, Ina Poser, Maria Novatchkova, Karl Mechtler, Anthony A. Hyman, Holger Stark, Brenda A Schulman, Jan-Michael Peters
APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex.
Nat Struct Mol Biol, 19(11) 1116-1123 (2012)
DOI

The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.

Elwy Okaz, Orlando Argüello-Miranda, Aliona Bogdanova, P K Vinod, Jesse J. Lipp, Zuzana Markova, Ievgeniia Zagoriy, Bela Novak, Wolfgang Zachariae
Meiotic Prophase Requires Proteolysis of M Phase Regulators Mediated by the Meiosis-Specific APC/C(Ama1).
Cell, 151(3) 603-618 (2012)
DOI

Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.

T-Y Dora Tang, Nicholas J Brooks, Christoph Jeworrek, Oscar Ces, Nicholas J Terrill, Roland Winter, Richard H Templer, John M Seddon
Hydrostatic pressure effects on the lamellar to gyroid cubic phase transition of monolinolein at limited hydration.
Langmuir, 28(36) 13018-13024 (2012)
DOI

Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.

Monalisa Mishra, Michaela Rentsch, Elisabeth Knust
Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli
Eur J Cell Biol, 91(9) 706-716 (2012)
PDF DOI

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.

Günther Gerisch, Britta Schroth-Diez, Annette Müller-Taubenberger, Mary Ecke
PIP3 waves and PTEN dynamics in the emergence of cell polarity.
Biophys J, 103(6) 1170-1178 (2012)
DOI

In a motile eukaryotic cell, front protrusion and tail retraction are superimposed on each other. To single out mechanisms that result in front to tail or in tail to front transition, we separated the two processes in time using cells that oscillate between a full front and a full tail state. State transitions were visualized by total internal reflection fluorescence microscopy using as a front marker PIP3 (phosphatidylinositol [3,4,5] tris-phosphate), and as a tail marker the tumor-suppressor PTEN (phosphatase tensin homolog) that degrades PIP3. Negative fluctuations in the PTEN layer of the membrane gated a local increase in PIP3. In a subset of areas lacking PTEN (PTEN holes), PIP3 was amplified until a propagated wave was initiated. Wave propagation implies that a PIP3 signal is transmitted by a self-sustained process, such that the temporal and spatial profiles of the signal are maintained during passage of the wave across the entire expanse of the cell membrane. Actin clusters were remodeled into a ring along the perimeter of the expanding PIP3 wave. The reverse transition of PIP3 to PTEN was linked to the previous site of wave initiation: where PIP3 decayed first, the entry of PTEN was primed.

Michael Groessl, H Luksch, Angela Rösen-Wolff, Andrej Shevchenko, Marc Gentzel
Profiling of the human monocytic cell secretome by quantitative label-free mass spectrometry identifies stimulus-specific cytokines and proinflammatory proteins.
Proteomics, 12(18) 2833-2842 (2012)
DOI

The immune response to pathogens or injury relies on the concerted release of cytokines and proteins with biological activity important for host protection, host defense, and wound healing. Consequently, the secretome of immune cells provides a promising resource for discovery of specific molecular markers and targets for pharmacological intervention. Here, we employ label-free MS for unbiased, quantitative profiling of the human monocytic cell secretome under different proinflammatory stimuli. The quantitative secretome profiles reveal the highly stimulus-dependent cellular response and differential, specific secretion of more than 200 proteins, including important proinflammatory proteins and cytokines.

Liliana Malinovska^*, Sonja Kroschwald^*, Matthias Munder^*, Doris Richter, Simon Alberti
Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates
Mol Biol Cell, 23(16) 3041-3056 (2012)
PDF DOI

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is currently not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein sorting factors of the Hook family termed Btn2 and Cur1 as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions, but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. Based on these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein sorting factors. Our model suggests that protein aggregation is not a haphazard process, but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.

Marine R-C Kraus, Anne Grapin-Botton
Patterning and shaping the endoderm in vivo and in culture.
Curr Opin Genet Dev, 22(4) 347-353 (2012)
DOI

The definitive endoderm (DE) was first defined as the innermost germ layer found in all metazoan embryos. During development, it gives rise to a vast array of specialized epithelial cell types lining the respiratory and digestive systems, and contributes to associated organs such as thyroid, thymus, lungs, liver, and pancreas. In the adult, the DE provides a protective barrier against the environment and assumes many essential functions including digestion, nutrient absorption, and glucose homeostasis. Since general endoderm formation and patterning have been reviewed recently in a comprehensive manner [1], we will only provide a brief summary of how extracellular signals and downstream transcription factors control endoderm patterning. We will then focus on emerging work addressing the chromatin remodeling events occurring during endoderm organ specification and discuss how these molecular tools can be used to engineer endodermal organs in vitro.

Sara K Olson, Garrett Greenan, Arshad Desai, Thomas Müller-Reichert, Karen Oegema
Hierarchical assembly of the eggshell and permeability barrier in C. elegans.
J Cell Biol, 198(4) 731-748 (2012)
DOI

In metazoans, fertilization triggers the assembly of an extracellular coat that constitutes the interface between the embryo and its environment. In nematodes, this coat is the eggshell, which provides mechanical rigidity, prevents polyspermy, and is impermeable to small molecules. Using immunoelectron microscopy, we found that the Caenorhabditis elegans eggshell was composed of an outer vitelline layer, a middle chitin layer, and an inner layer containing chondroitin proteoglycans. The switch between the chitin and proteoglycan layers was achieved by internalization of chitin synthase coincident with exocytosis of proteoglycan-containing cortical granules. Inner layer assembly did not make the zygote impermeable as previously proposed. Instead, correlative light and electron microscopy demonstrated that the permeability barrier was a distinct envelope that formed in a separate step that required fatty acid synthesis, the sugar-modifying enzyme PERM-1, and the acyl chain transfer enzyme DGTR-1. These findings delineate the hierarchy of eggshell assembly and define key molecular mechanisms at each step.

Jean-Léon Maître, Hélène Berthoumieux, Simon Frederik Gabriel Krens, Guillaume Salbreux, Frank Jülicher, Ewa Paluch^#, Carl-Philipp Heisenberg^#
Adhesion Functions in Cell Sorting by Mechanically Coupling the Cortices of Adhering Cells.
Science, 338(6104) 253-256 (2012)
DOI

Differential cell adhesion and cortex tension are thought to drive cell sorting by controlling cell-cell contact formation. Here, we show that cell adhesion and cortex tension have different mechanical functions in controlling progenitor cell-cell contact formation and sorting during zebrafish gastrulation. Cortex tension controls cell-cell contact expansion by modulating interfacial tension at the contact. In contrast, adhesion has little direct function in contact expansion but instead is needed to mechanically couple the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. The coupling function of adhesion is mediated by E-cadherin and limited by the mechanical anchoring of E-cadherin to the cortex. Thus, cell adhesion provides the mechanical scaffold for cell cortex tension to drive cell sorting during gastrulation.

Goran Malojčić^*, Eric R Geertsma^*, Maurice S Brozzo, Rudi Glockshuber
Mechanism of the prokaryotic transmembrane disulfide reduction pathway and its in vitro reconstitution from purified components.
Angew Chem Int Ed Engl, 51(28) 6900-6903 (2012)
DOI

Making your (Dsb) connection: the redox pathway bringing reducing equivalents from bacterial cytoplasm, across the inner membrane, to the three reductive Dsb pathways in the otherwise oxidizing periplasm (see scheme; TR=thioredoxin reductase, Trx=thioredoxin) is reconstituted from purified components. Transfer of reducing equivalents across the membrane is demonstrated and underlying mechanistic details are revealed.

Tzachi Hagai, Ágnes Tóth-Petróczy, Ariel Azia, Yaakov Levy
The origins and evolution of ubiquitination sites.
Mol Biosyst, 8(7) 1865-1877 (2012)
DOI

Protein ubiquitination is central to the regulation of various pathways in eukaryotes. The process of ubiquitination and its cellular outcome were investigated in hundreds of proteins to date. Despite this, the evolution of this regulatory mechanism has not yet been addressed comprehensively. Here, we quantify the rates of evolutionary changes of ubiquitination and SUMOylation (Small Ubiquitin-like MOdifier) sites. We estimate the time at which they first appeared, and compare them to acetylation and phosphorylation sites and to unmodified residues. We observe that the various modification sites studied exhibit similar rates. Mammalian ubiquitination sites are weakly more conserved than unmodified lysine residues, and a higher degree of relative conservation is observed when analyzing bona fide ubiquitination sites. Various reasons can be proposed for the limited level of excess conservation of ubiquitination, including shifts in locations of the sites, the presence of alternative sites, and changes in the regulatory pathways. We observe that disappearance of sites may be compensated by the presence of a lysine residue in close proximity, which is significant when compared to evolutionary patterns of unmodified lysine residues, especially in disordered regions. This emphasizes the importance of analyzing a window in the vicinity of functional residues, as well as the capability of the ubiquitination machinery to ubiquitinate residues in a certain region. Using prokaryotic orthologs of ubiquitinated proteins, we study how ubiquitination sites were formed, and observe that while sometimes sequence additions and rearrangements are involved, in many cases the ubiquitination machinery utilizes an already existing sequence without significantly changing it. Finally, we examine the evolution of ubiquitination, which is linked with other modifications, to infer how these complex regulatory modules have evolved. Our study gives initial insights into the formation of ubiquitination sites, their degree of conservation in various species, and their co-evolution with other posttranslational modifications.

Volker Bormuth^*, Bert Nitzsche^*, Felix Ruhnow, Aniruddha Mitra, Marko Storch, Burkhard Rammner, Jonathon Howard, Stefan Diez
The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias towards the left
Biophys J, 103(1 (Letter)) 4-6 (2012)
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Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.

Maria Carvalho^*, Julio Sampaio^*, Wilhelm Palm, Marko Brankatschk, Suzanne Eaton^#, Andrej Shevchenko^#
Effects of diet and development on the Drosophila lipidome
Mol Syst Biol, 8 Art. No. 600 (2012)
DOI

Cells produce tens of thousands of different lipid species, but the importance of this complexity in vivo is unclear. Analysis of individual tissues and cell types has revealed differences in abundance of individual lipid species, but there has been no comprehensive study comparing tissue lipidomes within a single developing organism. Here, we used quantitative shotgun profiling by high-resolution mass spectrometry to determine the absolute (molar) content of 250 species of 14 major lipid classes in 6 tissues of animals at 27 developmental stages raised on 4 different diets. Comparing these lipidomes revealed unexpected insights into lipid metabolism. Surprisingly, the fatty acids present in dietary lipids directly influence tissue phospholipid composition throughout the animal. Furthermore, Drosophila differentially regulates uptake, mobilization and tissue accumulation of specific sterols, and undergoes unsuspected shifts in fat metabolism during larval and pupal development. Finally, we observed striking differences between tissue lipidomes that are conserved between phyla. This study provides a comprehensive, quantitative and expandable resource for further pharmacological and genetic studies of metabolic disorders and molecular mechanisms underlying dietary response.

Christian Schröter, Saul Ares^*, Luis G. Morelli^*, Alina Isakova, Korneel Hens, Daniele Soroldoni, Martin Gajewski, Frank Jülicher, Sebastian J Maerkl, Bart Deplancke, Andrew C. Oates
Topology and dynamics of the zebrafish segmentation clock core circuit
PLoS Biol, 10(7) Art. No. e1001364 (2012)
Open Access DOI

During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a "dimer cloud" that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks.

Andreas Sagner, Matthias Merkel, Benoit Aigouy, Julia Gaebel, Marko Brankatschk, Frank Jülicher^#, Suzanne Eaton^#
Establishment of Global Patterns of Planar Polarity during Growth of the Drosophila Wing Epithelium.
Curr Biol, 22(14) 1296-1301 (2012)
DOI

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures [1-3]. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP [4, 5]. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally [6]. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.

C Schmees, Roberto Villaseñor, W Zheng, H Ma, Marino Zerial, Carl-Henrik Heldin, C Hellberg
Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V.
Mol Biol Cell, 23(13) 2571-2582 (2012)
PDF DOI

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

Maryam Aliee^*, Jens-Christian Röper^*, Katharina Landsberg, Constanze Pentzold, Thomas Widmann, Frank Jülicher^#, Christian Dahmann^#
Physical mechanisms shaping the Drosophila dorsoventral compartment boundary
Curr Biol, 22(11) 967-976 (2012)
DOI

Separating cells with distinct identities and fates by straight and sharp compartment boundaries is important for growth and pattern formation during animal development. The physical mechanisms shaping compartment boundaries, however, are not fully understood.

Asifa Akhtar, Karla M. Neugebauer
The nucleus and gene expression: the center of the cyclone
Curr Opin Cell Biol, 24(3) 293-295 (2012)
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Rico Barsacchi, Varadharajan Sundaramurthy, Kees Korbee, Jacques Neefjes, Tom H. M. Ottenhoff, Tiziana Scanu, Marino Zerial
Manipulating the Fight Between Human Host Cells and Intracellular Pathogens
In: Systems Microbiology: Current Topics and Applications. (Eds.) Brian D. Robertson, Brendan W. Wren, Norfolk, Caister Acad. Press (2012), 77-94 Ch. 5

Koichiro Uriu^#, Saul Ares, Andrew C. Oates, Luis G. Morelli^#
Optimal cellular mobility for synchronization arising from the gradual recovery of intercellular interactions.
Physical biology, 9(3) Art. No. 036006 (2012)
DOI

Cell movement and intercellular signaling occur simultaneously during the development of tissues, but little is known about how movement affects signaling. Previous theoretical studies have shown that faster moving cells favor synchronization across a population of locally coupled genetic oscillators. An important assumption in these studies is that cells can immediately interact with their new neighbors after arriving at a new location. However, intercellular interactions in cellular systems may need some time to become fully established. How movement affects synchronization in this situation has not been examined. Here, we develop a coupled phase oscillator model in which we consider cell movement and the gradual recovery of intercellular coupling experienced by a cell after movement, characterized by a moving rate and a coupling recovery rate, respectively. We find (1) an optimal moving rate for synchronization and (2) a critical moving rate above which achieving synchronization is not possible. These results indicate that the extent to which movement enhances synchrony is limited by a gradual recovery of coupling. These findings suggest that the ratio of time scales of movement and signaling recovery is critical for information transfer between moving cells.

Katja Kapp^*, Johannes Siemens^*, Hans-Ulrich Häring, Reiner Lammers
Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE.
Eur J Cell Biol, 91(9) 687-693 (2012)
PDF DOI

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.

Anja Zeigerer, Jerome Gilleron, Roman L Bogorad, Giovanni Marsico, Hidenori Nonaka, Sarah Seifert, Hila Epstein-Barash, Satya Kuchimanchi, Chang Geng Peng, Vera M Ruda, Perla Del Conte-Zerial, Jan G Hengstler, Yannis Kalaidzidis, Victor Koteliansky, Marino Zerial
Rab5 is necessary for the biogenesis of the endolysosomal system in vivo.
Nature, 485(7399) 465-470 (2012)
PDF DOI

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Nimesh Joseph, Andrea Hutterer, Ina Poser, Masanori Mishima
ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration
EMBO J, 31(11) 2604-2614 (2012)
DOI

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.

Alex T. Kalinka^#, Pavel Tomancak^#
The evolution of early animal embryos: conservation or divergence?
Trends Ecol Evol, 27(7) 385-393 (2012)
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Matthias Stein^#, Manohar Pilli, Sabine Bernauer, Bianca Habermann, Marino Zerial, Rebecca C Wade^#
The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.
PLoS ONE, 7(4) Art. No. e34870 (2012)
PDF DOI

Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.

Eugenio Fava^*, J Dehghany^*, Joke Ouwendijk, A Müller, Antje Niederlein, Paul Verkade, M Meyer-Hermann, Michele Solimena
Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.
Diabetologia, 55(4) 1013-1023 (2012)
DOI

Knowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size.

Sanjay Kumar, Hae Yong Yoo, Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts
Cell Cycle, 11(6) 1183-1194 (2012)
DOI

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.

Michal Segal, Ilya Soifer, Heike Petzold, Jonathon Howard, Michael Elbaum, Orly Reiner
Ndel1-derived peptides modulate bidirectional transport of injected beads in the squid giant axon.
Biol Open, 1(3) 220-231 (2012)
PDF DOI

Bidirectional transport is a key issue in cellular biology. It requires coordination between microtubule-associated molecular motors that work in opposing directions. The major retrograde and anterograde motors involved in bidirectional transport are cytoplasmic dynein and conventional kinesin, respectively. It is clear that failures in molecular motor activity bear severe consequences, especially in the nervous system. Neuronal migration may be impaired during brain development, and impaired molecular motor activity in the adult is one of the hallmarks of neurodegenerative diseases leading to neuronal cell death. The mechanisms that regulate or coordinate kinesin and dynein activity to generate bidirectional transport of the same cargo are of utmost importance. We examined how Ndel1, a cytoplasmic dynein binding protein, may regulate non-vesicular bidirectional transport. Soluble Ndel1 protein, Ndel1-derived peptides or control proteins were mixed with fluorescent beads, injected into the squid giant axon, and the bead movements were recorded using time-lapse microscopy. Automated tracking allowed for extraction and unbiased analysis of a large data set. Beads moved in both directions with a clear bias to the anterograde direction. Velocities were distributed over a broad range and were typically slower than those associated with fast vesicle transport. Ironically, the main effect of Ndel1 and its derived peptides was an enhancement of anterograde motion. We propose that they may function primarily by inhibition of dynein-dependent resistance, which suggests that both dynein and kinesin motors may remain engaged with microtubules during bidirectional transport.

Bettina C Kirchmaier, Kar Lai Poon, Thorsten Schwerte, Jan Huisken, Christoph Winkler, Benno Jungblut, Didier Y.R. Stainier, Thomas Brand
The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development.
Dev Biol, 363(2) 438-450 (2012)
DOI

The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)(s878)) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system.

Nancy Thompson, Emilie Gésina, Peter Scheinert, Philipp Bucher, Anne Grapin-Botton
RNA profiling and chromatin immunoprecipitation-sequencing reveal that PTF1a stabilizes pancreas progenitor identity via the control of MNX1/HLXB9 and a network of other transcription factors.
Mol Cell Biol, 32(6) 1189-1199 (2012)
DOI

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors.

Jean-Yves Tinevez, Guillaume Salbreux, Ewa Paluch
[The mechanics of the cellular division or how to split a sphere into two?].
Med Sci (Paris), 28(2) 144-146 (2012)
DOI

Iva Kelava, Isabel Reillo, Ayako Y Murayama, Alex T. Kalinka, Denise Stenzel, Pavel Tomancak, Fumio Matsuzaki, Cécile Lebrand, Erika Sasaki, Jens C Schwamborn, Hideyuki Okano, Wieland B. Huttner, Víctor Borrell
Abundant occurrence of basal radial glia in the subventricular zone of embryonic neocortex of a lissencephalic primate, the common marmoset Callithrix jacchus.
Cereb Cortex, 22(2) 469-481 (2012)
PDF DOI

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Andrew C. Oates, Luis G. Morelli, Saul Ares
Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock.
Development, 139(4) 625-639 (2012)
DOI

The segmentation clock is an oscillating genetic network thought to govern the rhythmic and sequential subdivision of the elongating body axis of the vertebrate embryo into somites: the precursors of the segmented vertebral column. Understanding how the rhythmic signal arises, how it achieves precision and how it patterns the embryo remain challenging issues. Recent work has provided evidence of how the period of the segmentation clock is regulated and how this affects the anatomy of the embryo. The ongoing development of real-time clock reporters and mathematical models promise novel insight into the dynamic behavior of the clock.

Elena Taverna, Christiane Haffner, Rainer Pepperkok, Wieland B. Huttner
A new approach to manipulate the fate of single neural stem cells in tissue.
Nat Neurosci, 15(2) 329-337 (2012)
DOI

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

Sebastian Fürthauer, Maaike Neeft, Stephan W. Grill, Karsten Kruse, Frank Jülicher
The Taylor-Couette motor: spontaneous flows of active polar fluids between two coaxial cylinders
New J Phys, 14(2) Art. No. e023001 (2012)
PDF DOI

We study the dynamics of active polar fluids in a Taylor–Couette geometry where the fluid is confined between two rotating coaxial cylinders. This system can spontaneously generate flow fields and thereby set the two cylinders into relative rotation either by spontaneous symmetry breaking or via asymmetric boundary conditions on the polarization field at the cylinder surfaces. In the presence of an externally applied torque, the system can act as a rotatory motor and perform mechanical work. The relation between the relative angular velocity of the cylinders and the externally applied torque exhibits rich behaviors such as dynamic instabilities and the coexistence of multiple stable steady states for certain ranges of parameter values and boundary conditions.

Mathias J. Gerl, Julio Sampaio, Lucie Kalvodova, Jean-Marc Verbavatz, Andrej Shevchenko, Cornelia Schroeder, Kai Simons
Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane
J Cell Biol, 196(2) 213-221 (2012)
PDF DOI

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin–Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

Shubha Vij^*, Jochen Rink^*, Hao Kee Ho, Deepak Babu, Michael Eitel, Vijayashankaranarayanan Narasimhan, Varnesh Tiku, Jody Westbrook, Bernd Schierwater, Sudipto Roy
Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program.
PLoS Genet, 8(11) Art. No. e1003019 (2012)
DOI

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.

Michal Surma, Christian Klose, Kai Simons
Lipid-dependent protein sorting at the trans-Golgi network
Biochim Biophys Acta, 1821(8, SI) 1059-1067 (2012)
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Maria Arantzazu Sanchez-Fernandez, Silvia Sbacchi, Miguel Correa-Tapia, Ronald Naumann, Julia Klemm, Pierre Chambon, Samiya Al-Robaiy, Manfred Blessing, Bernard Hoflack
Transgenic mice for a tamoxifen-induced, conditional expression of the cre recombinase in osteoclasts.
PLoS ONE, 7(5) Art. No. e37592 (2012)
PDF DOI

Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood.

Ursula Ehmer, Sandra Kalthoff, Bastian Fakundiny, Brigitte Pabst, Nicole Freiberg, Ronald Naumann, Michael P Manns, Christian P Strassburg
Gilbert syndrome redefined: A complex genetic haplotype influences the regulation of glucuronidation.
Hepatology, 55(6) 1912-1921 (2012)
DOI

Gilbert syndrome (GS) is characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage affecting about 10% of the white population. In GS the UGT1A1*28 variant reduces bilirubin conjugation by 70% and is associated with irinotecan and protease inhibitor side effects. Aim of this study was to characterize potential in vivo consequences of UGT1A gene variability in GS. Three hundred GS patients (UGT1A1*28 homozygous) and 249 healthy blood donors (HBD) were genotyped for UGT1A (UGT1A1*28, UGT1A3-66 T>C, UGT1A6*3a, UGT1A7*3) and transporter SNPs (SCLO1B1 p.V174A, SCLO1B1 p.N130D, ABCC2 p.I1324I, ABCC2 -24 UTR) using TaqMan-5'-nuclease-assays. A humanized transgenic UGT1A-SNP and corresponding wild-type mouse model were established carrying the GS-associated UGT1A variant haplotype. UGT1A transcript and protein expression, and transcriptional activation were studied in vivo. Homozygous UGT1A1*28 GS individuals were simultaneously homozygous for UGT1A3-66 T>C (91%), UGT1A6*2a (77%), and UGT1A7*3 (77%). Seventy-six percent of GS and only 9% of HBD were homozygous for the variant haplotype spanning 4 UGT1A genes. SCLO1B1 and ABCC2 single nucleotide polymorphisms (SNPs) showed no differences. In transgenic humanized UGT1A SNP and wild-type mice this UGT1A haplotype led to lower UGT1A mRNA expression and UGT1A protein synthesis. UGT1A transcriptional activation by dioxin, phenobarbital and endotoxin was significantly reduced in SNP mice. CONCLUSION: Our data redefine the genetic basis behind GS. In vivo data studying the genotype present in 76% of GS individuals suggest that transcription and transcriptional activation of glucuronidation genes responsible for conjugation and detoxification is directly affected leading to lower responsiveness. This study suggests that GS should be considered as a potential risk factor for drug toxicity. (HEPATOLOGY 2011.).

Birte Schrader, Sylvain Reboux, Ivo F. Sbalzarini
Choosing the best kernel: performance models for diffusion operators in particle methods
SIAM J Sci Comput, 34(3) 1607-1634 (2012)
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Minna-Liisa Änkö, Michaela Müller-McNicoll, Holger Brandl, Tomaz Curk, Crtomir Gorup, Ian Henry, Jernej Ule, Karla M. Neugebauer
The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes.
Genome Biol, 13(3) Art. No. R17 (2012)
PDF DOI

The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.

Maki Asami, Gregor A Pilz, Jovica Ninkovic, Leanne Godinho, Timm Schroeder, Wieland B. Huttner, Magdalena Götz
The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex
Development, 138(23) 5067-5078 (2011)
DOI

Successful brain development requires tight regulation of sequential symmetric and asymmetric cell division. Although Pax6 is known to exert multiple roles in the developing nervous system, its role in the regulation of cell division is unknown. Here, we demonstrate profound alterations in the orientation and mode of cell division in the cerebral cortex of mice deficient in Pax6 function (Pax6(Sey/Sey)) or after acute induced deletion of Pax6. Live imaging revealed an increase in non-vertical cellular cleavage planes, resulting in an increased number of progenitors with unequal inheritance of the apical membrane domain and adherens junctions in the absence of Pax6 function. This phenotype appears to be mediated by the direct Pax6 target Spag5, a microtubule-associated protein, reduced levels of which result in the replication of the Pax6 phenotype of altered cell division orientation. In addition, lack of Pax6 also results in premature delamination of progenitor cells from the apical surface due to an overall decrease in proteins mediating anchoring at the ventricular surface. Moreover, continuous long-term imaging in vitro revealed that Pax6-deficient progenitors generate daughter cells with asymmetric fates at higher frequencies. These data demonstrate a cell-autonomous role for Pax6 in regulating the mode of cell division independently of apicobasal polarity and cell-cell interactions. Taken together, our work reveals several direct effects that the transcription factor Pax6 has on the machinery that mediates the orientation and mode of cell division.

Anton A Poznyakovskiy, Thomas Zahnert, Yannis Kalaidzidis, Nikoloz Lazurashvili, Rolf Schmidt, Hans-Jürgen Hardtke, Björn Fischer, Yury M Yarin
A segmentation method to obtain a complete geometry model of the hearing organ.
Hear Res, 282(1-2) 25-34 (2011)
DOI

We present a method for obtaining a complete geometry model of the fluid chambers of cochlea (scalae) from tomography images. An accurate segmentation of cochlea is problematic due to the low contrast of the inner membranes of scalae. Our method of 3D segmentation is based on dynamic resampling of an original image stack to achieve a perpendicular cross-section of the scalae on all sections. Subsequently, perpendicular cross-section is being segmented using 2D active contours. The center of mass of the contour is extracted and used to predict further course of scalae centerline by Kalman filter. Cross-section contours are subsequently assembled to the total geometry model. This method has been applied to CT images, but we expect that it could be used for segmentation of strongly curved low-contrast tubular objects recorded with other tomography techniques.

Céline Bottier, Chiara Gabella, Benoît Vianay, Lara Buscemi, Ivo F. Sbalzarini, J-J Meister, A B Verkhovsky
Dynamic measurement of the height and volume of migrating cells by a novel fluorescence microscopy technique.
Lab on a chip, 11(22) 3855-3863 (2011)
PDF DOI

We propose a new technique to measure the volume of adherent migrating cells. The method is based on a negative staining where a fluorescent, non-cell-permeant dye is added to the extracellular medium. The specimen is observed with a conventional fluorescence microscope in a chamber of uniform height. Given that the fluorescence signal depends on the thickness of the emitting layer, the objects excluding the fluorescent dye (i.e., cells) appear dark, and the decrease of the fluorescent signal with respect to the background is expected to give information about the height and the volume of the object. Using a glass microfabricated pattern with steps of defined heights, we show that the drop in fluorescence intensity is indeed proportional to the height of the step and obtain calibration curves relating fluorescence intensity to height. The technique, termed the fluorescence displacement method, is further validated by comparing our measurements with the ones obtained by atomic force microscopy (AFM). We apply our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress. The fluorescence displacement technique allows fast and precise monitoring of cell height and volume, thus providing a valuable tool for characterizing the three-dimensional behaviour of migrating cells.

Nadine Muschalik, Elisabeth Knust
Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors
J Cell Sci, 124(21) 3715-3725 (2011)
PDF DOI

Photoreceptor morphogenesis in Drosophila requires remodelling of apico-basal polarity and adherens junctions (AJs), and includes cell shape changes, as well as differentiation and expansion of the apical membrane. The evolutionarily conserved transmembrane protein Crumbs (Crb) organises an apical membrane-associated protein complex that controls photoreceptor morphogenesis. Expression of the small cytoplasmic domain of Crb in crb mutant photoreceptor cells (PRCs) rescues the crb mutant phenotype to the same extent as the full-length protein. Here, we show that overexpression of the membrane-tethered cytoplasmic domain of Crb in otherwise wild-type photoreceptor cells has major effects on polarity and morphogenesis. Whereas early expression causes severe abnormalities in apico-basal polarity and ommatidial integrity, expression at later stages affects the shape and positioning of AJs. This result supports the importance of Crb for junctional remodelling during morphogenetic changes. The most pronounced phenotype observed upon early expression is the formation of ectopic apical membrane domains, which often develop into a complete second apical pole, including ectopic AJs. Induction of this phenotype requires members of the Par protein network. These data point to a close integration of the Crb complex and Par proteins during photoreceptor morphogenesis and underscore the role of Crb as an apical determinant.

Petra Stockinger, Jean-Léon Maître, Carl-Philipp Heisenberg
Defective neuroepithelial cell cohesion affects tangential branchiomotor neuron migration in the zebrafish neural tube
Development, 138(21) 4673-4683 (2011)
DOI

Facial branchiomotor neurons (FBMNs) in zebrafish and mouse embryonic hindbrain undergo a characteristic tangential migration from rhombomere (r) 4, where they are born, to r6/7. Cohesion among neuroepithelial cells (NCs) has been suggested to function in FBMN migration by inhibiting FBMNs positioned in the basal neuroepithelium such that they move apically between NCs towards the midline of the neuroepithelium instead of tangentially along the basal side of the neuroepithelium towards r6/7. However, direct experimental evaluation of this hypothesis is still lacking. Here, we have used a combination of biophysical cell adhesion measurements and high-resolution time-lapse microscopy to determine the role of NC cohesion in FBMN migration. We show that reducing NC cohesion by interfering with Cadherin 2 (Cdh2) activity results in FBMNs positioned at the basal side of the neuroepithelium moving apically towards the neural tube midline instead of tangentially towards r6/7. In embryos with strongly reduced NC cohesion, ectopic apical FBMN movement frequently results in fusion of the bilateral FBMN clusters over the apical midline of the neural tube. By contrast, reducing cohesion among FBMNs by interfering with Contactin 2 (Cntn2) expression in these cells has little effect on apical FBMN movement, but reduces the fusion of the bilateral FBMN clusters in embryos with strongly diminished NC cohesion. These data provide direct experimental evidence that NC cohesion functions in tangential FBMN migration by restricting their apical movement.

Wei Liu, Min Zi, Ronald Naumann, Susanne Ulm, Jiawei Jin, Domenico M Taglieri, Sukhpal Prehar, Junhong Gui, Hoyee Tsui, Rui-Ping Xiao, Ludwig Neyses, R John Solaro, Yunbo Ke, Elizabeth J Cartwright, Ming Lei, Xin Wang
Pak1 as a Novel Therapeutic Target for Antihypertrophic Treatment in the Heart.
Circulation, 124(24) 2702-2715 (2011)
DOI

Background-Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy.Methods and Results-Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation.Conclusions-These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Daniele Soroldoni, Andrew C. Oates
Live transgenic reporters of the vertebrate embryo's Segmentation Clock
Curr Opin Genet Dev, 21(5) 600-605 (2011)
DOI

Imaging rapidly changing gene expression during embryogenesis is a challenge for the development of probes and imaging techniques. The vertebrate Segmentation Clock is a genetic network that controls the subdivision of the elongating embryonic body axis into somites, the precursors of adult segmented structures, such as vertebrae. Because of its rapid oscillations, direct observation of gene expression in this system has proven difficult, and so is a benchmark for transgene design and imaging in vivo. Transgenic approaches using destabilized reporter cassettes in the mouse embryo have provided the first glimpses of this dynamic expression system. Nevertheless, improvements in temporal and spatial resolution, paired with the ability to make precise quantifications, will be necessary to connect observations and theory.

Claire Friel, Jonathon Howard
The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization.
EMBO J, 30(19) 3928-3939 (2011)
PDF DOI

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.

Ulrike Pässler, Margit Gruner, Sider Penkov, Teymuras V. Kurzchalia, Hans-Joachim Knölker
Synthesis of Ten Members of the Maradolipid Family; Novel Diacyltrehalose Glycolipids from Caenorhabditis elegans
SYNLETT, 22(17) 2482-2486 (2011)
DOI

Janina Görnemann, Charlotte Barrandon, Katja Hujer, Berthold Rutz, Guillaume Rigaut, Kimberly M. Kotovic, Céline Faux, Karla M. Neugebauer^#, Bertrand Séraphin^#
Cotranscriptional spliceosome assembly and splicing are independent of the Prp40p WW domain.
RNA, 17(12) 2119-2129 (2011)
PDF DOI

Complex cellular functions involve large networks of interactions. Pre-mRNA splicing and transcription are thought to be coupled by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). In yeast, the U1 snRNP subunit Prp40 was proposed to mediate cotranscriptional recruitment of early splicing factors through binding of its WW domains to the Pol II CTD. Here we investigate the role of Prp40 in splicing with an emphasis on the role of the WW domains, which might confer protein-protein interactions among the splicing and transcriptional machineries. Affinity purification revealed that Prp40 and Snu71 form a stable heterodimer that stably associates with the U1 snRNP only in the presence of Nam8, a known regulator of 5' splice site recognition. However, the Prp40 WW domains were dispensable for yeast viability. In their absence, no defect in splicing in vivo, U1 or U2 snRNP recruitment in vivo, or early splicing complex assembly in vitro was detected. We conclude that the WW domains of Prp40 do not mediate essential coupling between U1 snRNP and Pol II. Instead, delays in cotranscriptional U5 snRNP and Prp19 recruitment and altered spliceosome formation in vitro suggest that Prp40 WW domains assist in late steps of spliceosome assembly.

Helena Jambor, Christine Brunel, Anne Ephrussi
Dimerization of oskar 3' UTRs promotes hitchhiking for RNA localization in the Drosophila oocyte.
RNA, 17(12) 2049-2057 (2011)
PDF DOI

mRNA localization coupled with translational control is a highly conserved and widespread mechanism for restricting protein expression to specific sites within eukaryotic cells. In Drosophila, patterning of the embryo requires oskar mRNA transport to the posterior pole of the oocyte and translational repression prior to localization. oskar RNA splicing and the 3' untranslated region (UTR) are required for posterior enrichment of the mRNA. However, reporter RNAs harboring the oskar 3' UTR can localize by hitchhiking with endogenous oskar transcripts. Here we show that the oskar 3' UTR contains a stem-loop structure that promotes RNA dimerization in vitro and hitchhiking in vivo. Mutations in the loop that abolish in vitro dimerization interfere with reporter RNA localization, and restoring loop complementarity restores hitchhiking. Our analysis provides insight into the molecular basis of RNA hitchhiking, whereby localization-incompetent RNA molecules can become locally enriched in the cytoplasm, by virtue of their association with transport-competent RNAs.

Wilhelm Palm
The Lipoprotein Metabolism of Drosophila Melanogaster
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2011)

Sven Klose
Investigation of the signal peptide and establishment of a novel structure-function analysis of crumbs in drosophila melanogaster
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2011)

Jochen Rink^*^#, Hanh Thi-Kim Vu^*, Alejandro Sánchez Alvarado^#
The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling.
Development, 138(17) 3769-3780 (2011)
DOI

The maintenance of organs and their regeneration in case of injury are crucial to the survival of all animals. High rates of tissue turnover and nearly unlimited regenerative capabilities make planarian flatworms an ideal system with which to investigate these important processes, yet little is known about the cell biology and anatomy of their organs. Here we focus on the planarian excretory system, which consists of internal protonephridial tubules. We find that these assemble into complex branching patterns with a stereotyped succession of cell types along their length. Organ regeneration is likely to originate from a precursor structure arising in the blastema, which undergoes extensive branching morphogenesis. In an RNAi screen of signaling molecules, we identified an EGF receptor (Smed-EGFR-5) as a crucial regulator of branching morphogenesis and maintenance. Overall, our characterization of the planarian protonephridial system establishes a new paradigm for regenerative organogenesis and provides a platform for exploring its functional and evolutionary homologies with vertebrate excretory systems.

Hanchuan Peng, Fuhui Long, Ting Zhao, Eugene Myers
Proof-editing is the bottleneck of 3D neuron reconstruction: the problem and solutions.
Neuroinformatics, 9(2-3) 103-105 (2011)
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Jason Stumpff, Yaqing Du, Chauca A English, Zoltan Maliga, Michael Wagenbach, Charles L Asbury, Linda Wordeman^#, Ryoma Ohi^#
A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A.
Mol Cell, 43(5) 764-775 (2011)
PDF DOI

Metaphase chromosome positioning depends on Kif18A, a kinesin-8 that accumulates at and suppresses the dynamics of K-MT plus ends. By engineering Kif18A mutants that suppress MT dynamics but fail to concentrate at K-MT plus ends, we identify a mechanism that allows Kif18A to accumulate at K-MT plus ends to a level required to suppress chromosome movements. Enrichment of Kif18A at K-MT plus ends depends on its C-terminal tail domain, while the ability of Kif18A to suppress MT growth is conferred by the N-terminal motor domain. The Kif18A tail contains a second MT-binding domain that diffuses along the MT lattice, suggesting that it tethers the motor to the MT track. Consistently, the tail enhances Kif18A processivity and is crucial for it to accumulate at K-MT plus ends. The heightened processivity of Kif18A, conferred by its tail domain, thus promotes concentration of Kif18A at K-MT plus ends, where it suppresses their dynamics to control chromosome movements.

Anna Urbanska, Lukasz Sadowski, Yannis Kalaidzidis, Marta Miaczynska
Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment
Traffic, 12(9) 1227-1241 (2011)
DOI

APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.

Matilde Galli, Javier Muñoz, Vincent Portegijs, Mike Boxem, Stephan W. Grill, Albert J R Heck, Sander van den Heuvel
aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division.
Nat Cell Biol, 13(9) 1132-1138 (2011)
DOI

The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3-PAR-6-aPKC polarity complex. However, molecular interactions between polarity proteins and LIN-5-GPR-Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.

Jakub Sedzinski^*, Mate Biro^*, Annelie Oswald, Jean-Yves Tinevez, Guillaume Salbreux, Ewa Paluch
Polar actomyosin contractility destabilizes the position of the cytokinetic furrow.
Nature, 476(7361) 462-466 (2011)
DOI

Cytokinesis, the physical separation of daughter cells at the end of mitosis, requires precise regulation of the mechanical properties of the cell periphery. Although studies of cytokinetic mechanics mostly focus on the equatorial constriction ring, a contractile actomyosin cortex is also present at the poles of dividing cells. Whether polar forces influence cytokinetic cell shape and furrow positioning remains an open question. Here we demonstrate that the polar cortex makes cytokinesis inherently unstable. We show that limited asymmetric polar contractions occur during cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations, resulting in furrow displacement and aneuploidy. A theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. We further propose that membrane blebs, which commonly form at the poles of dividing cells and whose role in cytokinesis has long been enigmatic, stabilize cell shape by acting as valves releasing cortical contractility. Our findings reveal an inherent instability in the shape of the dividing cell and unveil a novel, spindle-independent mechanism ensuring the stability of cleavage furrow positioning.

Ivana Viktorinova, Lucie Kucerova, Marta Bohmova, Ian Henry, Marek Jindra, Petr Dolezal, Martina Zurovcova, Michal Zurovec
Characterization of two closely related α-amylase paralogs in the bark beetle, Ips typographus (L.).
Arch Insect Biochem Physiol, 77(4) 179-198 (2011)
DOI

Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding α-amylase in I. typographus were characterized and named AmyA and AmyB. Both α-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two α-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA.

Cihan Erkut, Sider Penkov, Hassan Khesbak, Daniela Vorkel, Jean-Marc Verbavatz, Karim Fahmy, Teymuras V. Kurzchalia
Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation.
Curr Biol, 21(15) 1331-1336 (2011)
DOI

Water is essential for life on Earth. In its absence, however, some organisms can interrupt their life cycle and temporarily enter an ametabolic state, known as anhydrobiosis [1]. It is assumed that sugars (in particular trehalose) are instrumental for survival under anhydrobiotic conditions [2]. However, the role of trehalose remained obscure because the corresponding evidence was purely correlative and based mostly on in vitro studies without any genetic manipulations of trehalose metabolism. In this study, we used C. elegans as a genetic model to investigate molecular mechanisms of anhydrobiosis. We show that the C. elegans dauer larva is a true anhydrobiote: under defined conditions it can survive even after losing 98% of its body water. This ability is correlated with a several fold increase in the amount of trehalose. Mutants unable to synthesize trehalose cannot survive even mild dehydration. Light and electron microscopy indicate that one of the major functions of trehalose is the preservation of membrane organization. Fourier-transform infrared spectroscopy of whole worms suggests that this is achieved by preserving homogeneous and compact packing of lipid acyl chains. By means of infrared spectroscopy, we can now distinguish a "dry, yet alive" larva from a "dry and dead" one.

Bastian Thaa, Ilya Levental, Andreas Herrmann, Michael Veit
Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation
Biochem J, 437(3) 389-397 (2011)
PDF DOI

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.

Nicola Bauer, Michaela Wilsch-Bräuninger, Jana Karbanová, Ana-Violeta Fonseca, Doreen Strauss, Daniel Freund, Christoph Thiele, Wieland B. Huttner, Martin Bornhäuser, Denis Corbeil
Haematopoietic stem cell differentiation promotes the release of prominin-1/CD133-containing membrane vesicles-a role of the endocytic-exocytic pathway
EMBO Mol Med, 3(7) 398-409 (2011)
DOI

The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein-lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.

Ivana Viktorinová, Len M Pismen, Benoit Aigouy, Christian Dahmann
Modelling planar polarity of epithelia: the role of signal relay in collective cell polarization
J R Soc Interface, 8(60) 1059-1063 (2011)
DOI

Collective cell polarization is an important characteristic of tissues. Epithelia commonly display cellular structures that are polarized within the plane of the tissue. Establishment of this planar cell polarity requires mechanisms that locally align polarized structures between neighbouring cells, as well as cues that provide global information about alignment relative to an axis of a tissue. In the Drosophila ovary, the cadherin Fat2 is required to orient actin filaments located at the basal side of follicle cells perpendicular to the long axis of the egg chamber. The mechanisms directing this orientation of actin filaments, however, remain unknown. Here we show, using genetic mosaic analysis, that fat2 is not essential for the local alignment of actin filaments between neighbouring cells. Moreover, we provide evidence that Fat2 is involved in the propagation of a cue specifying the orientation of actin filaments relative to the tissue axis. Monte Carlo simulations of actin filament orientation resemble the results of the genetic mosaic analysis, if it is assumed that a polarity signal can propagate from a signal source only through a connected chain of wild-type cells. Our results suggest that Fat2 is required for propagating global polarity information within the follicle epithelium through direct cell-cell contact. Our computational model might be more generally applicable to study collective cell polarization in tissues.

Shirin Pocha, Thomas Wassmer^#, Christian Niehage, Bernard Hoflack, Elisabeth Knust^#
Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs
Curr Biol, 21(13) 1111-1117 (2011)
PDF DOI

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

Zoltan Ungvari^*, Iain Ridgway^*, Eva E R Philipp^*, Courtney M Campbell, Philip McQuary, Tracy Chow, Miguel Coelho, Elizabeth S Didier, Sara Gelino, Marissa A Holmbeck, Insil Kim, Erik Levy, Danuta Sosnowska, William E Sonntag, Steven N Austad, Anna Csiszar
Extreme longevity is associated with increased resistance to oxidative stress in Arctica islandica, the longest-living non-colonial animal
J Gerontol A Biol Sci Med Sci, 66(7) 741-750 (2011)
DOI

We assess whether reactive oxygen species production and resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the ocean quahog Arctica islandica. We tested this hypothesis by comparing reactive oxygen species production, resistance to oxidative stress, antioxidant defenses, and protein damage elimination processes in long-lived A islandica with the shorter-lived hard clam, Mercenaria mercenaria. We compared baseline biochemical profiles, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Our data support the premise that extreme longevity in A islandica is associated with an attenuated cellular reactive oxygen species production. The observation of reduced protein carbonyl concentration in A islandica gill tissue compared with M mercenaria suggests that reduced reactive oxygen species production in long-living bivalves is associated with lower levels of accumulated macromolecular damage, suggesting cellular redox homeostasis may determine life span. Resistance to aging at the organismal level is often reflected in resistance to oxidative stressors at the cellular level. Following TBHP exposure, we observed not only an association between longevity and resistance to oxidative stress-induced mortality but also marked resistance to oxidative stress-induced cell death in the longer-living bivalves. Contrary to some expectations from the oxidative stress hypothesis, we observed that A islandica exhibited neither greater antioxidant capacities nor specific activities than in M mercenaria nor a more pronounced homeostatic antioxidant response following TBHP exposure. The study also failed to provide support for the exceptional longevity of A islandica being associated with enhanced protein recycling. Our findings demonstrate an association between longevity and resistance to oxidative stress-induced cell death in A islandica, consistent with the oxidative stress hypothesis of aging and provide justification for detailed evaluation of pathways involving repair of free radical-mediated macromolecular damage and regulation of apoptosis in the world's longest-living non-colonial animal.

Mohammed Mahamdeh, Citlali Perez Campos, Erik Schäffer
Under-filling trapping objectives optimizes the use of the available laser power in optical tweezers.
Opt express, 19(12) 11759-11768 (2011)

For optical tweezers, especially when used in biological studies, optimizing the trapping efficiency reduces photo damage or enables the generation of larger trapping forces. One important, yet not-well understood, tuning parameter is how much the laser beam needs to be expanded before coupling it into the trapping objective. Here, we measured the trap stiffness for 0.5-2 μm-diameter microspheres for various beam expansions. We show that the highest overall trapping efficiency is achieved by slightly under-filling a high-numerical aperture objective when using microspheres with a diameter corresponding to about the trapping-laser wavelength in the medium. The optimal filling ratio for the lateral direction depended on the microsphere size, whereas for the axial direction it was nearly independent. Our findings are in agreement with Mie theory calculations and suggest that apart from the choice of the optimal microsphere size, slightly under-filling the objective is key for the optimal performance of an optical trap.

Jonathon Howard^#, Stephan W. Grill^#, Justin Bois^#
Turing's next steps: the mechanochemical basis of morphogenesis.
Nat Rev Mol Cell Biol, 12(6) 400-406 (2011)
PDF DOI

Nearly 60 years ago, Alan Turing showed theoretically how two chemical species, termed morphogens, diffusing and reacting with each other can generate spatial patterns. Diffusion plays a crucial part in transporting chemical signals through space to establish the length scale of the pattern. When coupled to chemical reactions, mechanical processes - forces and flows generated by motor proteins - can also define length scales and provide a mechanochemical basis for morphogenesis.

Martin Loose, Karsten Kruse, Petra Schwille
Protein self-organization: lessons from the min system.
Ann Rev Biophys, 40 315-336 (2011)
DOI

One of the most fundamental features of biological systems is probably their ability to self-organize in space and time on different scales. Despite many elaborate theoretical models of how molecular self-organization can come about, only a few experimental systems of biological origin have so far been rigorously described, due mostly to their inherent complexity. The most promising strategy of modern biophysics is thus to identify minimal biological systems showing self-organized emergent behavior. One of the best-understood examples of protein self-organization, which has recently been successfully reconstituted in vitro, is represented by the oscillations of the Min proteins in Escherichia coli. In this review, we summarize the current understanding of the mechanism of Min protein self-organization in vivo and in vitro. We discuss the potential of the Min oscillations to sense the geometry of the cell and suggest that spontaneous protein waves could be a general means of intracellular organization. We hypothesize that cooperative membrane binding and unbinding, e.g., as an energy-dependent switch, may act as an important regulatory mechanism for protein oscillations and pattern formation in the cell.

Owen Randlett, Caren Norden, William A Harris
The vertebrate retina: a model for neuronal polarization in vivo.
Dev Neurobiol, 71(6) 567-583 (2011)
PDF DOI

The vertebrate retina develops rapidly from a proliferative neuroepithelium into a highly ordered laminated structure, with five distinct neuronal cell types. Like all neurons, these cells need to polarize in appropriate orientations order integrate their neuritic connections efficiently into functional networks. Its relative simplicity, amenability to in vivo imaging and experimental manipulation, as well as the opportunity to study varied cell types within a single tissue, make the retina a powerful model to uncover how neurons polarize in vivo. Here we review the progress that has been made thus far in understanding how the different retinal neurons transform from neuroepithelial cells into mature neurons, and how the orientation of polarization may be specified by a combination of pre-established intrinsic cellular polarity set up within neuroepithelial cells, and extrinsic cues acting upon these differentiating neurons.

Richard H Row, Jean-Léon Maître, Benjamin L Martin, Petra Stockinger, Carl-Philipp Heisenberg, David Kimelman
Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail
Dev Biol, 354(1) 102-110 (2011)
DOI

The process of gastrulation is highly conserved across vertebrates on both the genetic and morphological levels, despite great variety in embryonic shape and speed of development. This mechanism spatially separates the germ layers and establishes the organizational foundation for future development. Mesodermal identity is specified in a superficial layer of cells, the epiblast, where cells maintain an epithelioid morphology. These cells involute to join the deeper hypoblast layer where they adopt a migratory, mesenchymal morphology. Expression of a cascade of related transcription factors orchestrates the parallel genetic transition from primitive to mature mesoderm. Although the early and late stages of this process are increasingly well understood, the transition between them has remained largely mysterious. We present here the first high resolution in vivo observations of the blebby transitional morphology of involuting mesodermal cells in a vertebrate embryo. We further demonstrate that the zebrafish spadetail mutation creates a reversible block in the maturation program, stalling cells in the transition state. This mutation creates an ideal system for dissecting the specific properties of cells undergoing the morphological transition of maturing mesoderm, as we demonstrate with a direct measurement of cell-cell adhesion.

Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication.
J Cell Biol, 193(6) 995-1007 (2011)
DOI

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2-cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

Christine Moessinger, Lars Kuerschner, Johanna Spandl, Andrej Shevchenko, Christoph Thiele
Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine.
J Biol Chem, 286(24) 21330-21339 (2011)
DOI

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.

Daniela Roellig, Luis G. Morelli, Saul Ares, Frank Jülicher, Andrew C. Oates
SnapShot: the segmentation clock
Cell, 145(5) Art. No. 800-800.e1 (2011)
DOI

Magali Prigent, Emmanuelle Boy-Marcotte, Laurent Chesneau, Kimberley Gibson, Sophie Dupré-Crochet, Hélène Tisserand, Jean-Marc Verbavatz, Marie-Hélène Cuif
The RabGAP proteins Gyp5p and Gyl1p recruit the BAR domain protein Rvs167p for polarized exocytosis.
Traffic, 12(8) 1084-1097 (2011)
DOI

The RabGAP proteins Gyp5p and Gyl1p are involved in the control of polarized exocytosis at the small-bud stage in S. cerevisiae. Both Gyp5p and Gyl1p interact with the N-BAR domain protein Rvs167p, but the biological function of this interaction is unclear. We show here that Gyp5p and Gyl1p recruit Rvs167p to the small-bud tip, where it plays a role in polarized exocytosis. In gyp5Δgyl1Δ cells, Rvs167p is not correctly localized to the small-bud tip. Both a P473L mutation in the SH3 domain of Rvs167p and deletion of the proline-rich regions of Gyp5p and Gyl1p disrupt the interaction of Rvs167p with Gyp5p and Gyl1p and impair the localization of Rvs167p to the tips of small buds. We provide evidence for the accumulation of secretory vesicles in small buds of rvs167Δ cells and for defective Bgl2p secretion in rvs167Δ cultures enriched in small-budded cells at 13(°) C, implicating Rvs167p in polarized exocytosis. Moreover, both the accumulation of secretory vesicles in Rvs167p P473L cells cultured at 13(°) C and secretion defects in cells producing Gyp5p and Gyl1p without proline-rich regions strongly suggest that the function of Rvs167p in exocytosis depends on its ability to interact with Gyp5p and Gyl1p.

Kamil A Lipinski, Olga Puchta, Vineeth Surandranath, Marek Kudla, Pawel Golik
Revisiting the Yeast PPR Proteins - Application of an Iterative Hidden Markov Model Algorithm Reveals New Members of the Rapidly Evolving Family.
Mol Biol Evol, 28(10) 2935-2948 (2011)
PDF DOI

Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family, and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly to mitochondria and chloroplasts, and many were shown to modulate organellar genome expression on the posttranscriptional level. While the genomes of land plants encode hundreds of PPR proteins, only a few have been identified in Fungi and Metazoa. As the current PPR motif profiles are built mainly on the basis of the predominant plant sequences, they are unlikely to be optimal for detecting fungal and animal members of the family, and many putative PPR proteins in these genomes may remain undetected. In order to verify this hypothesis we designed a HMM-based bioinformatic tool called SCIPHER (Supervised Clustering-based Iterative Phylogenetic Hidden Markov Model algorithm for the Evaluation of tandem Repeat motif families) using sequence data from orthologous clusters from available yeast genomes. This approach allowed us to assign twelve new proteins in S. cerevisiae to the PPR family. Similarly, in other yeast species we obtained a five-fold increase in the detection of PPR motifs, compared to the previous tools. All the newly identified S. cerevisiae PPR proteins localize in the mitochondrion and are a part of the RNA processing interaction network. Furthermore, the yeast PPR proteins seem to undergo an accelerated divergent evolution. Analysis of single and double amino acid substitutions in the Dmr1 protein of S. cerevisiae suggests that cooperative interactions between motifs and pseudoreversion could be the force driving this rapid evolution.

Nathan Goehring, Carsten Hoege, Stephan W. Grill^#, Anthony A. Hyman^#
PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos.
J Cell Biol, 193(3) 583-594 (2011)
DOI

Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.

Corentin Laulier, Aurélia Barascu, Josée Guirouilh-Barbat, Gaëlle Pennarun, Catherine Le Chalony, François Chevalier, Gaëlle Palierne, Pascale Bertrand, Jean-Marc Verbavatz, Bernard S Lopez
Bcl-2 Inhibits Nuclear Homologous Recombination by Localizing BRCA1 to the Endomembranes.
Cancer Res, 71(10) 3590-3602 (2011)
PDF DOI

Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability. Cancer Res; 71(10); 3590-602. ©2011 AACR.

Alex T. Kalinka, Pavel Tomancak
linkcomm: an R package for the generation, visualization, and analysis of link communities in networks of arbitrary size and type.
Bioinformatics, 27(14) 2011-2012 (2011)
PDF DOI

SUMMARY: An essential element when analysing the structure, function, and dynamics of biological networks is the identification of communities of related nodes. An algorithm proposed recently enhances this process by clustering the links between nodes, rather than the nodes themselves, thereby allowing each node to belong to multiple overlapping or nested communities. The R package "linkcomm" implements this algorithm and extends it in several aspects: i) the clustering algorithm handles networks that are weighted, directed, or both weighted and directed; ii) several visualization methods are implemented that facilitate the representation of the link communities and their relationships; iii) a suite of functions are included for the downstream analysis of the link communities including novel community-based measures of node centrality; iv) the main algorithm is written in C++ and designed to handle networks of any size; v) several clustering methods are available for networks that can be handled in memory, and the number of communities can be adjusted by the user. AVAILABILITY: The program is freely available from the Comprehensive R Archive Network (http://cran.r-project.org/) under the terms of the GNU General Public License (version 2 or later). CONTACT: kalinka@mpi-cbg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Giovanni Marsico
Analysis of the interplay between endocytosis and cell morphology through the development of computational methods
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2011)

Marcus Jahnel, Martin Behrndt, Anita Jannasch, Erik Schäffer, Stephan W. Grill
Measuring the complete force field of an optical trap.
Opt Lett, 36(7) 1260-1262 (2011)
DOI

The use of optical traps to measure or apply forces on the molecular level requires a precise knowledge of the trapping force field. Close to the trap center, this field is typically approximated as linear in the displacement of the trapped microsphere. However, applications demanding high forces at low laser intensities can probe the light-microsphere interaction beyond the linear regime. Here, we measured the full nonlinear force and displacement response of an optical trap in two dimensions using a dual-beam optical trap setup with back-focal-plane photodetection. We observed a substantial stiffening of the trap beyond the linear regime that depends on microsphere size, in agreement with Mie theory calculations. Surprisingly, we found that the linear detection range for forces exceeds the one for displacement by far. Our approach allows for a complete calibration of an optical trap.

André Nadler, Christian Koch, Florian Brodhun, Jürgen A. Wehland, Kai Tittmann, Ivo Feussner, Ulf Diederichsen
Influence of substrate dideuteration on the reaction of the bifunctional heme enzyme psi factor producing oxygenase A (PpoA).
Chembiochem, 12(5) 728-737 (2011)
DOI

PpoA is a bifunctional enzyme that catalyzes the dioxygenation of unsaturated C18 fatty acids. The products of this reaction are termed psi factors and have been shown to play a crucial role in conferring a balance between sexual and asexual spore development as well as production of secondary metabolites in the fungus Aspergillus nidulans. Studies on the reaction mechanism revealed that PpoA uses two different heme domains to catalyze two subsequent reactions. Initially, the fatty acid substrate is dioxygenated at C8, yielding an 8-hydroperoxy fatty acid at the N-terminal domain. This reaction is catalyzed by a peroxidase/dioxygenase-type domain that exhibits many similarities to prostaglandin H2 synthases and involves a stereospecific homolytic hydrogen abstraction from C8 of the substrate. The C terminus harbors a heme thiolate P450 domain in which rearrangement of the 8-hydroperoxide to the final product, a 5,8-dihydroxy fatty acid, takes place. To obtain further information about the intrinsic kinetics and reaction mechanism of PpoA, we synthesized C5-dideutero- and C8-dideutero-oleic acid by a novel protocol that offers a straightforward synthesis without employing the toxic additive hexamethylphosphoramide (HMPA) during CC coupling reactions or mercury salts upon thioketal deprotection. These deuterated fatty acids were then employed for kinetic analysis under multiple-turnover conditions. The results indicate that the hydrogen abstraction at C8 is the rate-determining step of the overall reaction because we observed a KIE (V(H) /V(D) ) of ∼33 at substrate saturation that suggests extensive nuclear tunneling contributions for hydrogen transfer. Deuteration of the substrate at C5, however, had little effect on V(H) /V(D) but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate.

Monika Sokolowska, Honorata Czapinska, Matthias Bochtler
Hpy188I-DNA pre- and post-cleavage complexes-snapshots of the GIY-YIG nuclease mediated catalysis.
Nucleic Acids Res, 39(4) 1554-1564 (2011)
PDF DOI

The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.

Anja Nitzsche
Role of cohesin in the maintenance of embryonic stem cell identitiy
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2011)

Ewa Missol-Kolka, Jana Karbanová, Peggy Janich, Michael Haase, Christine A. Fargeas, Wieland B. Huttner, Denis Corbeil
Prominin-1 (CD133) is not restricted to stem cells located in the basal compartment of murine and human prostate.
Prostate, 71(3) 254-267 (2011)
PDF DOI

BACKGROUND: Rodent and human prominin-1 are expressed in numerous adult epithelia and somatic stem cells. A report has shown that human PROMININ-1 carrying the AC133 epitope can be used to identify rare prostate basal stem cells (Richardson et al., J Cell Sci 2004; 117:3539-3545). Here we re-investigated its general expression in male reproductive tract including mouse and human prostate and in prostate cancer samples using various anti-prominin-1 antibodies. METHODS: The expression was monitored by immunohistochemistry and blotting. Murine tissues were stained with 13A4 monoclonal antibody (mAb) whereas human samples were examined either with the AC133 mAb recognizing the AC133 glycosylation-dependent epitope or 80B258 mAb directed against the PROMININ-1 polypeptide. RESULTS: Mouse prominin-1 was detected at the apical domain of epithelial cells of ductus deferens, seminal vesicles, ampullary glands, and all prostatic lobes. In human prostate, immunoreactivity for 80B258, but not AC133 was revealed at the apical side of some epithelial (luminal) cells, in addition to the minute population of AC133/80B258-positive cells found in basal compartment. Examination of prostate adenocarcinoma revealed the absence of 80B258 immunoreactivity in the tumor regions. However, it was found to be up-regulated in luminal cells in the vicinity of the cancer areas. CONCLUSIONS: Mouse prominin-1 is widely expressed in prostate whereas in human only some luminal cells express it, demonstrating nevertheless that its expression is not solely associated with basal stem cells. In pathological samples, our pilot evaluation shows that PROMININ-1 is down-regulated in the cancer tissues and up-regulated in inflammatory regions. Prostate © 2010 Wiley-Liss, Inc.

Andrew C. Oates
What's all the noise about developmental stochasticity?
Development, 138(4) 601-607 (2011)
DOI

In October 2010, researchers from diverse backgrounds collided at the historic Cumberland Lodge (Windsor, UK) to discuss the role of randomness in cell and developmental biology. Organized by James Briscoe and Alfonso Marinez-Arias, The Company of Biologists' workshop was the latest in a series of meetings aimed at encouraging interdisciplinary interactions between biologists. This aim was reflected in talks at this workshop that ranged from the tissue to the cellular scale, and that integrated experimental and theoretical approaches to examining stochastic behavior in diverse systems.

Benoit Aigouy, Raphael Etournay, Andreas Sagner, Douglas B Staple, Reza Farhadifar, Jens-Christian Röper, Suzanne Eaton, Frank Jülicher
[Cell flow reorients planar polarity in the wing of Drosophila]
Med Sci (Paris), 27(2) 117-119 (2011)
DOI

William M Behnke-Parks, Jeremie Vendome, Barry Honig, Zoltan Maliga, Carolyn Moores, Steven S Rosenfeld
Loop L5 acts as a conformational latch in the mitotic kinesin Eg5.
J Biol Chem, 286(7) 5242-5253 (2011)
PDF DOI

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an α-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotide-induced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with α3 and in so doing controls the speed of movement and force generation in kinesin motors.

Martin P Stewart, Jonne H. Helenius, Yusuke Toyoda, Subramanian Ramanathan, Daniel J. Müller, Anthony A. Hyman
Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding.
Nature, 469(7329) 226-230 (2011)
PDF DOI

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.

Uenal Coskun, Kai Simons
Cell Membranes: The Lipid Perspective
Structure, 19(11) 1543-1548 (2011)
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Danming Tang, Yi Xiang, Stefano de Renzis, Jochen Rink, Gen Zheng, Marino Zerial, Yanzhuang Wang
The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle.
Nat Commun, 2 Art. No. 501 (2011)
PDF DOI

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.

Falko Riedel, Daniela Vorkel, Suzanne Eaton
Megalin-dependent yellow endocytosis restricts melanization in the Drosophila cuticle.
Development, 138(1) 149-158 (2011)
DOI

The cuticular exoskeleton of arthropods is a composite material comprising well-separated layers that differ in function and molecular constituents. Epidermal cells secrete these layers sequentially, synthesizing components of distal cuticle layers before proximal ones. Could the order of synthesis and secretion be sufficient to account for the precision with which cuticle components localize to specific layers? We addressed this question by studying the spatial restriction of melanization in the Drosophila wing. Melanin formation is confined to a narrow layer within the distal procuticle. Surprisingly, this tight localization depends on the multi-ligand endocytic receptor Megalin (Mgl). Mgl acts, in part, by promoting endocytic clearance of Yellow. Yellow is required for black melanin formation, and its synthesis begins as cuticle is secreted. Near the end of cuticle secretion, its levels drop precipitously by a mechanism that depends on Mgl and Rab5-dependent endocytosis. In the absence of Mgl, Yellow protein persists at higher levels and melanin granules form ectopically in more proximal layers of the procuticle. We propose that the tight localization of the melanin synthesis machinery to the distal procuticle depends not only on the timing of its synthesis and secretion, but also on the rapid clearance of these components before synthesis of subsequent cuticle layers.

Moritz Kreysing
Optics of the retina: novel tools new insights
Ph.D. Thesis, University of Cambridge, Cambridge, United Kingdom (2011)

Andrew Clark^#, Ewa Paluch^#
Mechanics and regulation of cell shape during the cell cycle.
In: Cell Cycle in Development. Results and Problems in Cell Differentiation, 53., Berlin;Heidelberg, Springer (2011), 31-73 Ch. 3
DOI

Many cell types undergo dramatic changes in shape throughout the cell cycle. For individual cells, a tight control of cell shape is crucial during cell division, but also in interphase, for example during cell migration. Moreover, cell cycle-related cell shape changes have been shown to be important for tissue morphogenesis in a number of developmental contexts. Cell shape is the physical result of cellular mechanical properties and of the forces exerted on the cell. An understanding of the causes and repercussions of cell shape changes thus requires knowledge of both the molecular regulation of cellular mechanics and how specific changes in cell mechanics in turn effect global shape changes. In this chapter, we provide an overview of the current knowledge on the control of cell morphology, both in terms of general cell mechanics and specifically during the cell cycle.

Ravi K R Marreddy, Joao P C Pinto, Justina C Wolters, Eric R Geertsma, Fabrizia Fusetti, Hjalmar P Permentier, Oscar P Kuipers, Jan Kok, B Poolman
The response of Lactococcus lactis to membrane protein production.
PLoS ONE, 6(8) Art. No. e24060 (2011)
Open Access DOI

The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach.

Malgorzata Firczuk^*, Marek Wojciechowski^*, Honorata Czapinska, Matthias Bochtler
DNA intercalation without flipping in the specific ThaI-DNA complex.
Nucleic Acids Res, 39(2) 744-754 (2011)
PDF DOI

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.

Claire Friel, Clive R Bagshaw, Jonathon Howard
Analysing the ATP turnover cycle of microtubule motors.
Methods Mol Biol, 777 177-192 (2011)
PDF DOI

Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5'-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubule interaction. In this chapter, a number of methods are outlined by which the ATP turnover cycle of a kinesin can be analysed with a particular focus on the use of fluorescently labelled ATP and ADP analogues as a means of isolating individual steps in the cycle. By analysing the ATP turnover cycle of a kinesin, both in solution and in the presence of microtubules, the change in nucleotide state triggered upon microtubule binding can be determined. This provides information vital to understanding the coupling of the chemical and mechanical cycles that is integral to the action of members of the kinesin superfamily.

Maria L Begasse, Anthony A. Hyman
The first cell cycle of the Caenorhabditis elegans embryo: spatial and temporal control of an asymmetric cell division.
In: Cell Cycle in Development. Results and Problems in Cell Differentiation, 53., Berlin;Heidelberg, Springer (2011), 109-133 Ch. 6
PDF DOI

Throughout the development of an organism, it is essential that the cell cycle machinery is fine-tuned to generate cells of different fate. A series of asymmetric cell divisions leads to lineage specification. The Caenorhabditis elegans embryo is an excellent system to study various aspects of the early embryonic cell cycle. The invariant nature of the rapid cell divisions is the key feature for studying the effects of small perturbations to a complex process such as the cell cycle. The thorough characterization of the asymmetric first cell division of the C. elegans embryo has given great insight on how the oscillations of the cell cycle coordinate with the cytoplasmic rearrangements that ultimately lead to two developmentally distinct daughter cells.

Xin Liang, Johnson Madrid, Henri Saleh, Jonathon Howard
NOMPC, a member of the TRP channel family, localizes to the tubular body and distal cilium of Drosophila campaniform and chordotonal receptor cells.
Cytoskeleton, 68(1) 1-7 (2011)
PDF DOI

Mechanoreception underlies the senses of touch, hearing and balance. An early event in mechanoreception is the opening of ion channels in response to mechanical force impinging on the cell. Here, we report antibody localization of NOMPC, a member of the transient receptor potential (TRP) ion channel family, to the tubular body of campaniform receptors in the halteres and to the distal regions of the cilia of chordotonal neurons in Johnston's organ, the sound-sensing organ of flies. Because NOMPC has been shown to be associated with the mechanotransduction process, our studies suggest that the transduction apparatus in both types of sensory cells is located in regions where a specialized microtubule-based cytoskeleton is in close proximity to an overlying cuticular structure. This localization suggests a transmission route of the mechanical stimulus to the cell. Furthermore, the commonality of NOMPC locations in the two structurally different receptor types suggests a conserved transduction apparatus involving both the intracellular cytoskeleton and the extracellular matrix.

Anja Nitzsche^*, Maciej Paszkowski-Rogacz^*, Filomena Matarese, Eva M Janssen-Megens, Nina C Hubner, Herbert Schulz, Ingrid de Vries, Li Ding, Norbert Huebner, Matthias Mann, Hendrik G Stunnenberg, Frank Buchholz
RAD21 cooperates with pluripotency transcription factors in the maintenance of embryonic stem cell identity
PLoS ONE, 6(5) Art. No. e19470 (2011)
DOI

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21--pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

Nasreddin Abolmaali, Robert Haase, A Koch, Daniel Zips, J Steinbach, Michael Baumann, Jörg Kotzerke, Klaus Zöphel
Two or four hour [¹⁸F]FMISO-PET in HNSCC. When is the contrast best?
Nuklearmedizin, 50(1) 22-27 (2011)
DOI

[¹⁸F]Fluoromisonidazole positron emission tomography (FMISO-PET) is a non invasive imaging technique that can assist detecting intra tumour regions of hypoxia. FMISO-PET evinces comparatively low signal-to-noise-ratio (SNR) and may be acquired dynamically or after different uptake times post injection (p.i.). The aim of this study was to identify, if static images acquired two hours (MISO2) or four hours (MISO4) p.i. reveal higher contrast.

María E Primo, Jean Jakoncic, Martín E Noguera, Valeria A Risso, Laura Sosa, Mauricio P Sica, Michele Solimena, Edgardo Poskus, Mario R Ermácora
Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain
PLoS ONE, 6(9) Art. No. e24191 (2011)
DOI

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

Rajesh Ramaswamy, Ivo F. Sbalzarini
A partial-propensity formulation of the stochastic simulation algorithm for chemical reaction networks with delays
J Chem Phys, 134(1) Art. No. 014106 (2011)
PDF DOI

Several real-world systems, such as gene expression networks in biological cells, contain coupled chemical reactions with a time delay between reaction initiation and completion. The non-Markovian kinetics of such reaction networks can be exactly simulated using the delay stochastic simulation algorithm (dSSA). The computational cost of dSSA scales with the total number of reactions in the network. We reduce this cost to scale at most with the smaller number of species by using the concept of partial reaction propensities. The resulting delay partial-propensity direct method (dPDM) is an exact dSSA formulation for well-stirred systems of coupled chemical reactions with delays. We detail dPDM and present a theoretical analysis of its computational cost. Furthermore, we demonstrate the implications of the theoretical cost analysis in two prototypical benchmark applications. The dPDM formulation is shown to be particularly efficient for strongly coupled reaction networks, where the number of reactions is much larger than the number of species.

Elisabeth Knust
An interview with Elisabeth Knust: President of the German Society for Developmental Biology
Development, 138(12) 2399-2400 (2011)
PDF DOI

Zlatko Smole, Nela Nikolic, Fran Supek, Tomislav Šmuc, Ivo F. Sbalzarini, Anita Krisko
Proteome sequence features carry signatures of the environmental niche of prokaryotes.
BMC Evol Biol, 11 26-26 (2011)
Open Access PDF DOI

Prokaryotic environmental adaptations occur at different levels within cells to ensure the preservation of genome integrity, proper protein folding and function as well as membrane fluidity. Although specific composition and structure of cellular components suitable for the variety of extreme conditions has already been postulated, a systematic study describing such adaptations has not yet been performed. We therefore explored whether the environmental niche of a prokaryote could be deduced from the sequence of its proteome. Finally, we aimed at finding the precise differences between proteome sequences of prokaryotes from different environments.

Ömer Demirel, Birte Schrader, Ivo F. Sbalzarini
A parallel particle method for solving the EEG source localization forward problem
In: Proc. 6th Intl. Symp. Health Informatics and Bioinformatics (HIBIT) (2011), Piscataway, N.J., IEEE (2011), 154-158
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Jonathon Howard
Motor proteins as nanomachines : the roles of thermal fluctuations in generating force and motion
In: Biological physics : Poincaré Seminar 2009. (Eds.) Bertrand Duplantier Progress in mathematical physics ; 60., Basel, Birkhäuser (2011), 47-59
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Motor proteins are enzymes that convert chemical energy derived from the hydrolysis of a small molecule called ATP into mechanical work used to power directed movement along cytoskeletal filaments inside cells. Motor proteins have essential biological functions such as driving the contraction of muscle, the beating of sperm and cilia, and the transport of intracellular cargoes. Motor proteins are also interesting from a physical point of view because they do what no man-made engines do: they transduce chemical energy directly to mechanical work without using heat or electrical energy as an intermediate. A central issue in the mechanism of this chemomechanical transduction by motor proteins concerns the roles played by thermal fluctuations, diffusion and Brownian motion. In this lecture I discuss several molecular models for motor proteins, including so-called ratchet models, and compare predictions of these models to experimental results for the microtubule-based motor protein kinesin. I argue that kinesin, which has two motor domains or “heads,” walks using a “hand-over-hand” mechanism such that at least one head is bound to the microtubule. Diffusion likely plays an essential role by facilitating the search of the unbound head for the next binding site, a distance 8 nm away. During this diffusive phase, the bound head supports the load ensuring that forward motion can still take place even against loads up to several piconewtons.

Konstantinos Tripsianes, Tobias Madl, Martin Machyna, Dimitrios Fessas, Clemens Englbrecht, Utz Fischer, Karla M. Neugebauer, Michael Sattler
Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins.
Nat Struct Mol Biol, 18(12) 1414-1420 (2011)
PDF DOI

Arginine dimethylation plays critical roles in the assembly of ribonucleoprotein complexes in pre-mRNA splicing and piRNA pathways. We report solution structures of SMN and SPF30 Tudor domains bound to symmetric and asymmetric dimethylated arginine (DMA) that is inherent in the RNP complexes. An aromatic cage in the Tudor domain mediates dimethylarginine recognition by electrostatic stabilization through cation-π interactions. Distinct from extended Tudor domains, dimethylarginine binding by the SMN and SPF30 Tudor domains is independent of proximal residues in the ligand. Yet, enhanced micromolar affinities are obtained by external cooperativity when multiple methylation marks are presented in arginine- and glycine-rich peptide ligands. A hydrogen bond network in the SMN Tudor domain, including Glu134 and a tyrosine hydroxyl of the aromatic cage, enhances cation-π interactions and is impaired by a mutation causing an E134K substitution associated with spinal muscular atrophy. Our structural analysis enables the design of an optimized binding pocket and the prediction of DMA binding properties of Tudor domains.

Yehuda Brody, Noa Neufeld, Nicole Bieberstein, Sebastien Z Causse, Eva-Maria Böhnlein, Karla M. Neugebauer, Xavier Darzacq, Yaron Shav-Tal
The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
PLoS Biol, 9(1) Art. No. e1000573 (2011)
Open Access PDF DOI

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.

Wayne Pereanu, Parvez Ahammad^#, Arnim Jenett^#, Eugene W Myers^#, James W Truman^#
Investigating Neural Circuitry Using a Compartment- Level Connectivity Map of the Adult Drosophila Brain
PLoS Biol, 9(1) 1-20 (2011)
Open Access PDF

An approach taken in Drosophila neurobiology has been to perturb genetically addressable sets of neurons and make inferences about the potential function of the affected neurons based on the observed phenotype. Much of this effort has been focused on grossly recognizable structures, such as the mushroom bodies, central complex substructures, antennal lobe, and recently, the antenno-mechanosensory motor center. However, all of these structures comprise only a quarter of the total neuropile volume of the central brain. The field currently lacks information about brain-wide connectivity that could be used as a guide to facilitate in-depth studies of the remaining volume of the central brain. Here we construct a coarse connectivity map of the adult fly brain using a combination of structural elements and developmental information from the semi-differentiated neurons at the late larval stage. The resolution of our map consists of a description of the interconnections between defined neuropile compartments, providing constraints on how information can flow through the central brain. Analysis of network topology and putative functional roles of compartments allow us to measure several network features and compare these to similarly studied nervous systems, including the macaque cortex and cat cortex. We used this analysis to generate and test a precise hypothesis about sensory modulation of a motor behavior. Our results reveal that the premetamorphic connections of the immature lineages prefigure the final pattern of connectivity of the mature brain. This latter finding suggests that brain connectivity can be studied in a development context, since the immature system is greatly reduced in its branching complexity. It is the hope that incorporation of behavioral results into the common structured framework of a connectivity network will allow novel and synthetic predictions, an approach that is likely to prove valuable for similar efforts in other model organisms.

Günther Gerisch, Mary Ecke, Dirk Wischnewski, Britta Schroth-Diez
Different modes of state transitions determine pattern in the Phosphatidylinositide-Actin system.
BMC Cell Biol, 12 Art. No. 42 (2011)
DOI

In a motile polarized cell the actin system is differentiated to allow protrusion at the front and retraction at the tail. This differentiation is linked to the phosphoinositide pattern in the plasma membrane. In the highly motile Dictyostelium cells studied here, the front is dominated by PI3-kinases producing PI(3,4,5)tris-phosphate (PIP3), the tail by the PI3-phosphatase PTEN that hydrolyses PIP3 to PI(4,5)bis-phosphate. To study de-novo cell polarization, we first depolymerized actin and subsequently recorded the spontaneous reorganization of actin patterns in relation to PTEN.

Nicola Maghelli, Iva M. Tolic-Norrelykke
Laser ablation of the microtubule cytoskeleton: setting up and working with an ablation system.
Methods Mol Biol, 777 261-271 (2011)
PDF DOI

Laser ablation is a powerful tool that can be used to study a variety of biological mechanisms. Microscopes with high optical performances are nowadays available, and lasers that could be used to perform ablations have become accessible to every laboratory. Setting up a laser ablation system is a relatively straightforward task; however, it requires some basic knowledge of optics. We illustrate the fundamental components of the experimental setup and describe the most common pitfalls and difficulties encountered when designing, setting up, and working with a laser ablation system.

Rajesh Ramaswamy, Ivo F. Sbalzarini
Intrinsic noise alters the frequency spectrum of mesoscopic oscillatory chemical reaction systems
Sci Rep, 1 Art. No. 154 (2011)
PDF DOI

Mesoscopic oscillatory reaction systems, for example in cell biology, can exhibit stochastic oscillations in the form of cyclic random walks even if the corresponding macroscopic system does not oscillate. We study how the intrinsic noise from molecular discreteness influences the frequency spectrum of mesoscopic oscillators using as a model system a cascade of coupled Brusselators away from the Hopf bifurcation. The results show that the spectrum of an oscillator depends on the level of noise. In particular, the peak frequency of the oscillator is reduced by increasing noise, and the bandwidth increased. Along a cascade of coupled oscillators, the peak frequency is further reduced with every stage and also the bandwidth is reduced. These effects can help understand the role of noise in chemical oscillators and provide fingerprints for more reliable parameter identification and volume measurement from experimental spectra.

Senthil Arumugam, Grzegorz Chwastek, Petra Schwille
Protein-membrane interactions: the virtue of minimal systems in systems biology
Wiley Interdiscip Rev Syst Biol Med, 3(3) 269-280 (2011)
DOI

The plasma membrane of cells can be viewed as a highly dynamic, regulated, heterogeneous environment with multiple functions. It constitutes the boundary of the cell, encapsulating all its components. Proteins interact with the membrane in many ways to accommodate essential processes, such as membrane trafficking, membrane protrusions, cytokinesis, signaling, and cell-cell communication. A vast amount of literature has already fostered our current understanding of membrane-protein interactions. However, many phenomena still remain to be understood, e.g., the exact mechanisms of how certain proteins cause or assist membrane transformations. Systems biology aims to predict biological processes on the basis of the set of molecules involved. Many key processes arise from interactions with the lipid membrane. Protein interactome maps do not consider such specific interactions, and thus cannot predict precise outcomes of the interactions of the involved proteins. These can only be inferred from experimental approaches. We describe examples of how an emergent behavior of protein-membrane interactions has been demonstrated by the use of minimal systems. These studies contribute to a deeper understanding of protein interactomes involving membranes and complement other approaches of systems biology.

Ronny Herzog, Dominik Schwudke, Kai Schuhmann, Julio Sampaio, Stefan R. Bornstein, Michael Schroeder, Andrej Shevchenko
A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language.
Genome Biol, 12(1) Art. No. R8 (2011)
DOI

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

Christian L. Müller, Ivo F. Sbalzarini
Global characterization of the CEC 2005 fitness landscapes using fitness-distance analysis
In: Proc. EvoStar (2011) Leture Notes in Computer Science, New York, Springer (2011), 294-303
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Christian L. Müller, Ivo F. Sbalzarini
A conjecture about an upper bound of the RMSD between linear chains
In: Proc. EuroCG (2011), Brussels, EuroCG (2011), 31-34
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Alicja K Antonczak^*, Zuzana Simova^*, Isaac T Yonemoto, Matthias Bochtler, Anna Piasecka, Honorata Czapinska, Andrea Brancale, Eric M Tippmann
Importance of single molecular determinants in the fidelity of expanded genetic codes.
Proc Natl Acad Sci U.S.A., 108(4) 1320-1325 (2011)
PDF DOI

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

Kai Simons
The Biology of Lipids : Trafficking, Regulation, and Function ; a subject collection from Cold Spring Harbor Perspectives in Biology
Cold Spring Harbor, N.Y, Cold Spring Harbor Laboratory Press (2011), 334 S.
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Monika I Mayr, Marko Storch, Jonathon Howard, Thomas U Mayer
A Non-Motor Microtubule Binding Site Is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A.
PLoS ONE, 6(11) Art. No. e27471 (2011)
PDF DOI

Members of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs.

Rajesh Ramaswamy, Ivo F. Sbalzarini, Nélido González-Segredo
Noise-induced modulation of the relaxation kinetics around a non-equilibrium steady state of non-linear chemical reaction networks
PLoS ONE, 6(1) Art. No. e16045 (2011)
PDF DOI

Stochastic effects from correlated noise non-trivially modulate the kinetics of non-linear chemical reaction networks. This is especially important in systems where reactions are confined to small volumes and reactants are delivered in bursts. We characterise how the two noise sources confinement and burst modulate the relaxation kinetics of a non-linear reaction network around a non-equilibrium steady state. We find that the lifetimes of species change with burst input and confinement. Confinement increases the lifetimes of all species that are involved in any non-linear reaction as a reactant. Burst monotonically increases or decreases lifetimes. Competition between burst-induced and confinement-induced modulation may hence lead to a non-monotonic modulation. We quantify lifetime as the integral of the time autocorrelation function (ACF) of concentration fluctuations around a non-equilibrium steady state of the reaction network. Furthermore, we look at the first and second derivatives of the ACF, each of which is affected in opposite ways by burst and confinement. This allows discriminating between these two noise sources. We analytically derive the ACF from the linear Fokker-Planck approximation of the chemical master equation in order to establish a baseline for the burst-induced modulation at low confinement. Effects of higher confinement are then studied using a partial-propensity stochastic simulation algorithm. The results presented here may help understand the mechanisms that deviate stochastic kinetics from its deterministic counterpart. In addition, they may be instrumental when using fluorescence-lifetime imaging microscopy (FLIM) or fluorescence-correlation spectroscopy (FCS) to measure confinement and burst in systems with known reaction rates, or, alternatively, to correct for the effects of confinement and burst when experimentally measuring reaction rates.

Rajesh Ramaswamy
Partial-propensity simulation algorithms for stochastic chemical kinetics and the role of fluctuations in mesoscopic reaction systems
Ph.D. Thesis, ETH Zurich, Department of Computer Science, Zurich, Switzerland (2011)
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Mark Gerstein^*, Zhi John Lu^*, Eric L. Van Nostrand^*, Chao Cheng^*, Bradley I. Arshinoff^*, Tao Liu^*, Kevin Y. Yip^*, Rebecca Robilotto^*, Andreas Rechtsteiner^*, Kohta Ikegami^*, Pedro Alves^*, Aurelien Chateigner^*, Marc Perry^*, Mitzi Morris^*, Raymond Auerbach^*, Xin Feng^*, Jing Leng^*, Anne Vielle^*, Wei Niu^*, Kahn Rhrissorrakrai^*, Ashish Agarwal, Roger P. Alexander, Galt Barber, Cathleen M Brdlik, Jennifer Brennan, Jeremy Jean Brouillet, Adrian Carr, Ming-Sin Cheung, Hiram Clawson, Sergio Contrino, Luke O. Dannenberg, Abby F. Dernburg, Arshad Desai, Lindsay Dick, Andréa Dosé, Jiang Du, Thea Egelhofer, Sevinc Ercan, Ghia Euskirchen, Brent Ewing, Elise A. Feingold, Reto Gassmann, Peter J. Good, Phil Green, Francois Gullier, Michelle Gutwein, Mark S. Guyer, Lukas Habegger, Ting Han, Jorja G. Henikoff, Stefan R. Henz, Angie Hinrichs, Heather Holster, Anthony A. Hyman, A. Leo Iniguez, Judith Janette, Morten Jensen, Masaomi Kato, W. James Kent, Ellen Kephart, Vishal Khivansara, Ekta Khurana, John K. Kim, Paulina Kolasinska-Zwierz, Eric C. Lai, Isabel Latorre, Amber Leahey, Suzanna E Lewis, Paul Lloyd, Lucas Lochovsky, Rebecca F. Lowdon, Yaniv Lubling, Rachel Lyne, Michael MacCoss, Sebastian D. Mackowiak, Marco Mangone, Sheldon McKay, Desirea Mecenas, Gennifer Merrihew, David M. Miller, Andrew Muroyama, John I. Murray, Siew-Loon Ooi, Hoang Pham, Taryn Phippen, Elicia A. Preston, Nikolaus Rajewski, Gunnar Rätsch, Heidi Rosenbaum, Joel Rozowksy, Kim Rutherford, Peter Ruzanov, Mihail Sarov, Rajkumar Sasidharan, Andrea Sboner, Paul Scheid, Eran Segal, Hyunjin Shin, Chong Shou, Frank J. Slack, Cindie Slightam, Richard Smith, William C. Spencer, E. O. Stinson, Scott Taing, Teruaki Takasaki, Dionne Vafeados, Ksenia Voronina, Guilin Wang, Nicole L. Washington, Christina M. Whittle, Beijing Wu, Koon-Kiu Yan, Georg Zeller, Zheng Zha, Mei Zhong, Xingliang Zhou, Julie Ahringer, Susan Strome, Kristin C. Gunsalus, Gos Micklem, X. Shirley Liu, Valerie Reinke, Stuart K Kim, LaDeana W Hillier, Steven Henikoff, Fabio Piano, Michael Snyder, Lincoln Stein, Jason D. Lieb, Robert H Waterston
Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project
Science, 330(6012) 1775-1787 (2010)
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We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Jakub Sedzinski
Mechanics of the polar cell cortex during cytokinesis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Alex T. Kalinka^*, Karolina M Varga^*, Dave T. Gerrard, Stephan Preibisch, David L. Corcoran, Julia Jarrells, Uwe Ohler, Casey M. Bergman, Pavel Tomancak
Gene expression divergence recapitulates the developmental hourglass model.
Nature, 468(7325) 811-814 (2010)
PDF DOI

The observation that animal morphology tends to be conserved during the embryonic phylotypic period (a period of maximal similarity between the species within each animal phylum) led to the proposition that embryogenesis diverges more extensively early and late than in the middle, known as the hourglass model. This pattern of conservation is thought to reflect a major constraint on the evolution of animal body plans. Despite a wealth of morphological data confirming that there is often remarkable divergence in the early and late embryos of species from the same phylum, it is not yet known to what extent gene expression evolution, which has a central role in the elaboration of different animal forms, underpins the morphological hourglass pattern. Here we address this question using species-specific microarrays designed from six sequenced Drosophila species separated by up to 40 million years. We quantify divergence at different times during embryogenesis, and show that expression is maximally conserved during the arthropod phylotypic period. By fitting different evolutionary models to each gene, we show that at each time point more than 80% of genes fit best to models incorporating stabilizing selection, and that for genes whose evolutionarily optimal expression level is the same across all species, selective constraint is maximized during the phylotypic period. The genes that conform most to the hourglass pattern are involved in key developmental processes. These results indicate that natural selection acts to conserve patterns of gene expression during mid-embryogenesis, and provide a genome-wide insight into the molecular basis of the hourglass pattern of developmental evolution.

Dario Piano, Sabah El Alaoui, Henryk J Korza, Renata Filipek, Izabela Sabala, Patrycja Haniewicz, Claudia Buechel, Daniele De Sanctis, Matthias Bochtler
Crystallization of the photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotiana tabacum.
Photosyn Res, 106(3) 221-226 (2010)
PDF DOI

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.

Markus Decker
Setting centrosome size in the early caenorhabditis elegans embryo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Daniele Soroldoni
Tracking the oscillations of high-fidelity BAC transgenes during zebrafish somitogenesis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Mirjam Mayer
Mechanics of the C. elegans cell cortex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Kai Simons
Engineering the Future with Cell Biology
Mol Biol Cell, 21(22) Art. No. 3822 (2010)
PDF DOI

Benoît Maugis, Jan Brugués, Pierre Nassoy, Nancy Guillen, Pierre Sens, François Amblard
Dynamic instability of the intracellular pressure drives bleb-based motility.
J Cell Sci, 123(Pt 22) 3884-3892 (2010)
DOI

We have demonstrated that the two- and three-dimensional motility of the human pathogenic parasite Entamoeba histolytica (Eh) depends on sustained instability of the intracellular hydrostatic pressure. This instability drives the cyclic generation and healing of membrane blebs, with typical protrusion velocities of 10-20 μm/second over a few hundred milliseconds and healing times of 10 seconds. The use of a novel micro-electroporation method to control the intracellular pressure enabled us to develop a qualitative model with three parameters: the rate of the myosin-driven internal pressure increase; the critical disjunction stress of membrane-cytoskeleton bonds; and the turnover time of the F-actin cortex. Although blebs occur randomly in space and irregularly time, they can be forced to occur with a defined periodicity in confined geometries, thus confirming our model. Given the highly efficient bleb-based motility of Eh in vitro and in vivo, Eh cells represent a unique model for studying the physical and biological aspects of amoeboid versus mesenchymal motility in two- and three-dimensional environments.

Lara O'Donnell^*, Stephanie Panier^*, Jan Wildenhain^*, Johnny M Tkach, Abdallah Al-Hakim, Marie-Claude Landry, Cristina Escribano-Diaz, Rachel K Szilard, Jordan T F Young, Meagan Munro, Marella D Canny, Nadine K Kolas, Wei Zhang, Shane M Harding, Jarkko Ylanko, Megan Mendez, Michael Mullin, Thomas Sun, Bianca Habermann, Alessandro Datti, Robert G Bristow, Anne-Claude Gingras, Michael D Tyers, Grant W Brown, Daniel Durocher
The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination.
Mol Cell, 40(4) 619-631 (2010)
PDF DOI

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.

Katharina Nakel, Sophia A Hartung, Fabien Bonneau, Christian R. Eckmann, Elena Conti
Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform.
RNA, 16(11) 2058-2067 (2010)
PDF DOI

Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-Å-resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2-KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein-protein interaction platform.

Jakob Suckale, Michele Solimena
The insulin secretory granule as a signaling hub.
Trends Endocrinol Metab, 21(10) 599-609 (2010)
PDF DOI

The insulin granule was previously thought of as merely a container, but accumulating evidence suggests that it also acts as a signaling node. Regulatory pathways intersect at but also originate from the insulin granule membrane. Examples include the small G-proteins Rab3a and Rab27a, which influence granule movement, and the transmembrane proteins (tyrosine phosphatase receptors type N) PTPRN and PTPRN2, which upregulate β-cell transcription and proliferation. In addition, many cosecreted compounds possess regulatory functions, often related to energy metabolism. For instance, ATP and γ-amino butyric acid (GABA) modulate insulin and glucagon secretion, respectively; C-peptide protects β-cells and kidney cells; and amylin reduces gastric emptying and food intake via the brain. In this paper, we review the current knowledge of the insulin granule proteome and discuss its regulatory functions.

Yanmei Liu, Jakob Suckale, Jimmy Masjkur, Maria Grazia Magro, Anja Steffen, Konstantinos Anastassiadis, Michele Solimena
Tamoxifen-independent recombination in the RIP-CreER mouse.
PLoS ONE, 5(10) Art. No. e13533 (2010)
PDF DOI

The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.

Robert R G Vries^*, Meritxell Huch^*, Hans Clevers
Stem cells and cancer of the stomach and intestine.
Mol Oncol, 4(5) 373-384 (2010)
DOI

Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This implies that at any point during tumour development any tumour cell can revert to a cancer stem cell after having gained a clonal advantage over the original cancer stem cell. Therefore, these models represent two sides of the same coin.

Pablo Oteiza^*, Mathias Köppen^*, Michael Krieg, Eduardo Pulgar, Cecilia Farias, Cristina Melo, Stephan Preibisch, Daniel J. Müller, Masazumi Tada, Steffen Hartel, Carl-Philipp Heisenberg, Miguel L Concha
Planar cell polarity signalling regulates cell adhesion properties in progenitors of the zebrafish laterality organ.
Development, 137(20) 3459-3468 (2010)
PDF DOI

Organ formation requires the precise assembly of progenitor cells into a functional multicellular structure. Mechanical forces probably participate in this process but how they influence organ morphogenesis is still unclear. Here, we show that Wnt11- and Prickle1a-mediated planar cell polarity (PCP) signalling coordinates the formation of the zebrafish ciliated laterality organ (Kupffer's vesicle) by regulating adhesion properties between organ progenitor cells (the dorsal forerunner cells, DFCs). Combined inhibition of Wnt11 and Prickle1a reduces DFC cell-cell adhesion and impairs their compaction and arrangement during vesicle lumen formation. This leads to the formation of a mis-shapen vesicle with small fragmented lumina and shortened cilia, resulting in severely impaired organ function and, as a consequence, randomised laterality of both molecular and visceral asymmetries. Our results reveal a novel role for PCP-dependent cell adhesion in coordinating the supracellular organisation of progenitor cells during vertebrate laterality organ formation.

Martina Huranová, Ivan Ivani, Ales Benda, Ina Poser, Yehuda Brody, Martin Hof, Yaron Shav-Tal, Karla M. Neugebauer, David Stanĕk
The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells.
J Cell Biol, 191(1) 75-86 (2010)
PDF DOI

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

Ilya Levental^*, Daniel Lingwood, Michal Grzybek, Uenal Coskun, Kai Simons^*
Palmitoylation regulates raft affinity for the majority of integral raft proteins
Proc Natl Acad Sci U.S.A., 107(51) 22050-22054 (2010)
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The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of T cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.

Albert Cardona, Stephan Saalfeld, Stephan Preibisch, Benjamin Schmid, Anchi Cheng, Jim Pulokas, Pavel Tomancák, Volker Hartenstein
An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.
PLoS Biol, 8(10) Art. No. e1000502 (2010)
Open Access PDF DOI

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

Mirjam Mayer, Martin Depken, Justin Bois, Frank Jülicher, Stephan W. Grill
Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows.
Nature, 467(7315) 617-621 (2010)
PDF DOI

Asymmetric cell divisions are essential for the development of multicellular organisms. To proceed, they require an initially symmetric cell to polarize. In Caenorhabditis elegans zygotes, anteroposterior polarization is facilitated by a large-scale flow of the actomyosin cortex, which directs the asymmetry of the first mitotic division. Cortical flows appear in many contexts of development, but their underlying forces and physical principles remain poorly understood. How actomyosin contractility and cortical tension interact to generate large-scale flow is unclear. Here we report on the subcellular distribution of cortical tension in the polarizing C. elegans zygote, which we determined using position- and direction-sensitive laser ablation. We demonstrate that cortical flow is associated with anisotropies in cortical tension and is not driven by gradients in cortical tension, which contradicts previous proposals. These experiments, in conjunction with a theoretical description of active cortical mechanics, identify two prerequisites for large-scale cortical flow: a gradient in actomyosin contractility to drive flow and a sufficiently large viscosity of the cortex to allow flow to be long-ranged. We thus reveal the physical requirements of large-scale intracellular cortical flow that ensure the efficient polarization of the C. elegans zygote.

Sandra Schubert, Klaus-Peter Knoch, Joke Ouwendijk, Shabaz Mohammed, Yury Bodrov, Melanie Jäger, Anke Altkrüger, Carolin Wegbrod, Marvin E Adams, Yong Kim, Stanley C. Froehner, Ole N Jensen, Yannis Kalaidzidis, Michele Solimena
Beta2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.
PLoS ONE, 5(9) Art. No. e12929 (2010)
PDF DOI

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.

Benoit Aigouy, Reza Farhadifar, Douglas B Staple, Andreas Sagner, Jens-Christian Röper, Frank Jülicher, Suzanne Eaton
Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila.
Cell, 142(5) 773-786 (2010)
PDF DOI

Planar cell polarity (PCP) proteins form polarized cortical domains that govern polarity of external structures such as hairs and cilia in both vertebrate and invertebrate epithelia. The mechanisms that globally orient planar polarity are not understood, and are investigated here in the Drosophila wing using a combination of experiment and theory. Planar polarity arises during growth and PCP domains are initially oriented toward the well-characterized organizer regions that control growth and patterning. At pupal stages, the wing hinge contracts, subjecting wing-blade epithelial cells to anisotropic tension in the proximal-distal axis. This results in precise patterns of oriented cell elongation, cell rearrangement and cell division that elongate the blade proximo-distally and realign planar polarity with the proximal-distal axis. Mutation of the atypical Cadherin Dachsous perturbs the global polarity pattern by altering epithelial dynamics. This mechanism utilizes the cellular movements that sculpt tissues to align planar polarity with tissue shape.

Marc Bickle
The beautiful cell: high-content screening in drug discovery.
Anal Bioanal Chem, 398(1) 219-226 (2010)
PDF DOI

The term "high-content screening" has become synonymous with imaging screens using automated microscopes and automated image analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments and analyzing the data have come to maturity, so the full benefits of high-content screening can now be realized. Those benefits are the capability of carrying out phenotypic multiparametric cellular assays in an unbiased, fully automated, and quantitative fashion. Automated microscopes and automated image analysis are being applied at all stages of the drug discovery and development pipeline. All major pharmaceutical companies have adopted the technology and it is in the process of being embraced broadly by the academic community. This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.

Marta Pabis, Noa Neufeld, Yaron Shav-Tal, Karla M. Neugebauer
Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20
Nucleus, 1(5) 412-421 (2010)
PDF DOI

The nuclear cap-binding complex (CBC) is a heterodimer composed of CBP20 and CBP80 subunits and has roles in the biogenesis of messenger RNAs (mRNAs), small nuclear RNAs (snRNAs) and microRNAs. CBP20 is a phylogenetically conserved protein that interacts with the 7-methyl guanosine (m7G) cap added to the 5’ end of all RNA polymerase II transcripts. CBP80 ensures high affinity binding of the cap by CBP20 and provides a platform for interactions with other factors. Here we characterize an alternative splice variant of CBP20, termed CBP20S. The CBP20S transcript has an in- frame deletion, leading to the translation of a protein lacking most of the RNA recognition motif (RRM). We show that CBP20S is conserved among mammalian species and is expressed in human cell lines and bone marrow cells. Unlike the full-length CBP20, CBP20S does not bind CBP80 or the m7G cap. Nevertheless, CBP20S does bind mRNA, is localized to an active transcription site and redistributed to nucleolar caps upon transcription inhibition. Our results suggest that this novel form CBP20S plays a role in transcription and/or RNA processing independent of CBP80 or the cap.

Cornelia G Spruijt, Stefanie J J Bartels, Arie B Brinkman, Jorrit V Tjeertes, Ina Poser, Hendrik G Stunnenberg, Michiel Vermeulen
CDK2AP1/DOC-1 is a bona fide subunit of the Mi-2/NuRD complex.
Mol Biosyst, 6(9) 1700-1706 (2010)
PDF DOI

The Mi-2/NuRD (NUcleosome Remodeling and histone Deacetylase) chromatin remodeling complex is a large heterogeneous multiprotein complex associated with transcriptional repression. Here we apply a SILAC based quantitative proteomics approach to show that all known Mi-2/NuRD complex subunits co-purify with Cyclin Dependent Kinase 2 Associated Protein1 (CDK2AP1), also known as Deleted in Oral Cancer 1 (DOC-1). DOC-1 displays in vitro binding affinity for methylated DNA as part of the meCpG binding MBD2/NuRD complex. In luciferase reporter assays, DOC-1 is a potent repressor of transcription. Finally, immunofluorescence experiments reveal co-localization between MBD2 and DOC-1 in mouse NIH-3T3 nuclei. Collectively, these results indicate that DOC-1 is a bona fide subunit of the Mi-2/NuRD chromatin remodeling complex.

Jeremy N. Pulvers, Jaroslaw Bryk, Jennifer L. Fish, Michaela Wilsch-Bräuninger, Yoko Arai, Dora Schreier, Ronald Naumann, Jussi Helppi, Bianca Habermann, Johannes Vogt, Robert Nitsch, Attila Toth, Wolfgang Enard, Svante Pääbo, Wieland B. Huttner
Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.
Proc Natl Acad Sci U.S.A., 107(38) 16595-16600 (2010)
PDF DOI

Mutations in ASPM (abnormal spindle-like microcephaly associated) cause primary microcephaly in humans, a disorder characterized by a major reduction in brain size in the apparent absence of nonneurological anomalies. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody, suggest that it regulates brain development by a cell division-related mechanism. Furthermore, evidence that positive selection affected ASPM during primate evolution has led to suggestions that such a function changed during primate evolution. Here, we report that in Aspm mutant mice, truncated Aspm proteins similar to those causing microcephaly in humans fail to localize to the midbody during M-phase and cause mild microcephaly. A human ASPM transgene rescues this phenotype but, interestingly, does not cause a gain of function. Strikingly, truncated Aspm proteins also cause a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by reduced fertility. These germline effects, too, are fully rescued by the human ASPM transgene, indicating that ASPM is functionally similar in mice and humans. Our findings broaden the spectrum of phenotypic effects of ASPM mutations and raise the possibility that positive selection of ASPM during primate evolution reflects its function in the germline.

Yvonne Gloor, Mario Schöne, Bianca Habermann, Ebru Ercan, Mike Beck, Grit Weselek, Thomas Müller-Reichert, Christiane Walch-Solimena
Interaction between Sec7p and Pik1p: the first clue for the regulation of a coincidence detection signal.
Eur J Cell Biol, 89(8) 575-583 (2010)
PDF DOI

Sec7p, a guanine nucleotide exchange factor, regulates the activation of small Arf GTPases, which function in the formation of distinct classes of transport carriers from the Golgi. The recruitment of a subset of Arf effectors depends on the cooperation between these GTPases and phosphatidylinositol 4-phosphate. Here, we show that the catalytic domain of Sec7p interacts with a conserved region of the Golgi phosphatidylinositol 4-kinase Pik1p. We found that Sec7p and Pik1p as well as its product, colocalize at the late Golgi. Gea1p/Gea2p, an alternative pair of Arf activators, do not bind to Pik1p and function on a different Golgi sub-compartment. Sec7p and Pik1p interact with each other and cooperate in the formation of clathrin-coated vesicles. This interaction reveals a distinct role for Sec7p among the Golgi Arf-GEFs and provides a working model for the coordinated generation of Arf-GTP and phosphatiylinositol 4-phosphate as dual signal for specific recruitment of clathrin coats to the late Golgi.

Jan Brugués, Benoît Maugis, Jaume Casademunt, Pierre Nassoy, François Amblard, Pierre Sens
Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration.
Proc Natl Acad Sci U.S.A., 107(35) 15415-15420 (2010)
DOI

Bleb-based cell motility proceeds by the successive inflation and retraction of large spherical membrane protrusions ("blebs") coupled with substrate adhesion. In addition to their role in motility, cellular blebs constitute a remarkable illustration of the dynamical interactions between the cytoskeletal cortex and the plasma membrane. Here we study the bleb-based motions of Entamoeba histolytica in the constrained geometry of a micropipette. We construct a generic theoretical model that combines the polymerization of an actin cortex underneath the plasma membrane with the myosin-generated contractile stress in the cortex and the stress-induced failure of membrane-cortex adhesion. One major parameter dictating the cell response to micropipette suction is the stationary cortex thickness, controlled by actin polymerization and depolymerization. The other relevant physical parameters can be combined into two characteristic cortex thicknesses for which the myosin stress (i) balances the suction pressure and (ii) provokes membrane-cortex unbinding. We propose a general phase diagram for cell motions inside a micropipette by comparing these three thicknesses. In particular, we theoretically predict and experimentally verify the existence of saltatory and oscillatory motions for a well-defined range of micropipette suction pressures.

Marko Brankatschk, Suzanne Eaton
Lipoprotein particles cross the blood-brain barrier in Drosophila.
J Neurosci, 30(31) 10441-10447 (2010)
PDF DOI

The blood-brain barrier (BBB) regulates passage of nutrients and signaling molecules from the circulation into the brain. Whether lipoproteins cross the BBB in vivo has been controversial, and no clear requirement for circulating lipoproteins in brain development has been shown. We address these issues in Drosophila, which has an functionally conserved BBB, and lipoproteins that resemble those of vertebrates. We show that the Drosophila lipoprotein lipophorin exists in two isoforms. Both isoforms cross the BBB, but accumulate on distinct subsets of cells within the brain. In addition to acting as a lipid carrier, lipophorin carries both sterol-linked and GPI-linked proteins into the circulation and transports them across the BBB. Finally, lipophorin promotes neuroblast proliferation by a mechanism that does not depend on delivery of dietary lipids. Transport of lipophorin and its cargo across the BBB represents a novel mechanism by which peripherally synthesized proteins might enter the brain and influence its development. Furthermore, lipid-linkage may be an efficient method to transport therapeutic molecules across the BBB.

Till Korten, Alf Månsson, Stefan Diez
Towards the Application of Cytoskeletal Motor Proteins in Molecular Detection and Diagnostic Devices
Curr Opin Biotechnol, 21(4) 477-488 (2010)
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Over the past ten years, great advancements have been made towards using biomolecular motors for nanotechnological applications. In particular, devices using cytoskeletal motor proteins for molecular transport are maturing. First efforts towards designing such devices used motor proteins attached to micro-structured substrates for the directed transport of microtubules and actin filaments. Soon thereafter, the specific capture, transport and detection of target analytes like viruses were demonstrated. Recently, spatial guiding of the gliding filaments was added to increase the sensitivity of detection and allow parallelization. Whereas molecular motor powered devices have not yet demonstrated performance beyond the level of existing detection techniques, the potential is great: Replacing microfluidics with transport powered by molecular motors allows integration of the energy source (ATP) into the assay solution. This opens up the opportunity to design highly integrated, miniaturized, autonomous detection devices. Such devices, in turn, may allow fast and cheap on-site diagnosis of diseases and detection of environmental pathogens and toxins.

Adam P. Kupinski, Thomas Müller-Reichert, Christian R. Eckmann
The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation.
Dev Biol, 344(2) 758-771 (2010)
PDF DOI

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.

Elwy Okaz
The role of proteolysis in regulation entry into the first divsion of meiosis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Julio Sampaio
The role of lipids in cellular architecture and function
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Leah Herrgen, Saul Ares, Luis G. Morelli, Christian Schröter, Frank Jülicher, Andrew C. Oates
Intercellular coupling regulates the period of the segmentation clock.
Curr Biol, 20(14) 1244-1253 (2010)
PDF DOI

Coupled biological oscillators can tick with the same period. How this collective period is established is a key question in understanding biological clocks. We explore this question in the segmentation clock, a population of coupled cellular oscillators in the vertebrate embryo that sets the rhythm of somitogenesis, the morphological segmentation of the body axis. The oscillating cells of the zebrafish segmentation clock are thought to possess noisy autonomous periods, which are synchronized by intercellular coupling through the Delta-Notch pathway. Here we ask whether Delta-Notch coupling additionally influences the collective period of the segmentation clock.

Thomas Müller-Reichert, Garrett Greenan, Eileen T. O'Toole, Martin Srayko
The elegans of spindle assembly.
Cell Mol Life Sci, 67(13) 2195-2213 (2010)
PDF DOI

The Caenorhabditis elegans one-cell embryo is a powerful system in which to study microtubule organization because this large cell assembles both meiotic and mitotic spindles within the same cytoplasm over the course of 1 h in a stereotypical manner. The fertilized oocyte assembles two consecutive acentrosomal meiotic spindles that function to reduce the replicated maternal diploid set of chromosomes to a single-copy haploid set. The resulting maternal DNA then unites with the paternal DNA to form a zygotic diploid complement, around which a centrosome-based mitotic spindle forms. The early C. elegans embryo is amenable to live-cell imaging and electron tomography, permitting a detailed structural comparison of the meiotic and mitotic modes of spindle assembly.

Giovanna Mottola, Anne-Kathrin Classen, Marcos González-Gaitán, Suzanne Eaton, Marino Zerial
A novel function for the Rab5 effector Rabenosyn-5 in planar cell polarity.
Development, 137(14) 2353-2364 (2010)
PDF DOI

In addition to apicobasal polarization, some epithelia also display polarity within the plane of the epithelium. To what extent polarized endocytosis plays a role in the establishment and maintenance of planar cell polarity (PCP) is at present unclear. Here, we investigated the role of Rabenosyn-5 (Rbsn-5), an evolutionarily conserved effector of the small GTPase Rab5, in the development of Drosophila wing epithelium. We found that Rbsn-5 regulates endocytosis at the apical side of the wing epithelium and, surprisingly, further uncovered a novel function of this protein in PCP. At early stages of pupal wing development, the PCP protein Fmi redistributes between the cortex and Rab5- and Rbsn-5-positive early endosomes. During planar polarization, Rbsn-5 is recruited at the apical cell boundaries and redistributes along the proximodistal axis in an Fmi-dependent manner. At pre-hair formation, Rbsn-5 accumulates at the bottom of emerging hairs. Loss of Rbsn-5 causes intracellular accumulation of Fmi and typical PCP alterations such as defects in cell packing, in the polarized distribution of PCP proteins, and in hair orientation and formation. Our results suggest that establishment of planar polarity requires the activity of Rbsn-5 in regulating both the endocytic trafficking of Fmi at the apical cell boundaries and hair morphology.

Carsten Hoege, Alexandru T. Constantinescu, Anne Schwager, Nathan W. Goehring, Prateek Kumar, Anthony A. Hyman
LGL can partition the cortex of one-cell Caenorhabditis elegans embryos into two domains.
Curr Biol, 20(14) 1296-1303 (2010)
PDF DOI

Many metazoan cell types are polarized by asymmetric partitioning of the conserved PAR (PAR-3/PAR-6/PKC-3) complex. Cortical domains containing this PAR complex are counterbalanced by opposing domains of varying composition. The tumor-suppressor protein LGL facilitates asymmetric localization of cell fate determinants, in part through modulating the activity of the PAR complex. However, the mechanisms by which LGL acts to maintain a cortical domain remain unclear. Here we identify Caenorhabditis elegans LGL in a biochemical complex with PAR proteins, which localize to the anterior cortex. But LGL itself localizes to the posterior cortex. We show that increasing the amounts of LGL can restrict localization of the PAR complex to an anterior cortical domain, even in the absence of PAR-2. Importantly, LGL must be phosphorylated on conserved residues to exert this function. LGL and the PAR complex can maintain two cortical domains that are sufficient to partition cell fate determinants. Our data suggest a mechanism of "mutual elimination" in which an LGL phosphorylation cycle regulates association of the PAR complex with the cortex: binding of LGL to the PAR complex at the interface of the two domains stimulates its phosphorylation by PKC-3, and the whole complex leaves the cortex.

Vineeth Surendranath, Janet Chusainow, Joachim Hauber, Frank Buchholz, Bianca Habermann
SeLOX--a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems.
Nucleic Acids Res, 38(Suppl. 2) 293-298 (2010)
PDF DOI

Site-specific recombinases have become a resourceful tool for genome engineering, allowing sophisticated in vivo DNA modifications and rearrangements, including the precise removal of integrated retroviruses from host genomes. In a recent study, a mutant form of Cre recombinase has been used to excise the provirus of a specific HIV-1 strain from the human genome. To achieve provirus excision, the Cre recombinase had to be evolved to recombine an asymmetric locus of recombination (lox)-like sequence present in the long terminal repeat (LTR) regions of a HIV-1 strain. One pre-requisite for this type of work is the identification of degenerate lox-like sites in genomic sequences. Given their nature-two inverted repeats flanking a spacer of variable length-existing search tools like BLAST or RepeatMasker perform poorly. To address this lack of available algorithms, we have developed the web-server SeLOX, which can identify degenerate lox-like sites within genomic sequences. SeLOX calculates a position weight matrix based on lox-like sequences, which is used to search genomic sequences. For computational efficiency, we transform sequences into binary space, which allows us to use a bit-wise AND Boolean operator for comparisons. Next to finding lox-like sites for Cre type recombinases in HIV LTR sequences, we have used SeLOX to identify lox-like sites in HIV LTRs for six yeast recombinases. We finally demonstrate the general usefulness of SeLOX in identifying lox-like sequences in large genomes by searching Cre type recombination sites in the entire human genome. SeLOX is freely available at http://selox.mpi-cbg.de/cgi-bin/selox/index.

Hesso Farhan, Markus W Wendeler, Sandra Mitrovic, Eugenio Fava, Yael Silberberg, Roded Sharan, Marino Zerial, Hans-Peter Hauri
MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening.
J Cell Biol, 189(6) 997-1011 (2010)
PDF DOI

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf-MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)-ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.

Chandrashekhar V. Kulkarni, T-Y Dora Tang, Annela M Seddon, John M Seddon, Oscar Ces, Richard H Templer
Engineering bicontinuous cubic structures at the nanoscale—the role of chain splay
Soft Matter, 6 3191-3194 (2010)

Judith Schenk
Interkinetic nuclear migration and centrosome positioning in mammalian neural progenitors : the role of non-muscle myosin II
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Christian Schenk^*, Henrik Bringmann^*, Anthony A. Hyman, Carrie R. Cowan
Cortical domain correction repositions the polarity boundary to match the cytokinesis furrow in C. elegans embryos.
Development, 137(10) 1743-1753 (2010)
PDF DOI

In asymmetrically dividing cells, a failure to coordinate cell polarity with the site of cell division can lead to cell fate transformations and tumorigenesis. Cell polarity in C. elegans embryos is defined by PAR proteins, which occupy reciprocal halves of the cell cortex. During asymmetric division, the boundary between the anterior and posterior PAR domains precisely matches the site of cell division, ensuring exclusive segregation of cell fate. The PAR domains determine the site of cell division by positioning the mitotic spindle, suggesting one means by which cell polarity and cell division might be coordinated. Here, we report that cell polarity and cell division are coordinated through an additional mechanism: the site of cell division repositions the PAR-2 boundary. Galpha-mediated microtubule-cortex interactions appear to direct cortical flows of PAR-2 and myosin toward the site of cell division, which acts as a PAR-2 and myosin sink. Embryos with defects in PAR-2 boundary correction undergo mis-segregation of cortical polarity and cytoplasmic determinants, suggesting that PAR domain correction might help prevent cell fate transformation.

Zhaiyi Zhang, Dominik Theler, Katarzyna H Kaminska, Michael Hiller, Pierre de la Grange, Rainer Pudimat, Ilona Rafalska, Bettina Heinrich, Janusz M Bujnicki, Frédéric H-T Allain, Stefan Stamm
The YTH domain is a novel RNA binding domain.
J Biol Chem, 285(19) 14701-14710 (2010)
PDF DOI

The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an alpha-helix/beta-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.

Sergey Lekomtsev, Julien Guizetti, Andrei I. Pozniakovsky, Daniel W Gerlich, Mark Petronczki
Evidence that the tumor-suppressor protein BRCA2 does not regulate cytokinesis in human cells.
J Cell Sci, 123(Pt 9) 1395-1400 (2010)
PDF DOI

Germline mutations in the tumor-suppressor gene BRCA2 predispose to breast and ovarian cancer. BRCA2 plays a well-established role in maintaining genome stability by regulating homologous recombination. BRCA2 has more recently been implicated in cytokinesis, the final step of cell division, but the molecular basis for this remains unknown. We have used time-lapse microscopy, recently developed cytokinesis assays and BAC recombineering (bacterial artificial chromosome recombinogenic engineering) to investigate the function and localization of BRCA2 during cell division. Our analysis suggests that BRCA2 does not regulate cytokinesis in human cells. Thus, cytokinesis defects are unlikely to contribute to chromosomal instability and tumorigenesis in BRCA2-related cancers.

Annett Kilic^*, Sven Klose^*, Bernhard Dobberstein, Elisabeth Knust, Katja Kapp
The Drosophila Crumbs signal peptide is unusually long and is a substrate for signal peptide peptidase.
Eur J Cell Biol, 89(6) 449-461 (2010)
PDF DOI

N-terminal signal sequences mediate nascent protein targeting to and protein insertion into the membrane of the endoplasmic reticulum. They are typically 15-30 amino acid residues long with a core hydrophobic region flanked by an N-terminal (n-) and a C-terminal region. Following cleavage by signal peptidase, some of the resulting signal peptides are further processed by signal peptide peptidase (SPP) and fragments are liberated into the cytosol. Such fragments can have independent, post-targeting functions affecting diverse cellular processes. We show that Drosophila melanogaster Crumbs, a transmembrane protein controlling cell polarity and morphogenesis, is synthesized with an 83 residues-long signal sequence. To our knowledge, this is currently the longest signal sequence described for an eukaryotic protein. The unusual length is caused by an extended n-region, but the extension does neither affect protein targeting nor signal sequence cleavage. The signal sequence is cleaved off and the resulting signal peptide, SP(Crb), is proteolytically processed by SPP, thus representing the first substrate described for the Drosophila enzyme. We further show that signal peptide fragments can be degraded by the proteasome. Expression of transgenes encoding tagged variants of Crumbs in Drosophila embryos suggests that the signal peptide is short-lived in vivo. Our findings support a model suggesting that besides generating fragments with post-targeting functions, SPP-mediated processing is the first step in the degradation of signal peptides.

József Jászai, Lilla M. Farkas, Christine A. Fargeas, Peggy Janich, Michael Haase, Wieland B. Huttner, Denis Corbeil
Prominin-2 is a novel marker of distal tubules and collecting ducts of the human and murine kidney.
Histochem Cell Biol, 133(5) 527-539 (2010)
PDF DOI

Prominin-1 (CD133) and its paralogue, prominin-2, are pentaspan membrane glycoproteins that are strongly expressed in the kidney where they have been originally cloned from. Previously, we have described the localization of prominin-1 in proximal tubules of the nephron. The spatial distribution of prominin-2, however, has not yet been documented in the kidney. We therefore examined the expression of this molecule along distinct tubular segments of the human and murine nephron using in situ hybridization and immunohistochemistry. Our findings indicated that human prominin-2 transcripts and protein were confined to distal tubules of the nephron including the thick ascending limb of Henle's loop and the distal convoluted tubule, the connecting duct and to the collecting duct system. Therein, this glycoprotein was enriched at the basolateral plasma membrane of the tubular epithelial cells with exception of the thick ascending limb where it was also found in the apical domain. This is in contrast with the exclusive apical localization of prominin-1 in epithelial cells of proximal nephron tubules. The distribution of murine prominin-2 transcripts was reminiscent of its human orthologue. In addition, a marked enrichment in the epithelium covering the papilla and in the urothelium of the renal pelvis was noted in mice. Finally, our biochemical analysis revealed that prominin-2 was released into the clinically healthy human urine as a constituent of small membrane vesicles. Collectively our data show the distribution and subcellular localization of prominin-2 within the kidney in situ and its release into the urine. Urinary detection of this protein might offer novel diagnostic approaches for studying renal diseases affecting distal segments of the nephron.

Denis Corbeil, Anne-Marie Marzesco, Michaela Wilsch-Bräuninger, Wieland B. Huttner
The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell differentiation.
FEBS Lett, 584(9) 1659-1664 (2010)
PDF DOI

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane protein concentrated in plasma membrane protrusions. In epithelial cells, notably neuroepithelial stem cells, prominin-1 is found in microvilli, the primary cilium and the midbody. These three types of apical membrane protrusions are subject to remodeling during (neuro)epithelial cell differentiation. The protrusion-specific localization of prominin involves its association with a distinct cholesterol-based membrane microdomain. Moreover, the three prominin-1-containing plasma membrane protrusions are the origin of at least two major subpopulations of prominin-1-containing extracellular membrane particles. Intriguingly, the release of these particles has been implicated in (neuro)epithelial cell differentiation.

Pavel Tomancák, Uwe Ohler
Mapping the complexity of transcription control in higher eukaryotes
Genome Biol, 11(4) Art. No. 115 (2010)
PDF DOI

Recent genomic analyses suggest the importance of combinatorial regulation by broadly expressed transcription factors rather than expression domains characterized by highly specific factors.

Ravi K R Marreddy, Eric R Geertsma, Hjalmar P Permentier, Joao P C Pinto, Jan Kok, B Poolman
Amino acid accumulation limits the overexpression of proteins in Lactococcus lactis.
PLoS ONE, 5(4) Art. No. e10317 (2010)
Open Access DOI

Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media.

Daniela Röllig
The role of the hes5 gene family in the evolution of the somitogenesis clock
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Dmitry Poteryaev^*, Sunando Datta^*, Karin Ackema, Marino Zerial, Anne Spang
Identification of the switch in early-to-late endosome transition.
Cell, 141(3) 497-508 (2010)
PDF DOI

Sequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.

Falk Butter, Dennis Kappei, Frank Buchholz^#, Michiel Vermeulen^#, Matthias Mann
A domesticated transposon mediates the effects of a single-nucleotide polymorphism responsible for enhanced muscle growth.
EMBO Rep, 11(4) 305-311 (2010)
PDF DOI

Single-nucleotide polymorphisms (SNPs) in the regulatory regions of the genome can have a profound impact on phenotype. The G3072A polymorphism in intron 3 of insulin-like growth factor 2 (IGF2) is implicated in higher muscle content and reduced fat in European pigs and is bound by a putative repressor. Here, we identify this repressor--which we call muscle growth regulator (MGR)--by using a DNA protein interaction screen based on quantitative mass spectrometry. MGR has a bipartite nuclear localization signal, two BED-type zinc fingers and is highly conserved between placental mammals. Surprisingly, the gene is located in an intron and belongs to the hobo-Ac-Tam3 transposase superfamily, suggesting regulatory use of a formerly parasitic element. In transactivation assays, MGR differentially represses the expression of the two SNP variants. Knockdown of MGR in C2C12 myoblast cells upregulates Igf2 expression and mild overexpression retards growth. Thus, MGR is the repressor responsible for enhanced muscle growth in the IGF2 G3072A polymorphism in commercially bred pigs.

Beate Neumann^*, Thomas Walter^*, Jean-Karim Hériché, Jutta Bulkescher, Holger Erfle, Christian Conrad, Phill Rogers, Ina Poser, Michael Held, Urban Liebel, Cihan Cetin, Frank Sieckmann, Gregoire Pau, Rolf Kabbe, Annelie Wünsche, Venkata Satagopam, Michael H A Schmitz, Catherine Chapuis, Daniel W Gerlich, Reinhard Schneider, Roland Eils, Wolfgang Huber, Jan-Michael Peters, Anthony A. Hyman, Richard Durbin, Rainer Pepperkok, Jan Ellenberg
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Nature, 464(7289) 721-727 (2010)
PDF DOI

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.

Nadja C Hübner, Lily Hui-Ching Wang, Manuel Kaulich, Patrick Descombes, Ina Poser, Erich A Nigg
Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs.
Chromosoma, 119(2) 149-165 (2010)
PDF DOI

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101-114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1-Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of "bypass suppression." In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling.

Marc Lenoir, Uenal Coskun, Michal Grzybek, Xinwang Cao, Sabine Barbara Buschhorn, Jonathan James, Kai Simons, Michael Overduin
Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domains.
EMBO Rep, 11(4) 279-284 (2010)
PDF DOI

The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4-phosphate (PtdIns(4)P) ligand and four-phosphate-adaptor protein (FAPP) modules have been defined. The micelle-bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full-length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.

Kai Simons
New beginnings: getting the kick I needed
Nat Cell Biol, 12(3) Art. No. 202 (2010)
PDF DOI

Abstract Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

Nathaniel Peters, Dahlia E Perez, Mi Hye Song, Yan Liu, Thomas Müller-Reichert, Cathy Caron, Kenneth J. Kemphues, Kevin F O'Connell
Control of mitotic and meiotic centriole duplication by the Plk4-related kinase ZYG-1.
J Cell Sci, 123(Pt 5) 795-805 (2010)
PDF DOI

Centriole duplication is of crucial importance during both mitotic and male meiotic divisions, but it is currently not known whether this process is regulated differently during the two modes of division. In Caenorhabditis elegans, the kinase ZYG-1 plays an essential role in both mitotic and meiotic centriole duplication. We have found that the C-terminus of ZYG-1 is necessary and sufficient for targeting to centrosomes and is important for differentiating mitotic and meiotic centriole duplication. Small truncations of the C-terminus dramatically lower the level of ZYG-1 at mitotic centrosomes but have little effect on the level of ZYG-1 at meiotic centrosomes. Interestingly, truncation of ZYG-1 blocks centrosome duplication in the mitotic cycle but leads to centrosome amplification in the meiotic cycle. Meiotic centriole amplification appears to result from the overduplication of centrioles during meiosis I and leads to the formation of multipolar meiosis II spindles. The extra centrioles also disrupt spermatogenesis by inducing the formation of supernumerary fertilization-competent spermatids that contain abnormal numbers of chromosomes and centrioles. Our data reveal differences in the regulation of mitotic and meiotic centrosome duplication, particularly with regard to ZYG-1 activity, and reveal an important role for centrosomes in spermatid formation.

René Martin, Eugeni V. Entchev, Teymuras V. Kurzchalia, Hans-Joachim Knölker
Steroid hormones controlling the life cycle of the nematode Caenorhabditis elegans: stereoselective synthesis and biology.
Org Biomol Chem, 8(4) 739-750 (2010)
PDF DOI

Cholesterol-derived hormones, the dafachronic acids, play a major role in controlling the life cycle and initiating dauer larva formation of the nematode Caenorhabditis elegans. This Perspective describes recent progress in the synthesis of these steroid hormones and their biological function.

Iva M. Tolic-Norrelykke
Force and length regulation in the microtubule cytoskeleton: lessons from fission yeast.
Curr Opin Cell Biol, 22(1) 21-28 (2010)
PDF DOI

How does a living cell deal with basic concepts of physics such as length and force? The cell has to measure distances and regulate forces to dynamically organize its interior. This is to a large extent based on microtubules (MTs) and motor proteins. Two concepts are emerging from recent studies as key to the positioning of cell components: preferred disassembly of longer MTs and preferred detachment of motors under high load force. The role of these concepts in nuclear centering and nuclear oscillations is coming to light from experimental and theoretical studies in fission yeast. These universal concepts are likely crucial for a variety of cell processes, including nuclear and mitotic spindle positioning, control of spindle length, and chromosome congression on the metaphase plate.

Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
Treslin collaborates with TopBP1 in triggering the initiation of DNA replication.
Cell, 140(3) 349-359 (2010)
PDF DOI

TopBP1 has important roles in both DNA replication and checkpoint regulation in vertebrates. We have identified a protein called Treslin that associates with TopBP1 in Xenopus egg extracts. Depletion of Treslin from egg extracts strongly inhibits chromosomal DNA replication. Binding of Treslin to chromatin in egg extracts occurs independently of TopBP1. However, loading of the initiator protein Cdc45 onto chromatin cannot take place in the absence of Treslin. Prior to the initiation of DNA replication, Treslin associates with TopBP1 in a Cdk2-dependent manner. Ablation of Treslin from human cells also strongly inhibits DNA replication. Taken together, these results indicate that Treslin and TopBP1 collaborate in the Cdk2-mediated loading of Cdc45 onto replication origins. Thus, Treslin regulates a pivotal step in the initiation of DNA replication in vertebrates.

Marcus Gentsch^*, Aneta Kaczmarczyk^*, Karin van Leeuwen, Martin de Boer, Magdalena Kaus-Drobek, Marie-Claire Dagher, Petra Kaiser, Peter D Arkwright, Manfred Gahr, Angela Rösen-Wolff, Matthias Bochtler, Elizabeth Secord, Pamela Britto-Williams, Gulam Mustafa Saifi, Anne Maddalena, Ghassan Dbaibo, Jacinta Bustamante, Jean-Laurent Casanova, Dirk Roos^*, Joachim Roesler^*
Alu-repeat-induced deletions within the NCF2 gene causing p67-phox-deficient chronic granulomatous disease (CGD).
Hum Mutat, 31(2) 151-158 (2010)
PDF DOI

Mutations that impair expression or function of the components of the phagocyte NADPH oxidase complex cause chronic granulomatous disease (CGD), which is associated with life-threatening infections and dysregulated granulomatous inflammation. In five CGD patients from four consanguineous families of two different ethnic backgrounds, we found similar genomic homozygous deletions of 1,380 bp comprising exon 5 of NCF2, which could be traced to Alu-mediated recombination events. cDNA sequencing showed in-frame deletions of phase zero exon 5, which encodes one of the tandem repeat motifs in the tetratricopeptide (TPR4) domain of p67-phox. The resulting shortened protein (p67Delta5) had a 10-fold reduced intracellular half-life and was unable to form a functional NADPH oxidase complex. No dominant negative inhibition of oxidase activity by p67Delta5 was observed. We conclude that Alu-induced deletion of the TPR4 domain of p67-phox leads to loss of function and accelerated degradation of the protein, and thus represents a new mechanism causing p67-phox-deficient CGD.

Jie Shen, Christian Dahmann, Gert Pflugfelder
Spatial discontinuity of optomotor-blind expression in the Drosophila wing imaginal disc disrupts epithelial architecture and promotes cell sorting.
BMC Dev Biol, 10 Art. No. 23 (2010)
PDF DOI

BACKGROUND: Decapentaplegic (Dpp) is one of the best characterized morphogens, required for dorso-ventral patterning of the Drosophila embryo and for anterior-posterior (A/P) patterning of the wing imaginal disc. In the larval wing pouch, the Dpp target gene optomotor-blind (omb) is generally assumed to be expressed in a step function above a certain threshold of Dpp signaling activity. RESULTS: We show that the transcription factor Omb forms, in fact, a symmetrical gradient on both sides of the A/P compartment boundary. Disruptions of the Omb gradient lead to a re-organization of the epithelial cytoskeleton and to a retraction of cells toward the basal membrane suggesting that the Omb gradient is required for correct epithelial morphology. Moreover, by analysing the shape of omb gain- and loss-of-function clones, we find that Omb promotes cell sorting along the A/P axis in a concentration-dependent manner. CONCLUSIONS: Our findings show that Omb distribution in the wing imaginal disc is described by a gradient rather than a step function. Graded Omb expression is necessary for normal cell morphogenesis and cell affinity and sharp spatial discontinuities must be avoided to allow normal wing development.

Garrett Greenan
Setting mitotic spindle length in the early Caenorhabditis elegans embryo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Nick Barker, Meritxell Huch, Pekka Kujala, Marc van de Wetering, Hugo J Snippert, Johan H van Es, Toshiro Sato, Daniel E Stange, Harry Begthel, Maaike van den Born, Esther Danenberg, Stieneke van den Brink, Jeroen Korving, Arie Abo, Peter J Peters, Nick Wright, Richard Poulsom, Hans Clevers
Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.
Cell Stem Cell, 6(1) 25-36 (2010)
DOI

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Jésica Díaz-Vera, Yézer G Morales, Juan R Hernández-Fernaud, Marcial Camacho, Mónica S Montesinos, Federico Calegari, Wieland B. Huttner, Ricardo Borges, José D Machado
Chromogranin B gene ablation reduces the catecholamine cargo and decelerates exocytosis in chromaffin secretory vesicles.
J Neurosci, 30(3) 950-957 (2010)
PDF DOI

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb(+/+) [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (approximately 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

Valeria Sordi, Raffaella Melzi, Alessia Mercalli, Roberta Formicola, Claudio Doglioni, Francesca Tiboni, Giuliana Ferrari, Rita Nano, Karolina Chwalek, Eckhard Lammert, Enzio Bonifacio, Danielle Borg, Lorenzo Piemonti
Mesenchymal cells appearing in pancreatic tissue culture are bone marrow-derived stem cells with the capacity to improve transplanted islet function.
Stem Cells, 28(1) 140-151 (2010)
PDF DOI

Adherent fibroblast-like cells have been reported to appear in cultures of human endocrine or exocrine pancreatic tissue during attempts to differentiate human beta cells from pancreatic precursors. A thorough characterization of these mesenchymal cells has not yet been completed, and there are no conclusive data about their origin.We demonstrated that the human mesenchymal cells outgrowing from cultured human pancreatic endocrine or exocrine tissue are pancreatic mesenchymal stem cells (pMSC) that propagate from contaminating pMSC. The origin of pMSC is partly extrapancreatic both in humans and mice, and by using green fluorescent protein (GFP(+)) bone marrow transplantation in the mouse model, we were able to demonstrate that these cells derive from the CD45(+) component of bone marrow. The pMSC express negligible levels of islet-specific genes both in basal conditions and after serum deprivation or exogenous growth factor exposure, and might not represent optimal candidates for generation of physiologically competent beta-cells. On the other hand, when cotransplanted with a minimal pancreatic islet mass, pMSC facilitate the restoration of normoglycemia and the neovascularization of the graft. These results suggest that pMSCs could exert an indirect role of "helper" cells in tissue repair processes.

Grzegorz Chojnowski, Katarzyna Breer, Marta Narczyk, Beata Wielgus-Kutrowska, Honorata Czapinska, Mariko Hashimoto, Sadao Hikishima, Tsutomu Yokomatsu, Matthias Bochtler, Agnieszka Girstun, Krzysztof Staron, Agnieszka Bzowska
1.45 A resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state.
Biochem Biophys Res Commun, 391(1) 703-708 (2010)
PDF DOI

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

Omar Awile, Ömer Demirel, Ivo F. Sbalzarini
Toward an Object-Oriented Core of the PPM Library
In: Numerical Analysis and Applied Mathematics : International Conference on Numerical Analysis and Applied Mathematics ; Rhodes, Greece, 19 - 25 September 2010 ; ICNAAM 2010 (2010)(Eds.) Theodore E. Simos AIP conference proceedings ; 1281, Melville, N.Y., AIP (2010), 1313-1316
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Denis Corbeil, Anne-Marie Marzesco, Christine A. Fargeas, Wieland B. Huttner
Prominin-1: a distinct cholesterol-binding membrane protein and the organisation of the apical plasma membrane of epithelial cells.
Subcell Biochem, 51 399-423 (2010)
DOI

The apical plasma membrane of polarized epithelial cells is composed of distinct subdomains, that is, planar regions and protrusions (microvilli, primary cilium), each of which are constructed from specific membrane microdomains. Assemblies containing the pentaspan glycoprotein prominin-1 and certain membrane lipids, notably cholesterol, are characteristic features of these microdomains in apical membrane protrusions. Here we highlight the recent findings concerning the molecular architecture of the apical plasma membrane of epithelial cells and its dynamics. The latter is illustrated by the budding and fission of prominin-1-containing membrane vesicles from apical plasma membrane protrusions, which is controlled, at least in part, by the level of membrane cholesterol and the cholesterol-dependent organization of membrane microdomains.

Katharina Landsberg
The role of cell bond tension in directing cell sorting at the anteroposterior compartment boundary in wing imaginal discs of Drosophila melanogaster
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2010)

Omar Awile, Anita Krisko, Ivo F. Sbalzarini, Bojan Zagrovic
Intrinsically disordered regions may lower the hydration free energy in proteins: a case study of nudix hydrolase in the bacterium Deinococcus radiodurans.
PLoS Comput Biol, 6(7) 1000854-1000854 (2010)
Open Access PDF DOI

The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the "surface-properties" of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity.

Rajesh Ramaswamy, Ivo F. Sbalzarini
A partial-propensity variant of the composition-rejection stochastic simulation algorithm for chemical reaction networks
J Chem Phys, 132(4) Art. No. 044102 (2010)
PDF DOI

We present the partial-propensity stochastic simulation algorithm with composition-rejection sampling (PSSA-CR). It is an exact formulation of the stochastic simulation algorithm (SSA) for well-stirred systems of coupled chemical reactions. The new formulation is a partial-propensity variant [R. Ramaswamy, N. Gonzalez-Segredo, and I. F. Sbalzarini, J. Chem. Phys. 130, 244104 (2009)] of the composition- rejection SSA [A. Slepoy, A. P. Thompson, and S. J. Plimpton, J. Chem. Phys. 128, 205101 (2008)]. The computational cost of this new formulation is bounded by a constant for weakly coupled reaction networks, and it increases at most linearly with the number of chemical species for strongly coupled reaction networks. PSSA-CR thus combines the advantages of partial-propensity methods and the composition-rejection SSA, providing favorable scaling of the computational cost for all classes of reaction networks.

Thomas Kurth, Jürgen Berger, Michaela Wilsch-Bräuninger, Susanne Kretschmar, Robert Cerny, Heinz Schwarz, Jan Löfberg, Thomas Piendl, Hans-Henning Epperlein
Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.
Methods Cell Biol, 96 395-423 (2010)
PDF DOI

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.

Thomas Widmann
The role of Dpp and Wingless signaling gradients in directing cell shape during Drosophila wing imaginal disc development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Ivana Viktorinová, Tina König, Karin Schlichting, Christian Dahmann
The cadherin Fat2 is required for planar cell polarity in the Drosophila ovary.
Development, 136(24) 4123-4132 (2009)
PDF DOI

Planar cell polarity is an important characteristic of many epithelia. In the Drosophila wing, eye and abdomen, establishment of planar cell polarity requires the core planar cell polarity genes and two cadherins, Fat and Dachsous. Drosophila Fat2 is a cadherin related to Fat; however, its role during planar cell polarity has not been studied. Here, we have generated mutations in fat2 and show that Fat2 is required for the planar polarity of actin filament orientation at the basal side of ovarian follicle cells. Defects in actin filament orientation correlate with a failure of egg chambers to elongate during oogenesis. Using a functional fosmid-based fat2-GFP transgene, we show that the distribution of Fat2 protein in follicle cells is planar polarized and that Fat2 localizes where basal actin filaments terminate. Mosaic analysis demonstrates that Fat2 acts non-autonomously in follicle cells, indicating that Fat2 is required for the transmission of polarity information. Our results suggest a principal role for Fat-like cadherins during the establishment of planar cell polarity.

Barbara Kind, Katrin Koehler, Mike Lorenz, Angela Huebner
The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope.
Biochem Biophys Res Commun, 390(2) 205-210 (2009)
PDF DOI

The nuclear pore complex (NPC) consists of approximately 30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

Nicholas Licata, Stephan W. Grill
The first-passage problem for diffusion through a cylindrical pore with sticky walls.
Eur Phys J E Soft Matter, 30(4) 439-447 (2009)
PDF DOI

We calculate the first-passage time distribution for diffusion through a cylindrical pore with sticky walls. A particle diffusively explores the interior of the pore through a series of binding and unbinding events with the cylinder wall. Through a diagrammatic expansion we obtain first-passage time statistics for the particle's exit from the pore. Connections between the model and nucleocytoplasmic transport in cells are discussed.

Mélisande Richard^*, Nadine Muschalik^*, Ferdi Grawe, Susann Ozüyaman, Elisabeth Knust
A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells.
Eur J Cell Biol, 88(12) 765-777 (2009)
PDF DOI

Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organizes an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy.

Katharina Landsberg^*, Reza Farhadifar^*, Jonas Ranft^*, Daiki Umetsu^*, Thomas Widmann, Thomas Bittig, Amani Said, Frank Jülicher, Christian Dahmann
Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary.
Curr Biol, 19(22) 1950-1955 (2009)
PDF DOI

Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development [1-6]. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues [1-6]. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. Here we quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. We found that mechanical tension is approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632 [7], an inhibitor of Rho-kinase whose main effector is Myosin II [8]. Simulations using a vertex model [9] demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. Our results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.

Albert Cardona, Stephan Saalfeld, Pavel Tomancák, Volker Hartenstein
Drosophila Brain Development: Closing the Gap between a Macroarchitectural and Microarchitectural Approach.
Cold Spring Harb Symp Quant Biol, 74 235-248 (2009)
PDF DOI

Neurobiologists address neural structure, development, and function at the level of "macrocircuits" (how different brain compartments are interconnected; what overall pattern of activity they produce) and at the level of "microcircuits" (how connectivity and physiology of individual neurons and their processes within a compartment determine the functional output of this compartment). Work in our lab aims at reconstructing the developing Drosophila brain at both levels. Macrocircuits can be approached conveniently by reconstructing the pattern of brain lineages, which form groups of neurons whose projections form cohesive fascicles interconnecting the compartments of the larval and adult brain. The reconstruction of microcircuits requires serial section electron microscopy, due to the small size of terminal neuronal processes and their synaptic contacts. Because of the amount of labor that traditionally comes with this approach, very little is known about microcircuitry in brains across the animal kingdom. Many of the problems of serial electron microscopy reconstruction are now solvable with digital image recording and specialized software for both image acquisition and postprocessing. In this chapter, we introduce our efforts to reconstruct the small Drosophila larval brain and discuss our results in light of the published data on neuropile ultrastructure in other animal taxa.

Marzuk Mahmud Kamal, Deryck Mills, Michal Grzybek, Jonathon Howard
Measurement of the membrane curvature preference of phospholipids reveals only weak coupling between lipid shape and leaflet curvature.
Proc Natl Acad Sci U.S.A., 106(52) 22245-22250 (2009)
PDF DOI

In biological processes, such as fission, fusion and trafficking, it has been shown that lipids of different shapes are sorted into regions with different membrane curvatures. This lipid sorting has been hypothesized to be due to the coupling between the membrane curvature and the lipid's spontaneous curvature, which is related to the lipid's molecular shape. On the other hand, theoretical predictions and simulations suggest that the curvature preference of lipids, due to shape alone, is weaker than that observed in biological processes. To distinguish between these different views, we have directly measured the curvature preferences of several lipids by using a fluorescence-based method. We prepared small unilamellar vesicles of different sizes with a mixture of egg-PC and a small mole fraction of N-nitrobenzoxadiazole (NBD)-labeled phospholipids or lysophospholipids of different chain lengths and saturation, and measured the NBD equilibrium distribution across the bilayer. We observed that the transverse lipid distributions depended linearly on membrane curvature, allowing us to measure the curvature coupling coefficient. Our measurements are in quantitative agreement with predictions based on earlier measurements of the spontaneous curvatures of the corresponding nonfluorescent lipids using X-ray diffraction. We show that, though some lipids have high spontaneous curvatures, they nevertheless showed weak curvature preferences because of the low values of the lipid molecular areas. The weak curvature preference implies that the asymmetric lipid distributions found in biological membranes are not likely to be driven by the spontaneous curvature of the lipids, nor are lipids discriminating sensors of membrane curvature.

Yanmei Liu, Hassan Mziaut, Anna Ivanova, Michele Solimena
beta-Cells at the crossroads: choosing between insulin granule production and proliferation.
Diabetes Obes Metab, 11(Suppl. 4) 54-64 (2009)
PDF DOI

Pancreatic beta-cells are the sole source of insulin, the major hormonal regulator of glycaemia. In physiological and pathological conditions with increased insulin demand, beta-cells adjust their insulin output either through increased insulin secretory granule (ISG) biogenesis and secretion, or hyperplasia. Failure of these compensatory mechanisms eventually results in hyperglycaemia and diabetes mellitus. Both of these major adaptive behaviours are positively regulated by several extrinsic factors, such as glucose, GLP-1, insulin and growth hormones (GH). Still unclear, however, it is how beta-cells in response to these stimuli opt for one or the other strategy at a given time. Here we review recent advances concerning the factors and pathways that enhance ISG biogenesis and beta-cell replication, and propose the existence of 'switch factors' that play a key role in regulating the shift between these two adaptive responses.

Dirk Benndorf, Carsten Vogt, Nico Jehmlich, Yvonne Schmidt, Henrik Thomas, Gary Woffendin, Andrej Shevchenko, Hans-Hermann Richnow, Martin von Bergen
Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments.
Biodegradation, 20(6) 737-750 (2009)
PDF DOI

BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community.

Marina Le Guédard, Jean-Jacques Bessoule, Valérie Boyer, Sophie Ayciriex, Gisèle Velours, Willem Kulik, Christer S. Ejsing, Andrej Shevchenko, Denis Coulon, René Lessire, Eric Testet
PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast.
FEBS J, 276(21) 6412-6424 (2009)
PDF DOI

In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either phosphatidylinositol is synthesized from distinct cytidine diphosphate-diacylglycerol molecules, or that, after its synthesis, it is modified by a hypothetical acyltransferase that incorporates saturated fatty acid into neo-synthesized molecules of phosphatidylinositol. We used database search methods to identify an acyltransferase that could catalyze such an activity. Among the various proteins that we studied, we found that Psi1p (phosphatidylinositol stearoyl incorporating 1 protein) is required for the incorporation of stearate into phosphatidylinositol because GC and MS analyses of psi1Delta lipids revealed an almost complete disappearance of stearic (but not of palmitic acid) at the sn-1 position of this phospholipid. Moreover, it was found that, whereas glycerol 3-phosphate, lysophosphatidic acid and 1-acyl lysophosphatidylinositol acyltransferase activities were similar in microsomal membranes isolated from wild-type and psi1Delta cells, microsomal membranes isolated from psi1Delta cells are devoid of the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity that is present in microsomal membranes isolated from wild-type cells. Moreover, after the expression of PSI1 in transgenic psi1Delta cells, the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity was recovered, and was accompanied by a strong increase in the stearic acid content of lysophosphatidylinositol. As previously suggested for phosphatidylinositol from animal cells (which contains almost exclusively stearic acid as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol.

Luke Buchanan, Mickaël Durand-Dubief, Assen Roguev, Cagri Sakalar, Brian Wilhelm, Annelie Strålfors, Anna Shevchenko, Rein Aasland, Andrej Shevchenko, Karl Ekwall, A. Francis Stewart
The Schizosaccharomyces pombe JmjC-protein, Msc1, prevents H2A.Z localization in centromeric and subtelomeric chromatin domains.
PLoS Genet, 5(11) Art. No. e1000726 (2009)
PDF DOI

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.

Dirk Helbing, Andreas Deutsch, Stefan Diez, Karsten Peters, Yannis Kalaidzidis, Kathrin Padberg-Gehle, Stefan Lämmer, Anders Johansson, Georg Breier, Frank Schulze, Marino Zerial
Biologistics and the struggle for efficiency: concepts and perspectives
Adv complex systems, 12(6) 533-548 (2009)
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The growth of world population, limitation of resources, economic problems, and environmental issues force engineers to develop increasingly efficient solutions for logistic systems. Pure optimization for efficiency, however, has often led to technical solutions that are vulnerable to variations in supply and demand, and to perturbations. In contrast, nature already provides a large variety of efficient, flexible, and robust logistic solutions. Can we utilize biological principles to design systems, which can flexibly adapt to hardly predictable, fluctuating conditions? We propose a bio-inspired "BioLogistics" approach to deduce dynamic organization processes and principles of adaptive self-control from biological systems, and to transfer them to man-made logistics (including nanologistics), using principles of modularity, self-assembly, self-organization, and decentralized coordination. Conversely, logistic models can help revealing the logic of biological processes at the systems level.

Thomas Widmann, Christian Dahmann
Wingless signaling and the control of cell shape in Drosophila wing imaginal discs.
Dev Biol, 334(1) 161-173 (2009)
PDF DOI

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer beta-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.

Alexander Picker, Florencia Cavodeassi, Anja Machate, Sabine Bernauer, Stefan Hans, Gembu Abe, Koichi Kawakami, Stephen W. Wilson, Michael Brand
Dynamic coupling of pattern formation and morphogenesis in the developing vertebrate retina.
PLoS Biol, 7(10) Art. No. e1000214 (2009)
Open Access PDF DOI

During embryonic development, pattern formation must be tightly synchronized with tissue morphogenesis to coordinate the establishment of the spatial identities of cells with their movements. In the vertebrate retina, patterning along the dorsal-ventral and nasal-temporal (anterior-posterior) axes is required for correct spatial representation in the retinotectal map. However, it is unknown how specification of axial cell positions in the retina occurs during the complex process of early eye morphogenesis. Studying zebrafish embryos, we show that morphogenetic tissue rearrangements during eye evagination result in progenitor cells in the nasal half of the retina primordium being brought into proximity to the sources of three fibroblast growth factors, Fgf8/3/24, outside the eye. Triple-mutant analysis shows that this combined Fgf signal fully controls nasal retina identity by regulating the nasal transcription factor Foxg1. Surprisingly, nasal-temporal axis specification occurs very early along the dorsal-ventral axis of the evaginating eye. By in vivo imaging GFP-tagged retinal progenitor cells, we find that subsequent eye morphogenesis requires gradual tissue compaction in the nasal half and directed cell movements into the temporal half of the retina. Balancing these processes drives the progressive alignment of the nasal-temporal retina axis with the anterior-posterior body axis and is controlled by a feed-forward effect of Fgf signaling on Foxg1-mediated cell cohesion. Thus, the mechanistic coupling and dynamic synchronization of tissue patterning with morphogenetic cell behavior through Fgf signaling leads to the graded allocation of cell positional identity in the eye, underlying retinotectal map formation.

Lukasz Wojtasz^*, Katrin Daniel^*, Ignasi Roig, Ewelina Bolcun-Filas, Hongzi Xu, Verawan Boonsanay, Christian R. Eckmann, Howard J Cooke, Maria Jasin, Scott Keeney, Michael J McKay, Attila Toth
Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins, are depleted from synapsed chromosome axes with the help of TRIP13 AAA-ATPase.
PLoS Genet, 5(10) Art. No. e1000702 (2009)
PDF DOI

Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins--HORMAD1 and HORMAD2--in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals.

Caren Norden^*, Stephen Young^*, Brian A Link, William A Harris
Actomyosin is the main driver of interkinetic nuclear migration in the retina.
Cell, 138(6) 1195-1208 (2009)
PDF DOI

Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.

Christian Lange, Wieland B. Huttner, Federico Calegari
Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.
Cell Stem Cell, 5(3) 320-331 (2009)
PDF DOI

During mouse embryonic development, neural progenitors lengthen the G1 phase of the cell cycle and this has been suggested to be a cause, rather than a consequence, of neurogenesis. To investigate whether G1 lengthening alone may cause the switch of cortical progenitors from proliferation to neurogenesis, we manipulated the expression of cdk/cyclin complexes and found that cdk4/cyclinD1 overexpression prevents G1 lengthening without affecting cell growth, cleavage plane, or cell cycle synchrony with interkinetic nuclear migration. Specifically, overexpression of cdk4/cyclinD1 inhibited neurogenesis while increasing the generation and expansion of basal (intermediate) progenitors, resulting in a thicker subventricular zone and larger surface area of the postnatal cortex originating from cdk4/cyclinD1-transfected progenitors. Conversely, lengthening of G1 by cdk4/cyclinD1-RNAi displayed the opposite effects. Thus, G1 lengthening is necessary and sufficient to switch neural progenitors to neurogenesis, and overexpression of cdk4/cyclinD1 can be used to increase progenitor expansion and, perhaps, cortical surface area.

René Martin, Eugeni V. Entchev, Frank Däbritz, Teymuras V. Kurzchalia, Hans-Joachim Knölker
Synthesis and Hormonal Activity of the (25S)-Cholesten-26-oic Acids – Potent Ligands for the DAF-12 Receptor in Caenorhabditis elegans
Eur J Org Chem, (22) 3703-3714 (2009)
PDF DOI

Using a highly stereoselective Evans aldol reaction for the introduction of the stereogenic center at C-25, we describe an efficient synthesis of the orthogonally diprotected (25S)-26-hydroxycholesterol 11. In a few synthetic steps, this crucial intermediate 11 has been converted into the four (25S)-cholesten-26-oic acids 1–4, which have been obtained in 12–15 steps and 19–53% overall yield based on commerciallyavailable 3β-hydroxychol-5-en-24-oic acid (5). Our biological studies of the compounds 1–4 reveal that (25S)-Δ7-dafachronic acid (1) represents the most active steroidal ligand for the hormonal receptor DAF-12 in Caenorhabditis elegans. Moreover, the aturated (25S)-dafachronic acid (3) represents a new ligand for this receptor and the (25S)-steroidal acids are more active as compared to their corresponding (25R)-counterparts.

Frank Buchholz
Engineering DNA processing enzymes for the postgenomic era.
Curr Opin Biotechnol, 20(4) 383-389 (2009)
PDF DOI

DNA has been a main focus of biomedical research ever since its discovery as the hereditary molecule. This discovery laid the foundation for scientists to begin both to elucidate and to manipulate its function. Since then many DNA processing enzymes have been discovered and many technologies have been developed to process and manipulate DNA with these enzymes. The sequencing of entire genomes has increased the demand for sophisticated DNA processing enzymes. This need is being addressed by engineering DNA processing enzymes using rational and evolutionary approaches to improve or design novel properties. It now appears feasible that engineered DNA processing enzymes will find utility in molecular medicine as tools for advanced genome engineering.

Natalia Bulgakova, Elisabeth Knust
The Crumbs complex: from epithelial-cell polarity to retinal degeneration.
J Cell Sci, 122(Pt 15) 2587-2596 (2009)
PDF DOI

The evolutionarily conserved Crumbs protein complex is a key regulator of cell polarity and cell shape in both invertebrates and vertebrates. The important role of this complex in normal cell function is illustrated by the finding that mutations in one of its components, Crumbs, are associated with retinal degeneration in humans, mice and flies. Recent results suggest that the Crumbs complex plays a role in the development of other disease processes that are based on epithelial dysfunction, such as tumorigenesis or the formation of cystic kidneys. Localisation of the complex is restricted to a distinct region of the apical plasma membrane that abuts the zonula adherens in epithelia and photoreceptor cells of invertebrates and vertebrates, including humans. In addition to the core components, a variety of other proteins can be recruited to the complex, depending on the cell type and/or developmental stage. Together with diverse post-transcriptional and post-translational mechanisms that regulate the individual components, this provides an enormous functional diversity and flexibility of the complex. In this Commentary, we summarise findings concerning the organisation and modification of the Crumbs complex, and the conservation of its constituents from flies to mammals. In addition, we discuss recent results that suggest its participation in various human diseases, including blindness and tumour formation.

Lucie Kalvodova^*, Julio Sampaio^*, Sandra Cordo, Christer S. Ejsing, Andrej Shevchenko, Kai Simons
The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry.
J Virol, 83(16) 7996-8003 (2009)
PDF DOI

Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.

Michael Cammer^*, Jean-Claude Gevrey^*, Mike Lorenz, Athanassios Dovas, John S Condeelis, Dianne Cox
The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo: a role for phosphatidylinositol 3-kinase and Cdc42.
J Biol Chem, 284(35) 23302-23311 (2009)
PDF DOI

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.

Michael Buchert, Charbel Darido, Ebba Lagerqvist, Anna Sedello, Chantal Cazevieille, Frank Buchholz, Jean-Francois Bourgaux, Julie Pannequin, Dominique Joubert, Frédéric Hollande
The symplekin/ZONAB complex inhibits intestinal cell differentiation by the repression of AML1/Runx1.
Gastroenterology, 137(1) 156-164 (2009)
PDF DOI

BACKGROUND & AIMS: Symplekin is a ubiquitously expressed protein involved in RNA polyadenylation and transcriptional regulation that localizes at tight junctions in epithelial cells. The association between symplekin and the Y-box transcription factor ZONAB activates proliferation in intestinal and kidney cells. We analyzed symplekin expression in human colonic crypts and investigated its function in differentiation. METHODS: Expression of differentiation markers and transcription factors was assessed in HT29-Cl.16E cells that expressed inducible symplekin short hairpin RNA or were transfected with ZONAB small interfering RNAs. Intestines of AML1(Delta/Delta) mice were stained with alcian blue and analyzed for expression of AML1/Runx1, GAPDH, KLF-4, and Muc-2. Mobility shift and chromatin immunoprecipitation were used to detect AML1 and ZONAB/DbpA binding to promoter regions of the Krüppel-like factor 4 (KLF4) and acute myeloid leukemia-1 (AML1) genes, respectively. RESULTS: The gradient of nuclear symplekin expression decreased from the proliferative toward the differentiated compartment of colonic crypts; symplekin down-regulation promoted the differentiation of HT29-Cl.16E colorectal carcinoma cells into goblet cells. Down-regulation of symplekin or ZONAB/Dbpa induced de novo expression of the transcription factor AML1/Runx1, thereby increasing the expression of KLF4 and promoting goblet cell differentiation. Furthermore, increased AML1 expression was required for the induction of goblet cell differentiation after symplekin down-regulation. KLF4 expression and goblet cell numbers were reduced in the intestines of AML1(Delta/Delta) mice, confirming the role of AML1 as a promoter of intestinal differentiation in vivo. CONCLUSIONS: Symplekin cooperates with ZONAB to negatively regulate intestinal goblet cell differentiation, acting by repression of AML1 and KLF4.

Riikka Hynynen^*, Monika Suchanek^*, Johanna Spandl, Nils Bäck, Christoph Thiele, Vesa M Olkkonen
OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids.
J Lipid Res, 50(7) 1305-1315 (2009)
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Oxysterol binding protein-related protein 2 (ORP2) is a member of the oxysterol binding protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K(d) 1.4 x 10(-8) M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 microM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [(14)C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.

Elke Bayha, Mette C Jørgensen, Palle Serup, Anne Grapin-Botton
Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis.
PLoS ONE, 4(6) 5845-5845 (2009)
Open Access DOI

Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.

Christian L. Müller, Ivo F. Sbalzarini, Wilfred F van Gunsteren, Bojan Zagrović, Philippe H Hünenberger
In the eye of the beholder: Inhomogeneous distribution of high-resolution shapes within the random-walk ensemble
J Chem Phys, 130(21) Art. No. 214904 (2009)
PDF DOI

The concept of high-resolution shapes (also referred to as folds or states, depending on the context) of a polymer chain plays a central role in polymer science, structural biology, bioinformatics, and biopolymer dynamics. However, although the idea of shape is intuitively very useful, there is no unambiguous mathematical definition for this concept. In the present work, the distributions of high-resolution shapes within the ideal random-walk ensembles with N=3,...,6 beads (or up to N=10 for some properties) are investigated using a systematic (grid-based) approach based on a simple working definition of shapes relying on the root-mean-square atomic positional deviation as a metric (i.e., to define the distance between pairs of structures) and a single cutoff criterion for the shape assignment. Although the random-walk ensemble appears to represent the paramount of homogeneity and randomness, this analysis reveals that the distribution of shapes within this ensemble, i.e., in the total absence of interatomic interactions characteristic of a specific polymer (beyond the generic connectivity constraint), is significantly inhomogeneous. In particular, a specific (densest) shape occurs with a local probability that is 1.28, 1.79, 2.94, and 10.05 times (N=3,...,6) higher than the corresponding average over all possible shapes (these results can tentatively be extrapolated to a factor as large as about 10(28) for N=100). The qualitative results of this analysis lead to a few rather counterintuitive suggestions, namely, that, e.g., (i) a fold classification analysis applied to the random-walk ensemble would lead to the identification of random-walk "folds;" (ii) a clustering analysis applied to the random-walk ensemble would also lead to the identification random-walk "states" and associated relative free energies; and (iii) a random-walk ensemble of polymer chains could lead to well-defined diffraction patterns in hypothetical fiber or crystal diffraction experiments. The inhomogeneous nature of the shape probability distribution identified here for random walks may represent a significant underlying baseline effect in the analysis of real polymer chain ensembles (i.e., in the presence of specific interatomic interactions). As a consequence, a part of what is called a polymer shape may actually reside just "in the eye of the beholder" rather than in the nature of the interactions between the constituting atoms, and the corresponding observation-related bias should be taken into account when drawing conclusions from shape analyses as applied to real structural ensembles.

Gero Fink^*, Lukasz Hajdo^*, Krzysztof J Skowronek, Cordula Reuther, Andrzej A Kasprzak, Stefan Diez
The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding.
Nat Cell Biol, 11(6) 717-723 (2009)
PDF DOI

During mitosis and meiosis, the bipolar spindle facilitates chromosome segregation through microtubule sliding as well as microtubule growth and shrinkage. Kinesin-14, one of the motors involved, causes spindle collapse in the absence of kinesin-5 (Refs 2, 3), participates in spindle assembly and modulates spindle length. However, the molecular mechanisms underlying these activities are not known. Here, we report that Drosophila melanogaster kinesin-14 (Ncd) alone causes sliding of anti-parallel microtubules but locks together (that is, statically crosslinks) those that are parallel. Using single molecule imaging we show that Ncd diffuses along microtubules in a tail-dependent manner and switches its orientation between sliding microtubules. Our results show that kinesin-14 causes sliding and expansion of an anti-parallel microtubule array by dynamic interactions through the motor domain on the one side and the tail domain on the other. This mechanism accounts for the roles of kinesin-14 in spindle organization.

J Thomas Hannich^*, Eugeni V. Entchev^*, Fanny Mende, Hristio Boytchev, René Martin, Vyacheslav Zagoriy, Gabriele Theumer, Isabelle Riezman, Howard Riezman, Hans-Joachim Knölker, Teymuras V. Kurzchalia
Methylation of the sterol nucleus by STRM-1 regulates dauer larva formation in Caenorhabditis elegans.
Dev Cell, 16(6) 833-843 (2009)
PDF DOI

In response to pheromone(s), Caenorhabditis elegans interrupts its reproductive life cycle and enters diapause as a stress-resistant dauer larva. This decision is governed by a complex system of neuronal and hormonal regulation. All the signals converge onto the nuclear hormone receptor DAF-12. A sterol-derived hormone, dafachronic acid (DA), supports reproductive development by binding to DAF-12 and inhibiting its dauer-promoting activity. Here, we identify a methyltransferase, STRM-1, that modulates DA levels and thus dauer formation. By modifying the substrates that are used for the synthesis of DA, STRM-1 can reduce the amount of hormone produced. Loss of STRM-1 function leads to elevated levels of DA and inefficient dauer formation. Sterol methylation was not previously recognized as a mechanism for regulating hormone activity. Moreover, the C-4 sterol nucleus methylation catalyzed by STRM-1 is unique to nematodes and thus could be a target for therapeutic strategies against parasitic nematode infections.

Monika Sokolowska, Honorata Czapinska, Matthias Bochtler
Crystal structure of the beta beta alpha-Me type II restriction endonuclease Hpy99I with target DNA.
Nucleic Acids Res, 37(11) 3799-3810 (2009)
PDF DOI

The beta beta alpha-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5'-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel beta-barrel and two beta 4 alpha 2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The beta beta alpha-Me region of the second beta 4 alpha 2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second beta 4 alpha 2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I-DNA co-crystal structure provides the first detailed illustration of the beta beta alpha-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).

Denis Corbeil^*, Angret Joester^*, Christine A. Fargeas, József Jászai, Jeremy Garwood, Andrea Hellwig, Hauke B Werner, Wieland B. Huttner
Expression of distinct splice variants of the stem cell marker prominin-1 (CD133) in glial cells.
Glia, 57(8) 860-874 (2009)
PDF DOI

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane glycoprotein specifically associated with plasma membrane protrusions. Prominin-1 is expressed by various stem and progenitor cells, notably neuroepithelial progenitors found in the developing embryonic brain. Here, we further investigated its expression in the murine brain. Biochemical analyses of brain membranes at early stages of development revealed the expression of two distinct splice variants of prominin-1, s1 and s3, which have different cytoplasmic C-terminal domains. The relative abundance of the s3 variant increased toward adulthood, whereas the opposite was observed for the s1 variant. Our combined in situ hybridization and immunohistochemistry revealed the expression of prominin-1 in a subpopulation of Olig-2-positive oligodendroglial cells present within white matter tracts of postnatal and adult brain. Furthermore, immunohistological and biochemical characterization suggested strongly that the s3 variant is a novel component of myelin. Consistent with this, the expression of prominin-1.s3 was significantly reduced in the brain of myelin-deficient mice. Finally, oligodendrocytes expressed selectively the s3 variant whereas GFAP-positive astrocytes expressed the s1 variant in primary glial cell cultures derived from embryonic brains. Collectively, our data demonstrate a complex expression pattern of prominin-1 molecules in developing adult brain. Given that prominin-1 is thought to act as an organizer of plasma membrane protrusions, they further suggest that a specific prominin-1 splice variant might play a role in morphogenesis and/or maintenance of the myelin sheath.

Pia Aanstad, Nicole Santos, Kevin C Corbit, Paul J Scherz, Le A Trinh, Willi Salvenmoser, Jan Huisken, Jeremy F Reiter, Didier Y.R. Stainier
The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling.
Curr Biol, 19(12) 1034-1039 (2009)
DOI

Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.

Jeremy N. Pulvers, Wieland B. Huttner
Brca1 is required for embryonic development of the mouse cerebral cortex to normal size by preventing apoptosis of early neural progenitors.
Development, 136(11) 1859-1868 (2009)
PDF DOI

The extent of apoptosis of neural progenitors is known to influence the size of the cerebral cortex. Mouse embryos lacking Brca1, the ortholog of the human breast cancer susceptibility gene BRCA1, show apoptosis in the neural tube, but the consequences of this for brain development have not been studied. Here we investigated the role of Brca1 during mouse embryonic cortical development by deleting floxed Brca1 using Emx1-Cre, which leads to conditional gene ablation specifically in the dorsal telencephalon after embryonic day (E) 9.5. The postnatal Brca1-ablated cerebral cortex was substantially reduced in size with regard to both cortical thickness and surface area. Remarkably, although the thickness of the cortical layers (except for the upper-most layer) was decreased, cortical layering as such was essentially unperturbed. High levels of apoptosis were found at E11.5 and E13.5, but dropped to near-control levels by E16.5. The apoptosis at the early stage of neurogenesis occurred in both BrdU pulse-labeled neural progenitors and the neurons derived therefrom. No changes were observed in the mitotic index of apical (neuroepithelial, radial glial) progenitors and basal (intermediate) progenitors, indicating that Brca1 ablation did not affect cell cycle progression. Brca1 ablation did, however, result in the nuclear translocation of p53 in neural progenitors, suggesting that their apoptosis involved activation of the p53 pathway. Our results show that Brca1 is required for the cerebral cortex to develop to normal size by preventing the apoptosis of early cortical progenitors and their immediate progeny.

Mirko Theis, Mikolaj Slabicki, Magno Junqueira, Maciej Paszkowski-Rogacz, Jana Sontheimer, Ralf Kittler, Anne-Kristin Heninger, Timo Glatter, Kristi Kruusmaa, Ina Poser, Anthony A. Hyman, Maria Teresa Pisabarro, Matthias Gstaiger, Rudolf Aebersold, Andrej Shevchenko, Frank Buchholz
Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division.
EMBO J, 28(10) 1453-1465 (2009)
PDF DOI

Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses.

Daniel Lingwood, Sebastian Schuck, Charles Ferguson, Mathias J. Gerl, Kai Simons
Generation of cubic membranes by controlled homotypic interaction of membrane proteins in the endoplasmic reticulum.
J Biol Chem, 284(18) 12041-12048 (2009)
PDF DOI

Cell membranes predominantly consist of lamellar lipid bilayers. When studied in vitro, however, many membrane lipids can exhibit non-lamellar morphologies, often with cubic symmetries. An open issue is how lipid polymorphisms influence organelle and cell shape. Here, we used controlled dimerization of artificial membrane proteins in mammalian tissue culture cells to induce an expansion of the endoplasmic reticulum (ER) with cubic symmetry. Although this observation emphasizes ER architectural plasticity, we found that the changed ER membrane became sequestered into large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy may be targeting irregular membrane shapes and/or aggregated protein. We suggest that membrane morphology can be controlled in cells.

Steffen Lawo, Mikhail Bashkurov, Michael Mullin, Mariana Gomez Ferreria, Ralf Kittler, Bianca Habermann, Andrea Tagliaferro, Ina Poser, James R A Hutchins, Björn Hegemann, Deborah Pinchev, Frank Buchholz, Jan-Michael Peters, Anthony A. Hyman, Anne-Claude Gingras, Laurence Pelletier
HAUS, the 8-subunit human Augmin complex, regulates centrosome and spindle integrity.
Curr Biol, 19(10) 816-826 (2009)
PDF DOI

BACKGROUND: The assembly of a robust microtubule-based mitotic spindle is a prerequisite for the accurate segregation of chromosomes to progeny. Spindle assembly relies on the concerted action of centrosomes, spindle microtubules, molecular motors, and nonmotor spindle proteins. RESULTS: Here we use an RNA-interference screen of the human centrosome proteome to identify novel regulators of spindle assembly. One such regulator is HAUS, an 8-subunit protein complex that shares homology to Drosophila Augmin. HAUS localizes to interphase centrosomes and to mitotic spindle microtubules, and its disruption induces microtubule-dependent fragmentation of centrosomes along with an increase in centrosome size. HAUS disruption results in the destabilization of kinetochore microtubules and the eventual formation of multipolar spindles. These severe mitotic defects are alleviated by codepletion of NuMA, indicating that both factors regulate opposing activities. HAUS disruption alters NuMA localization, suggesting that mislocalized NuMA activity contributes to the spindle and centrosome defects observed. CONCLUSION: The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.

Robin W. Klemm^*, Christer S. Ejsing^*, Michal Surma, Hermann-Josef Kaiser, Mathias J. Gerl, Julio Sampaio, Quentin de Robillard, Charles Ferguson, Tomasz J. Proszynski, Andrej Shevchenko, Kai Simons
Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network.
J Cell Biol, 185(4) 601-612 (2009)
PDF DOI

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

Hae Yong Yoo, Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM.
Mol Biol Cell, 20(9) 2351-2360 (2009)
PDF DOI

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs.

Li Ding, Maciej Paszkowski-Rogacz, Anja Nitzsche, Mikolaj Slabicki, Anne-Kristin Heninger, Ingrid de Vries, Ralf Kittler, Magno Junqueira, Andrej Shevchenko, Herbert Schulz, Norbert Hubner, Michael Xavier Doss, Agapios Sachinidis, Juergen Hescheler, Roberto Iacone, Konstantinos Anastassiadis, A. Francis Stewart, Maria Teresa Pisabarro, Antonio Caldarelli, Ina Poser, Mirko Theis, Frank Buchholz
A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.
Cell Stem Cell, 4(5) 403-415 (2009)
PDF DOI

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

Thomas Widmann, Christian Dahmann
Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1.
J Cell Sci, 122(Pt 9) 1362-1373 (2009)
PDF DOI

Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. Here, we show that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, we provide evidence that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC.

Tiago S. Balbuena, Vanildo Silveira, Magno Junqueira, Leonardo L C Dias, Claudete Santa-Catarina, Andrej Shevchenko, Eny I S Floh
Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian Pine (Araucaria angustifolia).
J Proteomics, 72(3) 337-352 (2009)
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Araucaria angustifolia is the only native conifer of economic importance in the Brazilian Atlantic Rainforest. Due to a clear-cutting form of exploitation this species has received the status of vulnerable. The aim of this work was to investigate and characterize changes in protein expression profile during seed development of this endangered species. For this, the proteome of developing seeds was characterized by 2-DE and LC-MS/MS. Ninety six proteins were confidently identified and classified according to their biological function and expression profile. Overaccumulated proteins in early seed development indicated a higher control on oxidative stress metabolism during this phase. In contrast, highly expressed proteins in late stages revealed an active metabolism, leading to carbon assimilation and storage compounds accumulation. Comprehensive protein expression profiles and identification of overaccumulated proteins provide new insights into the process of embryogenesis in this recalcitrant species. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.

Manuel Mendoza, Caren Norden, Kathrin Durrer, Harald Rauter, Frank Uhlmann, Yves Barral
A mechanism for chromosome segregation sensing by the NoCut checkpoint.
Nat Cell Biol, 11(4) 477-483 (2009)
PDF DOI

In Saccharomyces cerevisiae and HeLa cells, the NoCut checkpoint, which involves the chromosome passenger kinase Aurora B, delays the completion of cytokinesis in response to anaphase defects. However, how NoCut monitors anaphase progression has not been clear. Here, we show that retention of chromatin in the plane of cleavage is sufficient to trigger NoCut, provided that Aurora/Ipl1 localizes properly to the spindle midzone, and that the ADA histone acetyltransferase complex is intact. Furthermore, forcing Aurora onto chromatin was sufficient to activate NoCut independently of anaphase defects. These findings provide the first evidence that NoCut is triggered by the interaction of acetylated chromatin with the passenger complex at the spindle midzone.

Till Bretschneider^*, Kurt I. Anderson^*, Mary Ecke, Annette Müller-Taubenberger, Britta Schroth-Diez, Hellen C Ishikawa-Ankerhold, Günther Gerisch
The three-dimensional dynamics of actin waves, a model of cytoskeletal self-organization.
Biophys J, 96(7) 2888-2900 (2009)
PDF DOI

Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization.

Yoichi Kosodo, Wieland B. Huttner
Basal process and cell divisions of neural progenitors in the developing brain.
Dev Growth Differ, 51(3) 251-261 (2009)
PDF DOI

The basal process is an extension of certain types of neural progenitors during brain development; that is, the neuroepithelial and radial glial cells, which show radial orientation, emanating from their cell body. Originally, the basal process was considered to serve as a scaffold for the migration of newborn neurons, but recent observations obtained by advanced genetic manipulations and microscopic methods show that the basal process has additional roles. In this review, we first summarize the role of the radial glial basal process for neuronal migration and signaling and for the proper organization of the developing brain. We then focus on the emerging roles of the basal process during the division of neural progenitor cells, specifically the various modes of division of neuroepithelial and radial glial cells.

Aparna K Sapra, Minna-Liisa Änkö, Inna Grishina, Mike Lorenz, Marta Pabis, Ina Poser, Jarod Rollins, Eva-Marie Weiland, Karla M. Neugebauer
SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo.
Mol Cell, 34(2) 179-190 (2009)
PDF DOI

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.

Andrej Shevchenko, Christina Valcu, Magno Junqueira
Tools for exploring the proteomosphere.
J Proteomics, 72(2) 137-144 (2009)
PDF DOI

Homology-driven proteomics aims at exploring the proteomes of organisms with unsequenced genomes that, despite rapid genomic sequencing progress, still represent the overwhelming majority of species in the biosphere. Methodologies have been developed to enable automated LC-MS/MS identifications of unknown proteins, which rely on the sequence similarity between the fragmented peptides and reference database sequences from phylogenetically related species. However, because full sequences of matched proteins are not available and matching specificity is reduced, estimating protein abundances should become the obligatory element of homology-driven proteomics pipelines to circumvent the interpretation bias towards proteins from evolutionary conserved families.

Martin Depken, Eric A. Galburt, Stephan W. Grill
The origin of short transcriptional pauses.
Biophys J, 96(6) 2189-2193 (2009)
PDF DOI

RNA polymerases are protein molecular machines that transcribe genetic information from DNA into RNA. The elongation of the RNA molecule is frequently interrupted by pauses, the detailed nature of which remains controversial. Here we ask whether backtracking, the central mechanism behind long pauses, could also be responsible for short pauses normally attributed to the ubiquitous pause state. To this end, we model backtracking as a force-biased random walk, giving rise to a broad distribution of pause durations as observed in experiments. Importantly, we find that this single mechanism naturally generates two populations of pauses that are distinct both in duration and trajectory: long-time pauses with the expected behavior of diffusive backtracks, and a new class of short-time backtracks with characteristics similar to those of the ubiquitous pause. These characteristics include an apparent force insensitivity and immobility of the polymerase. Based on these results and a quantitative comparison to published pause trajectories measured with optical tweezers, we suggest that a significant fraction of short pauses are simply due to backtracking.

René Martin, Arndt W Schmidt, Gabriele Theumer, Tilo Krause, Eugeni V. Entchev, Teymuras V. Kurzchalia, Hans-Joachim Knölker
Synthesis and biological activity of the (25R)-cholesten-26-oic acids--ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans.
Org Biomol Chem, 7(5) 909-920 (2009)
PDF DOI

We describe the stereoselective transformation of diosgenin (4a) to (25R)-Delta(4)-dafachronic acid (1a),(25R)-Delta(7)-dafachronic acid (2a), and (25R)-cholestenoic acid (3a), which represent potential ligands forthe hormonal receptor DAF-12 in Caenorhabditis elegans. Key-steps of our synthetic approach are amodified Clemmensen reduction of diosgenin (4a) and a double bond shift from the 5,6- to the 7,8-position. In the 25R-series, the Delta(7)-dafachronic acid 2a exhibits the highest hormonal activity.

Christof Osman, Mathias Haag, Christoph Potting, Jonathan Rodenfels, Phat Vinh Dip, Felix T. Wieland, Britta Brügger, Benedikt Westermann, Thomas Langer
The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria.
J Cell Biol, 184(4) 583-596 (2009)
DOI

Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

Christopher Mulligan^*, Eric R Geertsma^*, Emmanuele Severi, David J Kelly, B Poolman, Gavin H Thomas
The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter.
Proc Natl Acad Sci U.S.A., 106(6) 1778-1783 (2009)
DOI

Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophilus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP.

Serena Zacchigna, Hideyasu Oh, Michaela Wilsch-Bräuninger, Ewa Missol-Kolka, József Jászai, Sandra Jansen, Naoyuki Tanimoto, Felix Tonagel, Mathias Seeliger, Wieland B. Huttner, Denis Corbeil, Mieke Dewerchin, Stefan Vinckier, Lieve Moons, Peter Carmeliet
Loss of the cholesterol-binding protein prominin-1/CD133 causes disk dysmorphogenesis and photoreceptor degeneration.
J Neurosci, 29(7) 2297-2308 (2009)
PDF DOI

Prominin-1/CD133 (Prom-1) is a commonly used marker of neuronal, vascular, hematopoietic and other stem cells, yet little is known about its biological role and importance in vivo. Here, we show that loss of Prom-1 results in progressive degeneration of mature photoreceptors with complete loss of vision. Despite the expression of Prom-1 on endothelial progenitors, photoreceptor degeneration was not attributable to retinal vessel defects, but caused by intrinsic photoreceptor defects in disk formation, outer segment morphogenesis, and associated with visual pigment sorting and phototransduction abnormalities. These findings shed novel insight on how Prom-1 regulates neural retinal development and phototransduction in vertebrates.

Jeremy N. Pulvers
The roles of the genes Aspm and Brcal in the development of the mouse neocortex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Stefanie Redemann
The role of the acto-myosin cortex for force-generation and spindle positioning in the C. elegans embryo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Daniel Lingwood
The activation of organization in cell membranes
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Charles R. Bradshaw
Improving the analysis of genome-wide screens through the detection of weakly conserved domains
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Marija Zanic, Jeffrey H. Stear, Jonathon Howard, Anthony A. Hyman
EB1 recognizes the nucleotide state of tubulin in the microtubule lattice.
PLoS ONE, 4(10) Art. No. e7585 (2009)
PDF DOI

Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.

Vladimir Mazurov
The properties of neural progenitors in the spinal cord during tail regeneration in Ambystoma mexicanum (axolotl)
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Fabio Demontis, Christian Dahmann
Characterization of the Drosophila ortholog of the human Usher Syndrome type 1G protein sans.
PLoS ONE, 4(3) Art. No. e4753 (2009)
PDF DOI

BACKGROUND: The Usher syndrome (USH) is the most frequent deaf-blindness hereditary disease in humans. Deafness is attributed to the disorganization of stereocilia in the inner ear. USH1, the most severe subtype, is associated with mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15, and sans. Myosin VIIa, harmonin, cadherin 23, and protocadherin 15 physically interact in vitro and localize to stereocilia tips in vivo, indicating that they form functional complexes. Sans, in contrast, localizes to vesicle-like structures beneath the apical membrane of stereocilia-displaying hair cells. How mutations in sans result in deafness and blindness is not well understood. Orthologs of myosin VIIa and protocadherin 15 have been identified in Drosophila melanogaster and their genetic analysis has identified essential roles in auditory perception and microvilli morphogenesis, respectively. PRINCIPAL FINDINGS: Here, we have identified and characterized the Drosophila ortholog of human sans. Drosophila Sans is expressed in tubular organs of the embryo, in lens-secreting cone cells of the adult eye, and in microvilli-displaying follicle cells during oogenesis. Sans mutants are viable, fertile, and mutant follicle cells appear to form microvilli, indicating that Sans is dispensable for fly development and microvilli morphogenesis in the follicle epithelium. In follicle cells, Sans protein localizes, similar to its vertebrate ortholog, to intracellular punctate structures, which we have identified as early endosomes associated with the syntaxin Avalanche. CONCLUSIONS: Our work is consistent with an evolutionary conserved function of Sans in vesicle trafficking. Furthermore it provides a significant basis for further understanding of the role of this Usher syndrome ortholog in development and disease.

Sven Vogel
The mechanism of meiotic nuclear oscillations in Schizosaccharomyces pombe
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

J Thomas Hannich
Lipid methylation in the development of Caenorhabditits elegans
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Marta Pabis
The cap-binding complex promotes co-transcriptional splicing in vivo
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Gaspare Benenati, Sider Penkov, Thomas Müller-Reichert, Eugeni V. Entchev, Teymuras V. Kurzchalia
Two cytochrome P450s in Caenorhabditis elegans are essential for the organization of eggshell, correct execution of meiosis and the polarization of embryo.
Mech Dev, 126(5-6) 382-393 (2009)
PDF DOI

The role of lipids in the process of embryonic development of Caenorhabditis elegans is still poorly understood. Cytochrome P450s, a class of lipid-modifying enzymes, are good candidates to be involved in the production or degradation of lipids essential for development. We investigated two highly similar cytochrome P450s in C. elegans, cyp-31A2 and cyp-31A3, that are homologs of the gene responsible for Bietti crystalline corneoretinal dystrophy in humans. Depletion of both cytochromes either by RNAi or using a double deletion mutant, led to the failure of establishing the correct polarity of the embryo and to complete the extrusion of the polar bodies during meiosis. In addition, the egg became osmotic sensitive and permeable to dyes. The phenotype of cyp-31A2 or cyp-31A3 is very similar to a class of mutants that have polarization and osmotic defects (POD), thus the genes were renamed to pod-7 and pod-8, respectively. Electron microscopic analysis demonstrated that the activity of pod-7/pod-8 is crucial for the proper assembly of the eggshell and, in particular, for the production of its lipid-rich layer. Using a complementation with lipid extracts, we show that POD-7/POD-8 function together with a NADPH cytochrome P450 reductase, coded by emb-8, and are involved in the production of lipid(s) required for eggshell formation.

Kuixing Zhang, Fangwen Rao, Brinda K. Rana, Jiaur R. Gayen, Federico Calegari, Angus King, Patrizia Rosa, Wieland B. Huttner, Mats Stridsberg, Manjula Mahata, Sucheta Vaingankar, Vafa Mahboubi, Rany M Salem, Juan L Rodriguez-Flores, Maple M Fung, Douglas W Smith, Nicholas J Schork, Michael G Ziegler, Laurent Taupenot, Sushil K Mahata, Daniel T O'Connor
Autonomic function in hypertension: Role of genetic variation at the catecholaminestorage vesicle protein chromogranin B (CHGB).
Circ Cardiovasc Genet, 2(1) 46-56 (2009)
PDF DOI

RATIONALE: Hypertension is a complex trait with deranged autonomic control of the circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common inter-individual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of the circulation, or blood pressure in the population? METHODS AND RESULTS: To probe inter-individual variability in CHGB, we systematically studied polymorphism across the locus by re-sequencing CHGB (~6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, UTRs) in n=160 subjects (2n=320 chromosomes) of diverse biogeographic ancestries. We identified 53 SNPs, of which 22 were common. We then studied n=1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5(th) %iles), typing 4 common polymorphisms spanning the ~14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5'/promoter region, and most prominent in men, and a peak effect in the proximal promoter at variant A-261T (A>T), accounting for ~8/~6 mmHg SBP/DBP in males. The promoter allele (A-261) that was a predictor of higher DBP and SBP was also associated with lower circulating/plasma CHGB concentration (CHGB(439-451) epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB(439-451), but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (-/-) ablation of the mouse Chgb gene; knockout mice displayed substantially increased BP, by ~20/~18 mmHg SBP/DBP, confirming the mechanistic basis of our findings in humans. CONCLUSIONS: We conclude that common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression, and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex-dependent. The results point to new molecular strategies for probing autonomic control of the circulation, and ultimately the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension.

Ines Kock, Natalia Bulgakova, Elisabeth Knust, Irmgard Sinning, Valérie Panneels
Targeting of Drosophila rhodopsin requires helix 8 but not the distal C-terminus.
PLoS ONE, 4(7) Art. No. e6101 (2009)
PDF DOI

BACKGROUND: The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. CONCLUSIONS/SIGNIFICANCE: Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres.

Helen Weavers^*, Silvia Prieto-Sánchez^*, Ferdi Grawe, Amparo Garcia-López, Ruben Artero, Michaela Wilsch-Bräuninger, Mar Ruiz-Gómez, Helen Skaer, Barry Denholm
The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm.
Nature, 457(7227) 322-326 (2009)
PDF DOI

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

Christer S. Ejsing, Julio Sampaio, Vineeth Surendranath, Eva Duchoslav, Kim Ekroos, Robin W. Klemm, Kai Simons, Andrej Shevchenko
Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.
Proc Natl Acad Sci U.S.A., 106(7) 2136-2141 (2009)
PDF DOI

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.

Boris Strilic
Molecular mechanism of vascular lumen formation in the dorsal aorta of mice
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Volker Bormuth
Optimized optical tweezers to study the mechanics of kinesin-8: stepping, slipping, protein friction
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Falko Riedel
Core members of the Drosophila melanogaster LDL receptor family and their function in wing development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Jens-Christian Röper
The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Christian L. Müller, Benedikt Baumgartner, Ivo F. Sbalzarini
Particle Swarm CMA Evolution Strategy for the Optimization of Multi-Funnel Landscapes
In: IEEE Congress on Evolutionary Computation : CEC 2009 ; Trondheim, Norway, 18 - 21 May 2009 (2009), Piscataway, N.J., IEEE (2009), 2685-2692
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Christian L. Müller, Benedikt Baumgartner, Georg Ofenbeck, Birte Schrader, Ivo F. Sbalzarini
pCMALib: a parallel FORTRAN 90 library for the evolution strategy with covariance matrix adaptation
In: Proc. ACM Genetic and Evolutionary Computation Conference (GECCO’09) (2009), New York, ACM (2009), 1-8
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Marat Gorivodsky, Mahua Mukhopadhyay, Michaela Wilsch-Bräuninger, Matthew Phillips, Andreas Teufel, Changmee Kim, Nasir Malik, Wieland B. Huttner, Heiner Westphal
Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain.
Dev Biol, 325(1) 24-32 (2009)
PDF DOI

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.

Julia Massier
siRNA screen for the identification of novel genes regulating lipid droplet metabolism in a human cell line
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2009)

Elizabeth Rach, Hsiang-Yu Yuan, William H Majoros, Pavel Tomancák, Uwe Ohler
Motif composition, conservation and condition-specificity of single and alternative transcription start sites in the Drosophila genome.
Genome Biol, 10(7) Art. No. R73 (2009)
PDF DOI

BACKGROUND: Transcription initiation is a key component in the regulation of gene expression. mRNA 5' full-length sequencing techniques have enhanced our understanding of mammalian transcription start sites (TSSs), revealing different initiation patterns on a genomic scale. RESULTS: To identify TSSs in Drosophila melanogaster, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs) and identified a high quality set of 5,665 TSSs for approximately 4,000 genes. We distinguished two initiation patterns: 'peaked' TSSs, and 'broad' TSS cluster groups. Peaked promoters were found to contain location-specific sequence elements; conversely, broad promoters were associated with non-location-specific elements. In alignments across other Drosophila genomes, conservation levels of sequence elements exceeded 90% within the melanogaster subgroup, but dropped considerably for distal species. Elements in broad promoters had lower levels of conservation than those in peaked promoters. When characterizing the distributions of ESTs, 64% of TSSs showed distinct associations to one out of eight different spatiotemporal conditions. Available whole-genome tiling array time series data revealed different temporal patterns of embryonic activity across the majority of genes with distinct alternative promoters. Many genes with maternally inherited transcripts were found to have alternative promoters utilized later in development. Core promoters of maternally inherited transcripts showed differences in motif composition compared to zygotically active promoters. CONCLUSIONS: Our study provides a comprehensive map of Drosophila TSSs and the conditions under which they are utilized. Distinct differences in motif associations with initiation pattern and spatiotemporal utilization illustrate the complex regulatory code of transcription initiation.

Christian L. Müller, Ivo F. Sbalzarini
A tunable real-world multi-funnel benchmark problem for evolutionary optimization (and why parallel island models might remedy the failure of CMA-ES on it)
In: Proc. Intl. Joint Conf. Computational Intelligence (IJCCI) (2009), INSTICC Press (2009), 248-253
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Eric Lécuyer, Pavel Tomancák
Mapping the gene expression universe.
Curr Opin Genet Dev, 18(6) 506-512 (2008)
PDF DOI

Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.

Mi Hye Song, L. Aravind, Thomas Müller-Reichert, Kevin F O'Connell
The conserved protein SZY-20 opposes the Plk4-related kinase ZYG-1 to limit centrosome size.
Dev Cell, 15(6) 901-912 (2008)
PDF DOI

Microtubules are organized by the centrosome, a dynamic organelle that exhibits changes in both size and number during the cell cycle. Here we show that SZY-20, a putative RNA-binding protein, plays a critical role in limiting centrosome size in C. elegans. SZY-20 localizes in part to centrosomes and in its absence centrosomes possess increased levels of centriolar and pericentriolar components including gamma-tubulin and the centriole duplication factors ZYG-1 and SPD-2. These enlarged centrosomes possess normal centrioles, nucleate more microtubules, and fail to properly direct a number of microtubule-dependent processes. Depletion of ZYG-1 restores normal centrosome size and function to szy-20 mutants, whereas loss of szy-20 suppresses the centrosome duplication defects in both zyg-1 and spd-2 mutants. Our results describe a pathway that determines centrosome size and implicate centriole duplication factors in this process.

Agnes Tóth-Petróczy, Christopher J Oldfield, István Simon, Yuichiro Takagi, A. Keith Dunker, Vladimir N Uversky, Monika Fuxreiter
Malleable machines in transcription regulation: the mediator complex.
PLoS Comput Biol, 4(12) Art. No. e1000243 (2008)
Open Access DOI

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.

Davide De Pietri Tonelli, Jeremy N. Pulvers, Christiane Haffner, Elizabeth P Murchison, Gregory J Hannon, Wieland B. Huttner
miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex.
Development, 135(23) 3911-3921 (2008)
PDF DOI

Neurogenesis during the development of the mammalian cerebral cortex involves a switch of neural stem and progenitor cells from proliferation to differentiation. To explore the possible role of microRNAs (miRNAs) in this process, we conditionally ablated Dicer in the developing mouse neocortex using Emx1-Cre, which is specifically expressed in the dorsal telencephalon as early as embryonic day (E) 9.5. Dicer ablation in neuroepithelial cells, which are the primary neural stem and progenitor cells, and in the neurons derived from them, was evident from E10.5 onwards, as ascertained by the depletion of the normally abundant miRNAs miR-9 and miR-124. Dicer ablation resulted in massive hypotrophy of the postnatal cortex and death of the mice shortly after weaning. Analysis of the cytoarchitecture of the Dicer-ablated cortex revealed a marked reduction in radial thickness starting at E13.5, and defective cortical layering postnatally. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny, and raise interesting perspectives as to the expansion of somatic stem cells.

René Martin, Frank Däbritz, Eugeni V. Entchev, Teymuras V. Kurzchalia, Hans-Joachim Knölker
Stereoselective synthesis of the hormonally active (25S)-delta7-dafachronic acid, (25S)-Delta4-dafachronic acid, (25S)-dafachronic acid, and (25S)-cholestenoic acid.
Org Biomol Chem, 6(23) 4293-4295 (2008)
PDF DOI

We report a stereoselective synthesis of the (25S)-cholestenoic-26-acids which are highly efficient ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans.

Lilla M. Farkas, Wieland B. Huttner
The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development.
Curr Opin Cell Biol, 20(6) 707-715 (2008)
PDF DOI

The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation.

Joao Matos^*, Jesse J. Lipp^*, Aliona Bogdanova, Sylvine Guillot, Elwy Okaz, Magno Junqueira, Andrej Shevchenko, Wolfgang Zachariae
Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I.
Cell, 135(4) 662-678 (2008)
PDF DOI

Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.

Jana Karbanová^*, Ewa Missol-Kolka^*, Ana-Violeta Fonseca, Christoph Lorra, Peggy Janich, Hana Hollerová, József Jászai, Jirí Ehrmann, Zdenek Kolár, Cornelia Liebers, Stefanie Arl, Danuse Subrtová, Daniel Freund, Jaroslav Mokry, Wieland B. Huttner, Denis Corbeil
The stem cell marker CD133 (Prominin-1) is expressed in various human glandular epithelia.
J Histochem Cytochem, 56(11) 977-993 (2008)
PDF DOI

Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Leah Herrgen
The regulation of segmentation clock period in zebrafish
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2008)

Moritz Kreysing, Tobias Kiessling, Anatol Fritsch, Christian Dietrich, Jochen Guck, Josef A Käs
The optical cell rotator.
Opt express, 16(21) 16984-16992 (2008)
Open Access PDF

The optical cell rotator (OCR) is a modified dual-beam laser trap for the holding and controlled rotation of suspended dielectric microparticles, such as cells. In contrast to optical tweezers, OCR uses two counter-propagating divergent laser beams, which are shaped and delivered by optical fibers. The rotation of a trapped specimen is carried out by the rotation of a dual-mode fiber, emitting an asymmetric laser beam. Experiments were performed on human erythrocytes, promyelocytic leukemia cells (HL60), and cell clusters (MCF-7). Since OCR permits the rotation of cells around an axis perpendicular to the optical axis of any microscope and is fully decoupled from imaging optics, it could be a suitable and expedient tool for tomographic microscopy.

Kai Simons
The misused impact factor.
Science, 322(5899) 165-165 (2008)
PDF DOI

Lilla M. Farkas, Christiane Haffner, Thomas Giger, Philipp Khaitovich, Katja Nowick, Carmen Birchmeier, Svante Pääbo, Wieland B. Huttner
Insulinoma-associated 1 has a panneurogenic role and promotes the generation and expansion of basal progenitors in the developing mouse neocortex.
Neuron, 60(1) 40-55 (2008)
PDF DOI

Basal (intermediate) progenitors are the major source of neurons in the mammalian neocortex. The molecular machinery governing basal progenitor biogenesis is unknown. Here, we show that the zinc-finger transcription factor Insm1 (insulinoma-associated 1) is expressed specifically in progenitors undergoing neurogenic divisions, has a panneurogenic role throughout the brain, and promotes basal progenitor formation in the neocortex. Mouse embryos lacking Insm1 contained half the number of basal progenitors and showed a marked reduction in cortical plate radial thickness. Forced premature expression of Insm1 in neuroepithelial cells resulted in their mitosis occurring at the basal (rather than apical) side of the ventricular zone and induced expression of the basal progenitor marker Tbr2. Remarkably, these cells remained negative for Tis21, a marker of neurogenic progenitors, and did not generate neurons but underwent self-amplification. Our data imply that Insm1 is involved in the generation and expansion of basal progenitors, a hallmark of neocortex evolution.

József Jászai, Christine A. Fargeas, Michael Haase, Lilla M. Farkas, Wieland B. Huttner, Denis Corbeil
Robust expression of Prominin-2 all along the adult male reproductive system and urinary bladder.
Histochem Cell Biol, 130(4) 749-759 (2008)
PDF DOI

Although the male reproductive system seems to be enriched in transcripts encoding for both Prominin genes, little is known about their spatial distribution in distinct segments of this organ system. This is especially true for the less-characterized second Prominin paralogue, Prominin-2. The present study, therefore, mainly examines the expression of Prominin-2 in male mice and reveals the existence of some crucial differences in the tissue compartmentalization of the two Prominin paralogues in the testis, epididymis, seminal vesicle, prostate and urinary bladder. Our in situ hybridization analysis demonstrates that the major domains of overlapping expression between the two Prominin genes are those compartments that are derived ontogenetically from the epigonadal mesonephric tubules, i.e. ductuli efferentes, or from the Wolffian-tube/ductus mesonephricus, for instance the corpus epididymidis and vesicula seminalis. In contrast, the sinus urogenitalis derivative urinary bladder epithelium expresses exclusively Prominin-2, but not Prominin-1 (CD133). The testis expresses only Prominin-1, not Prominin-2. In human prostate, we finally demonstrate that the expression of Prominin-2 (transcript and protein) is highly enriched in cells located in the basal compartment of the glandular epithelium where only a minute population was recently reported to be Prominin-1 positive. Taken together our data indicate that, except for the gonad, Prominin-2 is widely and abundantly expressed along the epithelia of various segments of the adult male genitourinary tract.

Jennifer L. Fish, Colette Dehay, Henry Kennedy, Wieland B. Huttner
Making bigger brains-the evolution of neural-progenitor-cell division.
J Cell Sci, 121(Pt 17) 2783-2793 (2008)
PDF DOI

Relative brain size differs markedly between species. This variation might ultimately result from differences in the cell biology of neural progenitors, which might underlie their different proliferative potential. On the basis of the cell-biological properties of neural progenitors of animals of varying brain size and complexity (namely, Drosophila melanogaster, rodents and primates), we hypothesize that the evolution of four related cell-biological features has contributed to increases in neuron number. Three of these features-the pseudostratification of the progenitor layer, the loss of (Inscuteable-mediated) mitotic-spindle rotation and the evolution of proteins (such as Aspm) that maintain the precision of symmetric progenitor division-affect the mode of cell division in the apically dividing progenitors of the ventricular zone. The fourth feature, however, concerns the evolution of the basally dividing progenitors of the subventricular zone. In rodents, these basal (or intermediate) progenitors lack cell polarity, whereas in primates a subpopulation of radial, presumably polarized, progenitors has evolved (outer-subventricular-zone progenitors). These cells undergo basal mitoses and are thought to retain epithelial characteristics. We propose the epithelial-progenitor hypothesis, which argues that evolutionary changes that promote the maintenance of epithelial features in neural progenitors, including outer-subventricular-zone progenitors, have been instrumental in the expansion of the cerebral cortex in primates.

Marisa P. McShane, Marino Zerial
Survival of the weakest: signaling aided by endosomes.
J Cell Biol, 182(5) 823-825 (2008)
PDF DOI

The tyrosine kinase receptor c-Met plays a key role in cell proliferation, morphogenesis, and motility in response to hepatocyte growth factor. C-Met is often altered in cancer and is a major target for therapeutic intervention. Despite knowing a great deal of the molecular machinery downstream of this receptor tyrosine kinase, the spatiotemporal regulation of c-Met signaling still remains elusive. In this issue of the Journal of Cell Biology, Kermorgant and Parker (Kermorgant, S. and P.J. Parker. 2008. J. Cell Biol. 182:855-863) provide evidence for a model in which the c-Met-activated STAT3 signal is mediated by endosomal trafficking. This study elegantly highlights how weak signals can be effectively transmitted to the nucleus by exploiting endosomal compartments, raising important mechanistic implications for the signaling research community.

Guillaume Charras, Ewa Paluch
Blebs lead the way: how to migrate without lamellipodia.
Nat Rev Mol Cell Biol, 9(9) 730-736 (2008)
PDF DOI

Blebs are spherical membrane protrusions that are produced by contractions of the actomyosin cortex. Blebs are often considered to be a hallmark of apoptosis; however, blebs are also frequently observed during cytokinesis and during migration in three-dimensional cultures and in vivo. For tumour cells and a number of embryonic cells, blebbing migration seems to be a common alternative to the more extensively studied lamellipodium-based motility. We argue that blebs should be promoted to a more prominent place in the world of cellular protrusions.

Iva M. Tolic-Norrelykke
Push-me-pull-you: how microtubules organize the cell interior.
Eur Biophys J, 37(7) 1271-1278 (2008)
PDF DOI

Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces.

Anton A Poznyakovskiy, Thomas Zahnert, Yannis Kalaidzidis, Rolf Schmidt, Björn Fischer, Johannes Baumgart, Yury M Yarin
The creation of geometric three-dimensional models of the inner ear based on micro computer tomography data.
Hear Res, 243(1-2) 95-104 (2008)
PDF DOI

The modeling of the mechanical process of hearing requires an accurate geometrical model of the inner ear (cochlea). The purpose of this study was the creation of a 3-D model of the fluid chambers of Guinea pig cochlea, which could serve as a basis for further mechanical modeling. Micro computer tomography used in this study is a noninvasive method to visualize bony structures. The visualization of the membranous labyrinth was achieved by additional staining of the specimen with OsO(4). The resulting stack of images has been transformed into a cylindrical coordinate system. To suppress noise on tomography images, a nonlinear smoothing method, anisotropic diffusion, were applied. A new approach has been proposed to estimate algorithm parameters automatically. Then, a segmentation using active contours (snakes) was performed. In this study, a new energy linking the contours on adjacent slices has been added to the standard approach. This compensates the inconsistencies between adjacent contours. The images segmented in this way were used as a basis for a 3-D reconstruction of the hearing organ.

Julien Mouysset, Alexandra Deichsel, Sandra Moser, Carsten Hoege, Anthony A. Hyman, Anton Gartner, Thorsten Hoppe
Cell cycle progression requires the CDC-48UFD-1/NPL-4 complex for efficient DNA replication.
Proc Natl Acad Sci U.S.A., 105(35) 12879-12884 (2008)
PDF DOI

Since cdc48 mutants were isolated by the first genetic screens for cell division cycle (cdc) mutants in yeast, the requirement of the chaperone-like ATPase Cdc48/p97 during cell division has remained unclear. Here, we discover an unanticipated function for Caenorhabditis elegans CDC-48 in DNA replication linked to cell cycle control. Our analysis of the CDC-48(UFD-1/NPL-4) complex identified a general role in S phase progression of mitotic cells essential for embryonic cell division and germline development of adult worms. These developmental defects result from activation of the DNA replication checkpoint caused by replication stress. Similar to loss of replication licensing factors, DNA content is strongly reduced in worms depleted for CDC-48, UFD-1, and NPL-4. In addition, these worms show decreased DNA synthesis and hypersensitivity toward replication blocking agents. Our findings identified a role for CDC-48(UFD-1/NPL-4) in DNA replication, which is important for cell cycle progression and genome stability.

Vitaly Zimyanin^*, Katsiaryna Belaya^*, Jacques Pecreaux, Michael J Gilchrist, Andrew Clark, Ilan Davis, Daniel St Johnston
In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization.
Cell, 134(5) 843-853 (2008)
PDF DOI

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.

Till Korten, Stefan Diez
Setting up roadblocks for kinesin-1: mechanism for the selective speed control of cargo carrying microtubules.
Lab on a chip, 8(9) 1441-1447 (2008)
PDF DOI

Motor-driven cytoskeletal filaments are versatile transport platforms for nanosized cargo in molecular sorting and nano-assembly devices. However, because cargo and motors share the filament lattice as a common substrate for their activity, it is important to understand the influence of cargo-loading on transport properties. By performing single-molecule stepping assays on biotinylated microtubules we found that individual kinesin-1 motors frequently stopped upon encounters with attached streptavidin molecules. Consequently, we attribute the deceleration of cargo-laden microtubules in gliding assays to an obstruction of kinesin-1 paths on the microtubule lattice rather than to 'frictional' cargo-surface interactions. We propose to apply this obstacle-caused slow-down of gliding microtubules in a novel molecular detection scheme: Using a mixture of two distinct microtubule populations that each bind a different kind of protein, the presence of these proteins can be detected via speed changes in the respective microtubule populations.

Masatake Kai, Carl-Philipp Heisenberg, Masazumi Tada
Sphingosine-1-phosphate receptors regulate individual cell behaviours underlying the directed migration of prechordal plate progenitor cells during zebrafish gastrulation.
Development, 135(18) 3043-3051 (2008)
PDF DOI

During vertebrate gastrulation, cells forming the prechordal plate undergo directed migration as a cohesive cluster. Recent studies revealed that E-cadherin-mediated coherence between these cells plays an important role in effective anterior migration, and that platelet-derived growth factor (Pdgf) appears to act as a guidance cue in this process. However, the mechanisms underlying this process at the individual cell level remain poorly understood. We have identified miles apart (mil) as a suppressor of defective anterior migration of the prospective prechordal plate in silberblick (slb)/wnt11 mutant embryos, in which E-cadherin-mediated coherence of cell movement is reduced. mil encodes Edg5, a sphingosine-1-phosphate (S1P) receptor belonging to a family of five G-protein-coupled receptors (S1PRs). S1P is a lipid signalling molecule that has been implicated in regulating cytoskeletal rearrangements, cell motility and cell adhesion in a variety of cell types. We examined the roles of Mil in anterior migration of prechordal plate progenitor cells and found that, in slb embryos injected with mil-MO, cells migrate with increased motility but decreased directionality, without restoring the coherence of cell migration. This indicates that prechordal plate progenitor cells can migrate effectively as individuals, as well as in a coherent cluster of cells. Moreover, we demonstrate that Mil regulates cell motility and polarisation through Pdgf and its intracellular effecter PI3K, but modulates cell coherence independently of the Pdgf/PI3K pathway, thus co-ordinating cell motility and coherence. These results suggest that the net migration of prechordal plate progenitors is determined by different parameters, including motility, persistence and coherence.

Magno Junqueira^*, Victor Spirin^*, Tiago S. Balbuena, Patrice Waridel, Vineeth Surendranath, Grigoriy Kryukov, Ivan Adzhubei, Henrik Thomas, Shamil Sunyaev, Andrej Shevchenko
Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification.
J Proteome Res, 7(8) 3382-3395 (2008)
PDF DOI

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

Pablo Oteíza^*, Mathias Köppen^*, Miguel L Concha, Carl-Philipp Heisenberg
Origin and shaping of the laterality organ in zebrafish.
Development, 135(16) 2807-2813 (2008)
PDF DOI

Handedness of the vertebrate body plan critically depends on transient embryonic structures/organs that generate cilia-dependent leftward fluid flow within constrained extracellular environments. Although the function of ciliated organs in laterality determination has been extensively studied, how they are formed during embryogenesis is still poorly understood. Here we show that Kupffer's vesicle (KV), the zebrafish organ of laterality, arises from a surface epithelium previously thought to adopt exclusively extra-embryonic fates. Live multi-photon confocal imaging reveals that surface epithelial cells undergo Nodal/TGFbeta signalling-dependent ingression at the dorsal germ ring margin prior to gastrulation, to give rise to dorsal forerunner cells (DFCs), the precursors of KV. DFCs then migrate attached to the overlying surface epithelium and rearrange into rosette-like epithelial structures at the end of gastrulation. During early somitogenesis, these epithelial rosettes coalesce into a single rosette that differentiates into the KV with a ciliated lumen at its apical centre. Our results provide novel insights into the morphogenetic transformations that shape the laterality organ in zebrafish and suggest a conserved progenitor role of the surface epithelium during laterality organ formation in vertebrates.

Magno Junqueira, Victor Spirin, Tiago S. Balbuena, Henrik Thomas, Ivan Adzhubei, Shamil Sunyaev, Andrej Shevchenko
Protein identification pipeline for the homology-driven proteomics.
J Proteomics, 71(3) 346-356 (2008)
PDF DOI

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ Orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download.

Karin Schlichting, Christian Dahmann
Hedgehog and Dpp signaling induce cadherin Cad86C expression in the morphogenetic furrow during Drosophila eye development.
Mech Dev, 125(8) 712-728 (2008)
PDF DOI

During Drosophila eye development, cell differentiation is preceded by the formation of a morphogenetic furrow, which progresses across the epithelium from posterior to anterior. Cells within the morphogenetic furrow are apically constricted and shortened along their apical-basal axis. However, how these cell shape changes and, thus, the progression of the morphogenetic furrow are controlled is not well understood. Here we show that cells simultaneously lacking Hedgehog and Dpp signal transduction fail to shorten and do not enter the morphogenetic furrow. Moreover, we have identified a gene, cadherin Cad86C, which is highly expressed in cells of the leading flank of the morphogenetic furrow. Ectopic activation of either the Hedgehog or Dpp signal transduction pathway results in elevated Cad86C expression. Conversely, simultaneous loss of both Hedgehog and Dpp signal transduction leads to decreased Cad86C expression. Finally, ectopic expression of Cad86C in either eye-antennal imaginal discs or wing imaginal discs results in apical constriction and shortening of cells. We conclude that Hedgehog and Dpp signaling promote the shortening of cells within the morphogenetic furrow. Induction of Cad86C expression might be one mechanism through which Hedgehog and Dpp promote these cell shape changes.

Alexander W. Bird, Anthony A. Hyman
Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A.
J Cell Biol, 182(2) 289-300 (2008)
PDF DOI

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.

Suzanne Eaton
Multiple roles for lipids in the Hedgehog signalling pathway.
Nat Rev Mol Cell Biol, 9(6) 437-445 (2008)
PDF DOI

The identification of endogenous sterol derivatives that modulate the Hedgehog (Hh) signalling pathway has begun to suggest testable hypotheses for the cellular biological functions of Patched, and for the lipoprotein association of Hh. Progress in the field of intracellular sterol trafficking has emphasized how tightly the distribution of intracellular sterol is controlled, and suggests that the synthesis of sterol derivatives can be influenced by specific sterol-delivery pathways. The combination of this field with Hh studies will rapidly give us a more sophisticated understanding of both the Hh signal-transduction pathway and the cell biology of sterol metabolism.

Natalia Bulgakova, Ozlem Kempkens, Elisabeth Knust
Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila.
J Cell Sci, 121(Pt 12) 2018-2026 (2008)
PDF DOI

Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells. Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. We have performed a structure-function analysis of Sdt by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. Our results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb-Sdt complex in adult photoreceptor cells.

Eugeni V. Entchev, Dominik Schwudke, Vyacheslav Zagoriy, Vitali Matyash, Aliona Bogdanova, Bianca Habermann, Lin Zhu, Andrej Shevchenko, Teymuras V. Kurzchalia
LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans.
J Biol Chem, 283(25) 17550-17560 (2008)
PDF DOI

LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.

Javier Martinez, Karla M. Neugebauer
El Sur también existe: processing RNA in the Argentine Patagonia. Meetings on 'Cell Biology, Signaling and Alternative Splicing' and 'Gene Expression and RNA Processing'.
EMBO Rep, 9(6) 516-520 (2008)
PDF DOI

Magdalena Strzelecka
The essential role of Cajal bodies in embryonic development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2008)

Lars Demmel^*, Maike Gravert^*, Ebru Ercan, Bianca Habermann, Thomas Müller-Reichert, Viktoria Kukhtina, Volker Haucke, Thorsten Baust, Marc Sohrmann, Yannis Kalaidzidis, Christian Klose, Mike Beck, Matthias Peter, Christiane Walch-Solimena
The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit.
Mol Biol Cell, 19(5) 1991-2002 (2008)
PDF DOI

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

Neil C Chi, Robin M Shaw, Benno Jungblut, Jan Huisken, Tania Ferrer, Rima Arnaout, Ian Scott, Dimitris Beis, Tong Xiao, Herwig Baier, Lily Y Jan, Martin Tristani-Firouzi, Didier Y.R. Stainier
Genetic and physiologic dissection of the vertebrate cardiac conduction system.
PLoS Biol, 6(5) 109-109 (2008)
Open Access DOI

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.

Thomas Müller-Reichert, Jana Mäntler, Martin Srayko, Eileen T. O'Toole
Electron microscopy of the early Caenorhabditis elegans embryo.
J Microsc, 230(Pt 2) 297-307 (2008)
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The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.

Rasa Sukackaite, Saulius Grazulis, Matthias Bochtler, Virginijus Siksnys
The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution.
J Mol Biol, 378(5) 1084-1093 (2008)
PDF DOI

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5'-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-A resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5'-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

Annette Schenck, Livia Goto-Silva, Claudio Collinet, Muriel Rhinn, Angelika Giner, Bianca Habermann, Michael Brand, Marino Zerial
The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development.
Cell, 133(3) 486-497 (2008)
PDF DOI

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

Gohta Goshima, Mirjam Mayer, Nan Zhang, Nico Stuurman, Ronald D Vale
Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle.
J Cell Biol, 181(3) 421-429 (2008)
PDF DOI

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.

Lawrence Rajendran, Anja Schneider, Georg Schlechtingen, Sebastian Weidlich, Jonas Ries, Tobias Braxmeier, Petra Schwille, Jörg B Schulz, Cornelia Schroeder, Mikael Simons, Gary Jennings, Hans-Joachim Knölker, Kai Simons
Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting.
Science, 320(5875) 520-523 (2008)
PDF DOI

beta-Secretase plays a critical role in beta-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a beta-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active beta-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting beta-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.

Michael Hiller, Karol Szafranski, Rileen Sinha, Klaus Huse, Swetlana Nikolajewa, Philip Rosenstiel, Stefan Schreiber, Rolf Backofen, Matthias Platzer
Assessing the fraction of short-distance tandem splice sites under purifying selection.
RNA, 14(4) 616-629 (2008)
PDF DOI

Many alternative splice events result in subtle mRNA changes, and most of them occur at short-distance tandem donor and acceptor sites. The splicing mechanism of such tandem sites likely involves the stochastic selection of either splice site. While tandem splice events are frequent, it is unknown how many are functionally important. Here, we use phylogenetic conservation to address this question, focusing on tandems with a distance of 3-9 nucleotides. We show that previous contradicting results on whether alternative or constitutive tandem motifs are more conserved between species can be explained by a statistical paradox (Simpson's paradox). Applying methods that take biases into account, we found higher conservation of alternative tandems in mouse, dog, and even chicken, zebrafish, and Fugu genomes. We estimated a lower bound for the number of alternative sites that are under purifying (negative) selection. While the absolute number of conserved tandem motifs decreases with the evolutionary distance, the fraction under selection increases. Interestingly, a number of frameshifting tandems are under selection, suggesting a role in regulating mRNA and protein levels via nonsense-mediated decay (NMD). An analysis of the intronic flanks shows that purifying selection also acts on the intronic sequence. We propose that stochastic splice site selection can be an advantageous mechanism that allows constant splice variant ratios in situations where a deviation in this ratio is deleterious.

Magdalena Lipka, Renata Filipek, Matthias Bochtler
Crystal structure and mechanism of the Staphylococcus cohnii virginiamycin B lyase (Vgb).
Biochemistry, 47(14) 4257-4265 (2008)
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The semisynthetic streptogramin antibiotic quinupristin/dalfopristin (trade name Synercid, Aventis Pharma) is a mixture of the A-type streptogramin dalfopristin and the B-type streptogramin quinupristin, a capped hexapeptide macrolactone. Quinupristin/dalfopristin was developed to combat multidrug resistant pathogens, but suffers from its own problems with drug resistance. Virginiamycin B lyase (Vgb) inactivates the quinupristin component of Synercid by lactone ring opening. Remarkably, the enzyme promotes this reaction by intramolecular beta-elimination without the involvement of a water molecule. Recently, structures of S. aureus Vgb in the presence and absence of substrate were reported and used together with detailed mutagenesis data to suggest a catalytic mechanism. Here, we report an independent determination of the S. cohnii Vgb crystal structure and a biochemical characterization of the enzyme. As expected, the S. cohnii and S. aureus Vgb structures and active sites are very similar. Moreover, both enzymes catalyze quinupristin lactone ring opening with similar rate constants, albeit perhaps with different dependencies on divalent metal ions. Replacement of the conserved active site residues His228, Glu268, or His270 with alanine reduces or abolishes S. cohnii Vgb activity. Residue Lys285 in S. cohnii Vgb is spatially equivalent to the S. aureus Vgb active site residue Glu284. A glutamate but not an alanine residue can substitute for the lysine without significant loss of activity.

Tobias Bollenbach^*, Periklis Pantazis^*, Anna Kicheva^*, Christian Bökel, Marcos González-Gaitán, Frank Jülicher
Precision of the Dpp gradient.
Development, 135(6) 1137-1146 (2008)
PDF DOI

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-beta-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spalt expression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

Anja Schneider^*, Lawrence Rajendran^*, Masanori Honsho, Matthias Gralle, Gerald Donnert, Fred Wouters, Stefan W Hell, Mikael Simons
Flotillin-dependent clustering of the amyloid precursor protein regulates its endocytosis and amyloidogenic processing in neurons.
J Neurosci, 28(11) 2874-2882 (2008)
PDF DOI

The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin- and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic beta-amyloid peptide (Abeta), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of Abeta. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing.

Hagen B Huttner, Peggy Janich, Martin Köhrmann, József Jászai, Florian Siebzehnrubl, Ingmar Blümcke, Meinolf Suttorp, Manfred Gahr, Daniela Kuhnt, Christopher Nimsky, Dietmar Krex, Gabriele Schackert, Kai Löwenbrück, Heinz Reichmann, Eric Jüttler, Werner Hacke, Peter D Schellinger, Stefan Schwab, Michaela Wilsch-Bräuninger, Anne-Marie Marzesco, Denis Corbeil
The stem cell marker prominin-1/CD133 on membrane particles in human cerebrospinal fluid offers novel approaches for studying central nervous system disease.
Stem Cells, 26(3) 698-705 (2008)
PDF DOI

Cerebrospinal fluid (CSF) is routinely used for diagnosing and monitoring neurological diseases. The CSF proteins used so far for diagnostic purposes (except for those associated with whole cells) are soluble. Here, we show that human CSF contains specific membrane particles that carry prominin-1/CD133, a neural stem cell marker implicated in brain tumors, notably glioblastoma. Differential and equilibrium centrifugation and detergent solubility analyses showed that these membrane particles were similar in physical properties and microdomain organization to small membrane vesicles previously shown to be released from neural stem cells in the mouse embryo. The levels of membrane particle-associated prominin-1/CD133 declined during childhood and remained constant thereafter, with a remarkably narrow range in healthy adults. Glioblastoma patients showed elevated levels of membrane particle-associated prominin-1/CD133, which decreased dramatically in the final stage of the disease. Hence, analysis of CSF for membrane particles carrying the somatic stem cell marker prominin-1/CD133 offers a novel approach for studying human central nervous system disease.

André Bachmann, Ferdi Grawe, Kevin Johnson, Elisabeth Knust
Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration.
Eur J Cell Biol, 87(3) 123-136 (2008)
PDF DOI

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.

Lars Demmel^*, Mike Beck^*, Christian Klose, Anne-Lore Schlaitz, Yvonne Gloor, Peggy P. Hsu, Jan Havlis, Andrej Shevchenko, Eberhard Krause, Yannis Kalaidzidis, Christiane Walch-Solimena
Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth.
Mol Biol Cell, 19(3) 1046-1061 (2008)
PDF DOI

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.

Jifeng Fei
Studies on the progeny of neuronal progenitors in the developing mouse brain using novel transgenic models of the Tis21 locus
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2008)

Anna Shevchenko, Assen Roguev, Daniel Schaft, Luke Buchanan, Bianca Habermann, Cagri Sakalar, Henrik Thomas, Nevan J Krogan, Andrej Shevchenko, A. Francis Stewart
Chromatin Central: towards the comparative proteome by accurate mapping of the yeast proteomic environment.
Genome Biol, 9(11) 167-167 (2008)
PDF DOI

BACKGROUND: Understanding the design logic of living systems requires the understanding and comparison of proteomes. Proteomes define the commonalities between organisms more precisely than genomic sequences. Because uncertainties remain regarding the accuracy of proteomic data, several issues need to be resolved before comparative proteomics can be fruitful. RESULTS: The Saccharomyces cerevisiae proteome presents the highest quality proteomic data available. To evaluate the accuracy of these data, we intensively mapped a proteomic environment, termed 'Chromatin Central', which encompasses eight protein complexes, including the major histone acetyltransferases and deacetylases, interconnected by twelve proteomic hyperlinks. Using sequential tagging and a new method to eliminate background, we confirmed existing data but also uncovered new subunits and three new complexes, including ASTRA, which we suggest is a widely conserved aspect of telomeric maintenance, and two new variations of Rpd3 histone deacetylase complexes. We also examined the same environment in fission yeast and found a very similar architecture based on a scaffold of orthologues comprising about two-thirds of all proteins involved, whereas the remaining one-third is less constrained. Notably, most of the divergent hyperlinks were found to be due to gene duplications, hence providing a mechanism for the fixation of gene duplications in evolution. CONCLUSIONS: We define several prerequisites for comparative proteomics and apply them to examine a proteomic environment in unprecedented detail. We suggest that high resolution mapping of proteomic environments will deliver the highest quality data for comparative proteomics.

André Bachmann, Margarete Draga, Ferdi Grawe, Elisabeth Knust
On the role of the MAGUK proteins encoded by Drosophila varicose during embryonic and postembryonic development.
BMC Dev Biol, 8 55-55 (2008)
PDF DOI

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs) form a family of scaffolding proteins, which are often associated with cellular junctions, such as the vertebrate tight junction, the Drosophila septate junction or the neuromuscular junction. Their capacity to serve as platforms for organising larger protein assemblies results from the presence of several protein-protein interaction domains. They often appear in different variants suggesting that they also mediate dynamic changes in the composition of the complexes. RESULTS: Here we show by electron microscopic analysis that Drosophila embryos lacking varicose function fail to develop septate junctions in the tracheae and the epidermis. In the embryo and in imaginal discs varicose expresses two protein isoforms, which belong to the MAGUK family. The two isoforms can be distinguished by the presence or absence of two L27 domains and are differentially affected in different varicose alleles. While the short isoform is essential for viability, the long isoform seems to have a supportive function. Varicose proteins co-localise with Neurexin IV in pleated septate junctions and are necessary, but not sufficient for its recruitment. The two proteins interact in vitro by the PDZ domain of Varicose and the four C-terminal amino acids of Neurexin IV. Postembryonic reduction of varicose function by expressing double-stranded RNA affects pattern formation and morphogenesis of the wing and the development of normal-shaped and -sized eyes. CONCLUSION: Expression of two Varicose isoforms in embryonic epithelia and imaginal discs suggests that the composition of Varicose-mediated protein scaffolds at septate junctions is dynamic, which may have important implications for the modulation of their function.

Magalie Lebreton
New outlooks on the transport of cholesterol in Caenorhabditis elegans
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2008)

André Bachmann, Elisabeth Knust
The use of P-Element Transposons to Generate Transgenic Flies
In: Drosophila: Methods and Protocols. (Eds.) Christian Dahmann Methods in molecular biology, 420., Totowa, USA, Humana Press (2008), 61-77 Ch. 4
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Fei Zhu^*, Steffen Lawo^*, Alexander W. Bird, Deborah Pinchev, Alison Ralph, Constance Richter, Thomas Müller-Reichert, Ralf Kittler, Anthony A. Hyman, Laurence Pelletier
The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis.
Curr Biol, 18(2) 136-141 (2008)
PDF DOI

Centrosomes are the major microtubule-organizing centers of mammalian cells. They are composed of a centriole pair and surrounding microtubule-nucleating material termed pericentriolar material (PCM). Bipolar mitotic spindle assembly relies on two intertwined processes: centriole duplication and centrosome maturation. In the first process, the single interphase centrosome duplicates in a tightly regulated manner so that two centrosomes are present in mitosis. In the second process, the two centrosomes increase in size and microtubule nucleation capacity through PCM recruitment, a process referred to as centrosome maturation. Failure to properly orchestrate centrosome duplication and maturation is inevitably linked to spindle defects, which can result in aneuploidy and promote cancer progression. It has been proposed that centriole assembly during duplication relies on both PCM and centriole proteins, raising the possibility that centriole duplication depends on PCM recruitment. In support of this model, C. elegans SPD-2 and mammalian NEDD-1 (GCP-WD) are key regulators of both these processes. SPD-2 protein sequence homologs have been identified in flies, mice, and humans, but their roles in centrosome biogenesis until now have remained unclear. Here, we show that Cep192, the human homolog of C. elegans and D. melanogaster SPD-2, is a major regulator of PCM recruitment, centrosome maturation, and centriole duplication in mammalian cells. We propose a model in which Cep192 and Pericentrin are mutually dependent for their localization to mitotic centrosomes during centrosome maturation. Both proteins are then required for NEDD-1 recruitment and the subsequent assembly of gamma-TuRCs and other factors into fully functional centrosomes.

Jakob Suckale, Michele Solimena
Pancreas islets in metabolic signaling - focus on the beta-cell.
Front Biosci, 13 7156-7171 (2008)
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The Islets of Langerhans form a nutrient sensing network spread throughout the pancreas. They are tightly connected to the source organ, the intestine, and the target organs--liver, muscle, and fat cells. The expression of a unique set of proteins enables beta cells, the most frequent islet cell type, to detect elevated blood glucose levels and secrete insulin accordingly. Clustered beta-cells achieve tighter regulation of glucose-induced insulin secretion by coordination through cell surface proteins. They also adjust their secretory capacity and flow to avoid being damaged. The immediate reaction of the beta cell to nutrients is regulated by translational mechanisms, while longer term adaptations involve changes in transcription. Glucose increases overall protein synthesis in beta-cells but selectively boosts translation of some secretory proteins including insulin. This may be mediated through recognition of RNA motifs in the untranslated regions of those messengers. If essential molecular components of this nutrient sensing system are broken or fail due to repeated stress, beta cells malfunction, which on a larger scale manifest as diseases like diabetes mellitus.

Alessio Attardo, Federico Calegari, Wulf Haubensak, Michaela Wilsch-Bräuninger, Wieland B. Huttner
Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny.
PLoS ONE, 3(6) 2388-2388 (2008)
PDF DOI

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.

Rebecca A. Green, Anjon Audhya, Andrei I. Pozniakovsky, Alexander Dammermann, Hayley Pemble, Joost Monen, Nathan Portier, Anthony A. Hyman, Arshad Desai, Karen Oegema
Expression and imaging of fluorescent proteins in the C. elegans gonad and early embryo
In: Fluorescent Proteins. (Eds.) Kevin F. Sullivan Methods in cell biology, 85., Amsterdam, Netherlands, Elsevier (2008), 179-218 Ch. 9
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The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.

Jessy Lardon, Denis Corbeil, Wieland B. Huttner, Zhidong Ling, Luc Bouwens
Stem cell marker prominin-1/AC133 is expressed in duct cells of the adult human pancreas.
Pancreas, 36(1) 1-6 (2008)
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OBJECTIVES: Many efforts are spent in identifying stem cells in adult pancreas because these could provide a source of beta cells for cell-based therapy of type 1 diabetes. Prominin-1, particularly its specific glycosylation-dependent AC133 epitope, is expressed on stem/progenitor cells of various human tissues and can be used to isolate them. We, therefore, examined its expression in adult human pancreas. METHODS: To detect prominin-1 protein, monoclonal antibody CD133/1 (AC133 clone), which recognizes the AC133 epitope, and the alphahE2 antiserum, which is directed against the human prominin-1 polypeptide, were used. Prominin-1 RNA expression was analyzed by real-time polymerase chain reaction. RESULTS: We report that all duct-lining cells of the pancreas express prominin-1. Most notably, the cells that react with the alphahE2 antiserum also react with the AC133 antibody. After isolation and culture of human exocrine cells, we found a relative increase in prominin-1 expression both at protein and RNA expression level, which can be explained by an enrichment of cells with ductal phenotype in these cultures. CONCLUSIONS: Our data show that pancreatic duct cells express prominin-1 and surprisingly reveal that its particular AC133 epitope is not an exclusive stem and progenitor cell marker.

Khaled Khairy, Jijin Foo, Jonathon Howard
Shapes of red blood cells: comparison of 3D confocal images with the bilayer-couple model
Cell Mol Bioeng, 1(2-3) 173-181 (2008)
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Martin Kragl, Dunja Knapp, Eugeniu Nacu, Shahryar Khattak, Esther Schnapp, Hans-Henning Epperlein, Elly M. Tanaka
Novel insights into the flexibility of cell and positional identity during urodele limb regeneration
In: Control and regulation of stem cells (2008)(Eds.) Bruce Stillmann Cold Spring Harbor symposia on quantitative biology ; 73, Cold Spring Harbor, N.Y, Cold Spring Harbor Laboratory Press (2008), 583-592
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The ability of diverse metazoans to regenerate whole-body structures was first described systematically by Spallanzani in 1768 and continues to fascinate biologists today. Given the current interest in stem cell biology and its therapeutic potential, examples of vertebrate regeneration garner strong interest. Among regeneration-competent vertebrates such as the fish, frog, and salamander, the salamander is particularly impressive because it can regenerate the entire limb and tail as well as various internal organs as an adult (Goss 1969). This spectacular natural phenomenon leads us to ask what cellular properties allow regeneration and what prevents this phenomenon in other vertebrates. From this perspective, it is imperative to know whether the stem cells in regenerating limbs harbor particularly special traits such as a higher plasticity in cell fate compared to tissue stem cells in other organisms. Flexibility in cell fate needs to be considered with respect not only to tissue identity, but also to patterning because limb amputation causes cells in a particular limb segment to form more distal limb elements. How positional identity is encoded in stem cells and how it is controlled to produce only the missing portion of the limb are also questions of fundamental importance.

Julia Franziska Winter
Genome-wide analysis of membrane trafficking in polarised epithelial cells in the C. elegans intestine
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2008)

Klaus Huse, Stefan Taudien, Marco Groth, Philip Rosenstiel, Karol Szafranski, Michael Hiller, Jochen Hampe, Kerstin Junker, Jorg Schubert, Stefan Schreiber, Gerd Birkenmeier, Michael Krawczak, Matthias Platzer
Genetic variants of the copy number polymorphic beta-defensin locus are associated with sporadic prostate cancer.
Tumour Biol, 29(2) 83-92 (2008)
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Prostate cancer represents the cancer with the highest worldwide prevalence in men. Chromosome 8p23 has shown suggestive genetic linkage to early-onset familial prostate cancer and is frequently deleted in cancer cells of the urogenital tract. Within this locus some beta-defensin genes (among them DEFB4, DEFB103, DEFB104) are localized, which are arranged in a gene cluster shown to exhibit an extensive copy number variation in the population. This structural variation considerably hampers genetic studies. In a new approach considering both sequence as well as copy number variations we aimed to compare the defensin locus at 8p23 in prostate cancer patients and controls.

Ching-Ju Tsai, Christer S. Ejsing, Andrej Shevchenko, Christine Ziegler
The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum.
J Struct Biol, 160(3) 275-286 (2007)
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The osmoregulated and chill-sensitive glycine-betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0-18:1, and that a phosphatidyl glycerol (PG) 16:0-18:1 rich preparation prevents formation of pseudo crystals.

Reza Farhadifar^*, Jens-Christian Röper^*, Benoit Aigouy, Suzanne Eaton, Frank Jülicher
The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.
Curr Biol, 17(24) 2095-2104 (2007)
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BACKGROUND: Epithelial junctional networks assume packing geometries characterized by different cell shapes, neighbor number distributions and areas. The development of specific packing geometries is tightly controlled; in the Drosophila wing epithelium, cells convert from an irregular to a hexagonal array shortly before hair formation. Packing geometry is determined by developmental mechanisms that likely control the biophysical properties of cells and their interactions. RESULTS: To understand how physical cellular properties and proliferation determine cell-packing geometries, we use a vertex model for the epithelial junctional network in which cell packing geometries correspond to stable and stationary network configurations. The model takes into account cell elasticity and junctional forces arising from cortical contractility and adhesion. By numerically simulating proliferation, we generate different network morphologies that depend on physical parameters. These networks differ in polygon class distribution, cell area variation, and the rate of T1 and T2 transitions during growth. Comparing theoretical results to observed cell morphologies reveals regions of parameter space where calculated network morphologies match observed ones. We independently estimate parameter values by quantifying network deformations caused by laser ablating individual cell boundaries. CONCLUSIONS: The vertex model accounts qualitatively and quantitatively for the observed packing geometry in the wing disc and its response to perturbation by laser ablation. Epithelial packing geometry is a consequence of both physical cellular properties and the disordering influence of proliferation. The occurrence of T2 transitions during network growth suggests that elimination of cells from the proliferating disc epithelium may be the result of junctional force balances.

Céline Elie-Caille, Fedor F. Severin, Jonne H. Helenius, Jonathon Howard, Daniel J. Müller, Anthony A. Hyman
Straight GDP-tubulin protofilaments form in the presence of taxol.
Curr Biol, 17(20) 1765-1770 (2007)
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Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration.

Nuran Ozcan, Christer S. Ejsing, Andrej Shevchenko, Andrej Lipski, Susanne Morbach, Reinhard Krämer
Osmolality, temperature, and membrane lipid composition modulate the activity of betaine transporter BetP in Corynebacterium glutamicum.
J Bacteriol, 189(20) 7485-7496 (2007)
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The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier, BetP, is instantly activated in response to an increasing cytoplasmic K(+) concentration. Importantly, it is also activated by chill stress independent of osmotic stress. We show that the activation of BetP by both osmotic stress and chill stress is altered in C. glutamicum cells grown at and adapted to low temperatures. BetP from cold-adapted cells is less sensitive to osmotic stress. In order to become susceptible for chill activation, cold-adapted cells in addition needed a certain amount of osmotic stimulation, indicating that there is cross talk of these two types of stimuli at the level of BetP activity. We further correlated the change in BetP regulation properties in cells grown at different temperatures to changes in the lipid composition of the plasma membrane. For this purpose, the glycerophospholipidome of C. glutamicum grown at different temperatures was analyzed by mass spectrometry using quantitative multiple precursor ion scanning. The molecular composition of glycerophospholipids was strongly affected by the growth temperature. The modulating influence of membrane lipid composition on BetP function was further corroborated by studying the influence of artificial modulation of membrane dynamics by local anesthetics and the lack of a possible influence of internally accumulated betaine on BetP activity.

Stephanie Schonegg, Alexandru T. Constantinescu, Carsten Hoege, Anthony A. Hyman
The Rho GTPase-activating proteins RGA-3 and RGA-4 are required to set the initial size of PAR domains in Caenorhabditis elegans one-cell embryos.
Proc Natl Acad Sci U.S.A., 104(38) 14976-14981 (2007)
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Caenorhabditis elegans embryos establish cortical domains of PAR proteins of reproducible size before asymmetric cell division. The ways in which the size of these domains is set remain unknown. Here we identify the GTPase-activating proteins (GAPs) RGA-3 and RGA-4, which regulate the activity of the small GTPase RHO-1. rga-3/4(RNAi) embryos have a hypercontractile cortex, and the initial relative size of their anterior and posterior PAR domains is altered. Thus, RHO-1 activity appears to control the level of cortical contractility and concomitantly the size of cortical domains. These data support the idea that in C. elegans embryos the initial size of the PAR domains is set by regulating the contractile activity of the acto-myosin cytoskeleton through the activity of RHO-1. RGA-3/4 have functions different from CYK-4, the other known GAP required for the first cell division, showing that different GAPs cooperate to control the activity of the acto-myosin cytoskeleton in the first cell division of C. elegans embryos.

Adriana Katz, Patrice Waridel, Andrej Shevchenko, Uri Pick
Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis.
Mol Cell Proteomics, 6(9) 1459-1472 (2007)
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The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.

Monika Sokolowska^*, Magdalena Kaus-Drobek^*, Honorata Czapinska, Gintautas Tamulaitis, Virginijus Siksnys, Matthias Bochtler
Restriction endonucleases that resemble a component of the bacterial DNA repair machinery.
Cell Mol Life Sci, 64(18) 2351-2357 (2007)
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It has long been known that most Type II restriction endonucleases share a conserved core fold and similar active-sites. The same core folding motif is also present in the MutH protein, a component of the bacterial DNA mismatch repair machinery. In contrast to most Type II restriction endonucleases, which assemble into functional dimers and catalyze double-strand breaks, MutH is a monomer and nicks hemimethylated DNA. Recent biochemical and crystallographic studies demonstrate that the restriction enzymes BcnI and MvaI share many additional features with MutH-like proteins, but not with most other restriction endonucleases. The structurally similar monomers all recognize approximately symmetric target sequences asymmetrically. Differential sensitivities to slight substrate asymmetries, which could be altered by protein engineering, determine whether the enzymes catalyze only single-strand nicks or double-strand breaks.

Ingmar H Riedel-Kruse, Claudia Müller, Andrew C. Oates
Synchrony dynamics during initiation, failure, and rescue of the segmentation clock.
Science, 317(5846) 1911-1915 (2007)
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The "segmentation clock" is thought to coordinate sequential segmentation of the body axis in vertebrate embryos. This clock comprises a multicellular genetic network of synchronized oscillators, coupled by intercellular Delta-Notch signaling. How this synchrony is established and how its loss determines the position of segmentation defects in Delta and Notch mutants are unknown. We analyzed the clock's synchrony dynamics by varying strength and timing of Notch coupling in zebra-fish embryos with techniques for quantitative perturbation of gene function. We developed a physical theory based on coupled phase oscillators explaining the observed onset and rescue of segmentation defects, the clock's robustness against developmental noise, and a critical point beyond which synchrony decays. We conclude that synchrony among these genetic oscillators can be established by simultaneous initiation and self-organization and that the segmentation defect position is determined by the difference between coupling strength and noise.

Thomas J. F. Nieland, Jared T Shaw, Firoz A Jaipuri, Zoltan Maliga, Jay L Duffner, Angela N Koehler, Monty Krieger
Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI.
J Lipid Res, 48(8) 1832-1845 (2007)
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Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC(50) approximately 1 microM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor alpha, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals.

Sandra Berger^*, Natalia Bulgakova^*, Ferdi Grawe, Kevin Johnson, Elisabeth Knust
Unraveling the genetic complexity of Drosophila stardust during photoreceptor morphogenesis and prevention of light-induced degeneration.
Genetics, 176(4) 2189-2200 (2007)
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Drosophila Stardust, a membrane-associated guanylate kinase (MAGUK), recruits the transmembrane protein Crumbs and the cytoplasmic proteins DPATJ and DLin-7 into an apically localized protein scaffold. This evolutionarily conserved complex is required for epithelial cell polarity in Drosophila embryos and mammalian cells in culture. In addition, mutations in Drosophila crumbs and DPATJ impair morphogenesis of photoreceptor cells (PRCs) and result in light-dependent retinal degeneration. Here we show that stardust is a genetically complex locus. While all alleles tested perturb epithelial cell polarity in the embryo, only a subset of them affects morphogenesis of PRCs or induces light-dependent retinal degeneration. Alleles retaining particular postembryonic functions still express some Stardust protein in pupal and/or adult eyes. The phenotypic complexity is reflected by the expression of distinct splice variants at different developmental stages. All proteins expressed in the retina contain the PSD95, Discs Large, ZO-1 (PDZ), Src homology 3 (SH3), and guanylate kinase (GUK) domain, but lack a large region in the N terminus encoded by one exon. These results suggest that Stardust-based protein scaffolds are dynamic, which is not only mediated by multiple interaction partners, but in addition by various forms of the Stardust protein itself.

Monika Suchanek, Riikka Hynynen, Gerd Wohlfahrt, Markku Lehto, Marie Johansson, Hannu Saarinen, Anna Radzikowska, Christoph Thiele, Vesa M Olkkonen
The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket.
Biochem J, 405(3) 473-480 (2007)
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OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.

Thomas Wendl^*, Dejan Adzic^*, Jeffrey J Schoenebeck, Steffen Scholpp, Michael Brand, Deborah Yelon, Klaus B Rohr
Early developmental specification of the thyroid gland depends on han-expressing surrounding tissue and on FGF signals.
Development, 134(15) 2871-2879 (2007)
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The thyroid is an endocrine gland in all vertebrates that develops from the ventral floor of the anterior pharyngeal endoderm. Unravelling the molecular mechanisms of thyroid development helps to understand congenital hypothyroidism caused by the absence or reduction of this gland in newborn humans. Severely reduced or absent thyroid-specific developmental genes concomitant with the complete loss of the functional gland in the zebrafish hands off (han, hand2) mutant reveals the han gene as playing a novel, crucial role in thyroid development. han-expressing tissues surround the thyroid primordium throughout development. Fate mapping reveals that, even before the onset of thyroid-specific developmental gene expression, thyroid precursor cells are in close contact with han-expressing cardiac lateral plate mesoderm. Grafting experiments show that han is required in surrounding tissue, and not in a cell-autonomous manner, for thyroid development. Loss of han expression in the branchial arches and arch-associated cells after morpholino knock-down of upstream regulator genes does not impair thyroid development, indicating that other han-expressing structures, most probably cardiac mesoderm, are responsible for the thyroid defects in han mutants. The zebrafish ace (fgf8) mutant has similar thyroid defects as han mutants, and chemical suppression of fibroblast growth factor (FGF) signalling confirms that this pathway is required for thyroid development. FGF-soaked beads can restore thyroid development in han mutants, showing that FGFs act downstream of or in parallel to han. These data suggest that loss of FGF-expressing tissue in han mutants is responsible for the thyroid defects.

Grzegorz Chojnowski, Matthias Bochtler
The statistics of the highest E value.
Acta Crystallogr A, 63(Pt 4) 297-305 (2007)
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In a previous publication, the Gumbel-Fisher-Tippett (GFT) extreme-value analysis has been applied to investigate the statistics of the intensity of the strongest reflection in a thin resolution shell. Here, a similar approach is applied to study the distribution, expectation value and standard deviation of the highest normalized structure-factor amplitude (E value). As before, acentric and centric reflections are treated separately, a random arrangement of scattering atoms is assumed, and E-value correlations are neglected. Under these assumptions, it is deduced that the highest E value is GFT distributed to a good approximation. Moreover, it is shown that the root of the expectation value of the highest ;normalized' intensity is not only an upper limit for the expectation value of the highest E value but also a very good estimate. Qualitatively, this can be attributed to the sharpness of the distribution of the highest E value. Although the formulas were derived with various simplifying assumptions and approximations, they turn out to be useful also for real small-molecule and protein crystal structures, for both thin and thick resolution shells. The only limitation is that low-resolution data (below 2.5 A) have to be excluded from the analysis. These results have implications for the identification of outliers in experimental diffraction data.

Christina Eugster, Daniela Panáková, Ali Mahmoud, Suzanne Eaton
Lipoprotein-heparan sulfate interactions in the Hh pathway.
Dev Cell, 13(1) 57-71 (2007)
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The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Here we show that Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens.

Robert D. Lynch, Stacy A Francis, Karin M. McCarthy, Elizabeth Casas, Christoph Thiele, Eveline E Schneeberger
Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin.
Exp Cell Res, 313(12) 2597-2610 (2007)
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Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [(3)H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.

Thomas Ohrt
Elucidating the intracellular mechanisms of RNA interference by single molecule fluorescence microscopy and spectroscopy
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Yvonne Gloor
The Arf GTPase eschange factor Sec7p interaction network: unraveling the crosstalk between key regulators of Golgi transport
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Cecile Leduc, Felix Ruhnow, Jonathon Howard, Stefan Diez
Detection of fractional steps in cargo movement by the collective operation of kinesin-1 motors.
Proc Natl Acad Sci U.S.A., 104(26) 10847-10852 (2007)
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The stepping behavior of single kinesin-1 motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in small groups when they transport cellular cargo. Until now, the cooperative interactions between motors in such groups were poorly understood. A fundamental question is whether two or more motors that move the same cargo step in synchrony, producing the same step size as a single motor, or whether the step size of the cargo movement varies. To answer this question, we performed in vitro gliding motility assays, where microtubules coated with quantum dots were driven over a glass surface by a known number of kinesin-1 motors. The motion of individual microtubules was then tracked with nanometer precision. In the case of transport by two kinesin-1 motors, we found successive 4-nm steps, corresponding to half the step size of a single motor. Dwell-time analysis did not reveal any coordination, in the sense of alternate stepping, between the motors. When three motors interacted in collective transport, we identified distinct forward and backward jumps on the order of 10 nm. The existence of the fractional steps as well as the distinct jumps illustrate a lack of synchronization and has implications for the analysis of motor-driven organelle movement investigated in vivo.

Monika Sokolowska, Magdalena Kaus-Drobek, Honorata Czapinska, Gintautas Tamulaitis, Roman H Szczepanowski, Claus Urbanke, Virginijus Siksnys, Matthias Bochtler
Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric complex with target DNA.
J Mol Biol, 369(3) 722-734 (2007)
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Restriction endonuclease BcnI cleaves duplex DNA containing the sequence CC/SGG (S stands for C or G, / designates a cleavage position) to generate staggered products with single nucleotide 5'-overhangs. Here, we show that BcnI functions as a monomer that interacts with its target DNA in 1:1 molar ratio and report crystal structures of BcnI in the absence and in the presence of DNA. In the complex with DNA, BcnI makes specific contacts with all five bases of the target sequence and not just with a half-site, as the protomer of a typical dimeric restriction endonuclease. Our data are inconsistent with BcnI dimerization and suggest that the enzyme introduces double-strand breaks by sequentially nicking individual DNA strands, although this remains to be confirmed by kinetic experiments. BcnI is remotely similar to the DNA repair protein MutH and shares approximately 20% sequence identity with the restriction endonuclease MvaI, which is specific for the related sequence CC/WGG (W stands for A or T). As expected, BcnI is structurally similar to MvaI and recognizes conserved bases in the target sequence similarly but not identically. BcnI has a unique machinery for the recognition of the central base-pair.

Ria H Duurkens, Menno B Tol, Eric R Geertsma, Hjalmar P Permentier, Dirk Jan Slotboom
Flavin binding to the high affinity riboflavin transporter RibU.
J Biol Chem, 282(14) 10380-10386 (2007)
DOI

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B(2)) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655-5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K(d) for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.

Anne Grapin-Botton, Daniel B Constam
Evolution of the mechanisms and molecular control of endoderm formation.
Mech Dev, 124(4) 253-278 (2007)
DOI

Endoderm differentiation and movements are of fundamental importance not only for subsequent morphogenesis of the digestive tract but also to enable normal patterning and differentiation of mesoderm- and ectoderm-derived organs. This review defines the tissues that have been called endoderm in different species, their cellular origin and their movements. We take a comparative approach to ask how signaling pathways leading to embryonic and extraembryonic endoderm differentiation have emerged in different organisms, how they became integrated and point to specific gaps in our knowledge that would be worth filling. Lastly, we address whether the gastrulation movements that lead to endoderm internalization are coupled with its differentiation.

Sunita S Shankaran^*, Dirk Sieger^*, Christian Schröter, Carmen Czepe, Marie-Christin Pauly, Mary A Laplante, Thomas S. Becker, Andrew C. Oates, Martin Gajewski
Completing the set of h/E(spl) cyclic genes in zebrafish: her12 and her15 reveal novel modes of expression and contribute to the segmentation clock.
Dev Biol, 304(2) 615-632 (2007)
PDF DOI

Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta-Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta-Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM.

Anne Grapin-Botton, Harry Heimberg, Frédéric Lemaigre
The genetic programme of pancreatic beta-cells: basic science for the development of beta-cell therapy. Workshop on programming pancreatic beta-cells.
EMBO Rep, 8(4) 322-326 (2007)
DOI

Alla Synytska, Manfred Stamm, Stefan Diez, Leonid Ionov
Simple and fast method for the fabrication of switchable bicomponent micropatterned polymer surfaces.
Langmuir, 23(9) 5205-5209 (2007)
PDF DOI

We report on the fabrication of micropatterned polymer surfaces that allow the reversible inversion of surface topography, charge, and wettability. Micropatterned surfaces were prepared by grafting two oppositely charged polyelectrolytes (poly(acrylic acid) and poly(2-vinylpyridine)) using a combination of photolithography, "lift off", and "grafting to" techniques. The switchable surfaces are of interest in microprinting and for the design of microfluidic devices and programmed protein adsorption.

Jesse J. Lipp, Toru Hirota, Ina Poser, Jan-Michael Peters
Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes.
J Cell Sci, 120(Pt 7) 1245-1255 (2007)
DOI

The assembly of mitotic chromosomes is controlled by condensin complexes. In vertebrates, condensin I binds to chromatin in prometaphase, confers rigidity to chromosomes and enables the release of cohesin complexes from chromosome arms, whereas condensin II associates with chromosomes in prophase and promotes their condensation. Both complexes are essential for chromosome segregation in anaphase. Although the association of condensins with chromatin is important for the assembly and segregation of mitotic chromosomes, it is poorly understood how this process is controlled. Here we show that the mitotic kinase Aurora B regulates the association of condensin I, but not the interaction of condensin II with chromatin. Quantitative time-lapse imaging of cells expressing GFP-tagged condensin subunits revealed that Aurora B is required for efficient loading of condensin I onto chromosomes in prometaphase and for maintenance of the complex on chromosomes in later stages of mitosis. The three non-SMC subunits of condensin I are Aurora B substrates in vitro and their mitosis-specific phosphorylation depends on Aurora B in vivo. Our data indicate that Aurora B contributes to chromosome rigidity and segregation by promoting the binding of condensin I to chromatin. We have also addressed how Aurora B might mediate the dissociation of cohesin from chromosome arms.

Eric A. Galburt^*, Stephan W. Grill^*, Anna Wiedmann, Lucyna Lubkowska, Jason Choy, Eva Nogales, Mikhail Kashlev, Carlos Bustamante
Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner.
Nature, 446(7137) 820-823 (2007)
PDF DOI

RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.

Isabel Wasserscheid, Ulrich Thomas, Elisabeth Knust
Isoform-specific interaction of Flamingo/Starry Night with excess Bazooka affects planar cell polarity in the Drosophila wing.
Dev Dyn, 236(4) 1064-1071 (2007)
PDF DOI

Epithelia display two types of polarity, apical-basal and planar cell polarity (PCP), and both are crucial for morphogenesis and organogenesis. PCP signaling pathways comprise transmembrane proteins, such as Flamingo/Starry Night, and cytoplasmic, membrane-associated proteins such as Dishevelled. During establishment of PCP in the Drosophila wing, PCP proteins accumulate apically in distinct "cortical domains" on proximal and distal plasma membranes. This finding suggests that their localized function depends on prior definition of apicobasal polarity. Here, we show that overexpression of Bazooka, a PDZ-domain protein essential for apicobasal polarity in the embryo, perturbs development of PCP, but has no effect on apicobasal polarity. The PCP phenotype is associated with a failure to restrict Flamingo/Starry night to the proximal and distal plasma membranes of the wing epithelium. We further demonstrate that flamingo expresses two differentially spliced RNAs in wing imaginal discs, which encode two isoforms of the atypical cadherin Flamingo. The predominant Starry night-type form contains a PDZ-binding motif, which mediates binding to Bazooka in vitro. Pull-down assays support the occurrence of such an interaction in wing imaginal discs. The results suggest that interaction between the apicobasal and planar cell polarity systems has to be tightly coordinated to ensure proper morphogenesis of the wing disc epithelium.

Torben Lessmann, Michele G Leuenberger, Sascha Menninger, Meritxell Lopez-Canet, Oliver Müller, Stefan Hümmer, Jenny Bormann, Kerstin Korn, Eugenio Fava, Marino Zerial, Thomas U Mayer, Herbert Waldmann
Natural product-derived modulators of cell cycle progression and viral entry by enantioselective oxa Diels-Alder reactions on the solid phase.
Chem Biol, 14(4) 443-451 (2007)
PDF DOI

The underlying frameworks of natural product classes with multiple biological activities can be regarded as biologically selected and prevalidated starting points in vast chemical structure space in the development of compound collections for chemical biology and medicinal chemistry research. For the synthesis of natural product-derived and -inspired compound collections, the development of enantioselective transformations in a format amenable to library synthesis, e.g., on the solid support, is a major and largely unexplored goal. We report on the enantioselective solid-phase synthesis of a natural product-inspired alpha,beta-unsaturated delta-lactone collection and its investigation in cell-based screens monitoring cell cycle progression and viral entry into cells. The screens identified modulators of both biological processes at a high hit rate. The screen for inhibition of viral entry opens up avenues of research for the identification of compounds with antiviral activity.

Sven Vogel^*, Isabel Raabe^*, Aygul Dereli, Nicola Maghelli, Iva M. Tolic-Norrelykke
Interphase microtubules determine the initial alignment of the mitotic spindle.
Curr Biol, 17(5) 438-444 (2007)
PDF DOI

In the fission yeast Schizosaccharomyces pombe, interphase microtubules (MTs) position the nucleus [1, 2], which in turn positions the cell-division plane [1, 3]. It is unclear how the spindle orients, with respect to the predetermined division plane, to ensure that the chromosomes are segregated across this plane. It has been proposed that, during prometaphase, the astral MT interaction with the cell cortex aligns the spindle with the cell axis [4] and also participates in a spindle orientation checkpoint (SOC), which delays entry into anaphase as long as the spindle is misaligned [5-7]. Here, we trace the position of the spindle throughout mitosis in a single-cell assay. We find no evidence for the SOC. We show that the spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired. We propose a new role for interphase microtubules: through interaction with the spindle pole body, interphase microtubules determine the initial alignment of the spindle in the subsequent cell division.

Robert G. Parton, Kai Simons
The multiple faces of caveolae.
Nat Rev Mol Cell Biol, 8(3) 185-194 (2007)
PDF DOI

Caveolae are a highly abundant but enigmatic feature of mammalian cells. They form remarkably stable membrane domains at the plasma membrane but can also function as carriers in the exocytic and endocytic pathways. The apparently diverse functions of caveolae, including mechanosensing and lipid regulation, might be linked to their ability to respond to plasma membrane changes, a property that is dependent on their specialized lipid composition and biophysical properties.

Sophia von der Hardt, Jeroen Bakkers, Adi Inbal, Lara Carvalho, Lilianna Solnica-Krezel, Carl-Philipp Heisenberg, Matthias Hammerschmidt
The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion.
Curr Biol, 17(6) 475-487 (2007)
PDF DOI

BACKGROUND: Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. RESULTS: Via bead implantations, we show that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca(2+)/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. CONCLUSIONS: We propose that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula.

Matthias Bochtler, Grzegorz Chojnowski
The highest reflection intensity in a resolution shell.
Acta Crystallogr A, 63(Pt 2) 146-155 (2007)
PDF DOI

The Gumbel-Fisher-Tippett (GFT) extreme-value analysis is applied to evaluate the distribution, expectation value and standard deviation of the intensity J of the strongest reflection in a given resolution shell in the X-ray diffraction pattern of a crystal with many scattering atoms in the unit cell. For convenience, intensities are measured in units of the average reflection intensity in the resolution shell and, for simplicity, centric and acentric reflections are treated separately. For acentric reflections, the expectation value mu and standard deviation sigma of J are mu = ln n + gamma and sigma = pi/6(1/2), where n is the number of crystallographically independent reflections in the resolution shell and gamma approximately 0.577 is the Euler-Mascheroni constant. For centric reflections with expectation value 1 for the intensity, the corresponding expressions are mu = 2(ln n + gamma) - ln(pi ln n) and sigma = 2pi/6(1/2) - pi/(6(1/2) ln n). Extensive numerical simulations show that these formulas are excellent approximations for random atom configurations at all resolutions, and good approximations for real protein crystal structures in the resolution range between 2.5 and 1.0 A.

Angelika Kippert^*, Katarina Trajkovic^*, Lawrence Rajendran, Jonas Ries, Mikael Simons
Rho regulates membrane transport in the endocytic pathway to control plasma membrane specialization in oligodendroglial cells.
J Neurosci, 27(13) 3560-3570 (2007)
PDF DOI

Differentiation of oligodendrocytes is associated with dramatic changes in plasma membrane structure, culminating in the formation of myelin membrane sheaths. Previous results have provided evidence that regulation of endocytosis may represent a mechanism to control myelin membrane growth. Immature oligodendrocytes have a high rate of clathrin-independent endocytosis for the transport of membrane to late endosomes/lysosomes (LE/Ls). After maturation and receiving signals from neurons, endocytosis is reduced and transport of membrane from LE/Ls to the plasma membrane is triggered. Here, we show that changes in Rho GTPase activity are responsible for switching between these two modes of membrane transport. Strikingly, Rho inactivation did not only reduce the transport of cargo to LE/L but also increased the dynamics of LE/L vesicles. Furthermore, we provide evidence that Rho inactivation results in the condensation of the plasma membrane in a polarized manner. In summary, our data reveal a novel role of Rho: to regulate the flow of membrane and to promote changes in cell surface structure and polarity in oligodendroglial cells. We suggest that Rho inactivation is required to trigger plasma membrane specialization in oligodendrocytes.

Kerstin A Johansson, Umut Dursun, Nathalie Jordan, Guoqiang Gu, Friedrich Beermann, Gérard Gradwohl, Anne Grapin-Botton
Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.
Dev Cell, 12(3) 457-465 (2007)
DOI

All pancreatic endocrine cells, producing glucagon, insulin, somatostatin, or PP, differentiate from Pdx1+ progenitors that transiently express Neurogenin3. To understand whether the competence of pancreatic progenitors changes over time, we generated transgenic mice expressing a tamoxifen-inducible Ngn3 fusion protein under the control of the pdx1 promoter and backcrossed the transgene into the ngn3(-/-) background, devoid of endogenous endocrine cells. Early activation of Ngn3-ER(TM) almost exclusively induced glucagon+ cells, while depleting the pool of pancreas progenitors. As from E11.5, Pdx1+ progenitors became competent to differentiate into insulin+ and PP+ cells. Somatostatin+ cells were generated from E14.5, while the competence to make glucagon+ cells was dramatically decreased. Hence, pancreas progenitors, similar to retinal or cortical progenitors, go through competence states that each allow the generation of a subset of cell types. We further show that the progenitors acquire competence to generate late-born cells in a mechanism that is intrinsic to the epithelium.

Vitaly Zimyanin, Nick Lowe, Daniel St Johnston
An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte.
Curr Biol, 17(4) 353-359 (2007)
PDF DOI

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. Here, we show that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA.

Hans-Henning Epperlein, Mark A J Selleck, Daniel Meulemans, Levan Mchedlishvili, Robert Cerny, Lidia Sobkow, Marianne Bronner-Fraser
Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo.
Dev Dyn, 236(2) 389-403 (2007)
PDF DOI

Using cell markers and grafting, we examined the timing of migration and developmental potential of trunk neural crest cells in axolotl. No obvious differences in pathway choice were noted for DiI-labeling at different lateral or medial positions of the trunk neural folds in neurulae, which contributed not only to neural crest but also to Rohon-Beard neurons. Labeling wild-type dorsal trunks at pre- and early-migratory stages revealed that individual neural crest cells migrate away from the neural tube along two main routes: first, dorsolaterally between the epidermis and somites and, later, ventromedially between the somites and neural tube/notochord. Dorsolaterally migrating crest primarily forms pigment cells, with those from anterior (but not mid or posterior) trunk neural folds also contributing glia and neurons to the lateral line. White mutants have impaired dorsolateral but normal ventromedial migration. At late migratory stages, most labeled cells move along the ventromedial pathway or into the dorsal fin. Contrasting with other anamniotes, axolotl has a minor neural crest contribution to the dorsal fin, most of which arises from the dermomyotome. Taken together, the results reveal stereotypic migration and differentiation of neural crest cells in axolotl that differ from other vertebrates in timing of entry onto the dorsolateral pathway and extent of contribution to some derivatives.

Veronique Dubreuil, Anne-Marie Marzesco, Denis Corbeil, Wieland B. Huttner, Michaela Wilsch-Bräuninger
Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1.
J Cell Biol, 176(4) 483-495 (2007)
PDF DOI

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1-enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1-bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation.

Jeremy N. Pulvers, Judith Schenk, Yoko Arai, Jifeng Fei, Kanako Saito, Wieland B. Huttner
On the origin of neurons.
Genome Biol, 8(7) 311-311 (2007)
PDF DOI

Ganka Nikolova^*, Boris Strilic^*, Eckhard Lammert
The vascular niche and its basement membrane.
Trends Cell Biol, 17(1) 19-25 (2007)
PDF DOI

Over the past few years, scientists have realized that many cellular and developmental processes, including pancreatic beta-cell growth and differentiation, stem cell and progenitor cell proliferation and cancer cell metastasis, occur in what are known as 'vascular niches'. Despite increasing numbers of reports on these niches, few common mechanisms have been identified to explain their various effects. Here, we define the term 'vascular niche' and suggest that a common and conserved feature of this niche is to provide a basement membrane to cells that are unable to form their own. We further propose that these cells require a vascular niche when they retain a high degree of plasticity.

Ingmar H. Riedel^*, Andreas Hilfinger^*, Jonathon Howard^#, Frank Jülicher^#
How molecular motors shape the flagellar beat
HFSP J, 1(1) 192-208 (2007)
PDF DOI

Cilia and eukaryotic ﬂagella are slender cellular appendages whose regular beating propels cells and microorganisms through aqueous media. The beat is an oscillating pattern of propagating bends generated by dynein motor proteins. A key open question is how the activity of the motors is coordinated in space and time. To elucidate the nature of this coordination we inferred the mechanical properties of the motors by analyzing the shape of beating sperm: Steadily beating bull sperm were imaged and their shapes were measured with high precision using a Fourier averaging technique. Comparing our experimental data with wave forms calculated for different scenarios of motor coordination we found that only the scenario of interdoublet sliding regulating motor activity gives rise to satisfactory ﬁts. We propose that the microscopic origin of such “sliding control” is the load dependent detachment rate of motors. Agreement between observed and calculated wave forms was obtained only if signiﬁcant sliding between microtubules occurred at the base. This suggests a novel mechanism by which changes in basal compliance could reverse the direction of beat propagation. We conclude that the ﬂagellar beat patterns are determined by an interplay of the basal properties of the axoneme and the mechanical feedback of dynein motors.

Nicola Bauer
New insights into the plasma membrane dynamics of rare stem cells by studying the prominin molecule
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Amani Said
Mechanisms underlying cell sorting at the A-P compartment boundary in the Drosophila wing
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Sebastien Charneau^*, Magno Junqueira^*, Camila M. Costa, Daniele L. Pires, Ellen S. Fernandes, Ana C. Bussacos, Marcelo V. de Sousa, Carlos A. O. Ricart, Andrej Shevchenko, Antonio R. L. Teixeira
The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors
Int J Mass Spectrom, 268(2 - 3 ) 265-276 (2007)
PDF DOI

The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC–MS/MS analysis using a LTQ–Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug’s blood-feeding.

Jennifer L. Fish
The evolution of neuronal progenitor cell division in mammals: the role of the abnormal spindle-like microcephaly associated (Aspm) protein and epithelial cell polarity
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Henrik Bringmann
Experiments concerning the mechanisms of cytokinesis in Caenorhabditis elegans embryos
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)
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In my thesis I aimed to contribute to the understanding of the

Christer S. Ejsing
Molecular characterization of the lipidome by mass spectrometry
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)
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Naomi Foster
The involvement of ARF6 in rapid membrane recycling during Drosophila spermatocyte cytokinesis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)

Cytokinesis involves constriction of the cell at the equator. Without

Anne-Lore Schlaitz, Martin Srayko, Alexander Dammermann, Sophie Quintin, Natalie Wielsch, Ian MacLeod, Quentin de Robillard, Andrea Zinke, John R. Yates, Thomas Müller-Reichert, Andrej Shevchenko, Karen Oegema, Anthony A. Hyman
The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly.
Cell, 128(1) 115-127 (2007)
PDF DOI

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.

Lara Carvalho
The role of yolk syncytial layer and blastoderm movements during gastrulation in zebrafish
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)
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Anna Natalia Grzyb
Characterisation of Y-Box protein 3 (MSY3) in the developing murine central nervous system
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)
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Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

Martin Kragl
Studying the Patterning Mechanisms and Cell Fates during Limb Regeneration in Ambystoma mexicanum
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2007)
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We studied patterning mechanisms and cell fates during limb regeneration in the axolotl. 1) It is crucial to understand the earliest events of patterning. Since it is technically challenging to study early events, we established single cell PCR. This new tool will allow us to obtain novel insight into the initial steps of limb patterning. 2)We have examined the roles of different tissues regarding their fates and features of proximo- distal patterning. Our strategy was to transplant GFP+ skin, skeleton, muscle and Schwann cells from transgenic donors to limbs of wild type hosts, amputate through the graft and analyze fluorescent progeny combined with the use of molecular markers. Our results revealed that different subpopulations of blastema cells exist regarding two aspects. First, we found that progeny of skin and skeleton have some tissue specific memory since they did not give rise to muscle lineages. However, cells of the skin contributed to other mesenchymal tissues like cartilage or tendons, while the majority of skeleton- derived cells undergoes self- renewal. Second, we performed one cellular and two molecular assays to investigate what tissues generate cells that exhibit features of proximo- distal patterning. Both assays revealed that Schwann cell- derived progeny do not display such features while progeny of skin, skeleton and muscle did. Therefore, we conclude that the blastema is a heterogeneous mix of cells regarding tissue lineages and features of proximo- distal patterning.

Karol Szafranski, Stefanie Schindler, Stefan Taudien, Michael Hiller, Klaus Huse, Niels Jahn, Stefan Schreiber, Rolf Backofen, Matthias Platzer
Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns.
Genome Biol, 8(8) 154-154 (2007)
Open Access PDF DOI

Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen.

Michael M Mwangi, Shang Wei Wu^#, Yanjiao Zhou^#, Krzysztof Sieradzki^#, Herminia de Lencastre^#, Paul Richardson^#, David Bruce^#, Edward Rubin^#, Eugene W Myers^#, Eric D Siggia^#, Alexander Tomasz^#
Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing
Proc Natl Acad Sci U.S.A., 104(22) 9451-9456 (2007)
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Timothy Erickson, Steffen Scholpp, Michael Brand, Cecilia B Moens, Andrew Jan Waskiewicz
Pbx proteins cooperate with Engrailed to pattern the midbrain-hindbrain and diencephalic-mesencephalic boundaries.
Dev Biol, 301(2) 504-517 (2007)
PDF DOI

Pbx proteins are a family of TALE-class transcription factors that are well characterized as Hox co-factors acting to impart segmental identity to the hindbrain rhombomeres. However, no role for Pbx in establishing more anterior neural compartments has been demonstrated. Studies done in Drosophila show that Engrailed requires Exd (Pbx orthologue) for its biological activity. Here, we present evidence that zebrafish Pbx proteins cooperate with Engrailed to compartmentalize the midbrain by regulating the maintenance of the midbrain-hindbrain boundary (MHB) and the diencephalic-mesencephalic boundary (DMB). Embryos lacking Pbx function correctly initiate midbrain patterning, but fail to maintain eng2a, pax2a, fgf8, gbx2, and wnt1 expression at the MHB. Formation of the DMB is also defective as shown by a caudal expansion of diencephalic epha4a and pax6a expression into midbrain territory. These phenotypes are similar to the phenotype of an Engrailed loss-of-function embryo, supporting the hypothesis that Pbx and Engrailed act together on a common genetic pathway. Consistent with this model, we demonstrate that zebrafish Engrailed and Pbx interact in vitro and that this interaction is required for both the eng2a overexpression phenotype and Engrailed's role in patterning the MHB. Our data support a novel model of midbrain development in which Pbx and Engrailed proteins cooperatively pattern the mesencephalic region of the neural tube.

Eric Marois, Suzanne Eaton
RNAi in the Hedgehog signaling pathway: pFRiPE, a vector for temporally and spatially controlled RNAi in Drosophila
In: Hedgehog signaling protocols. (Eds.) Jamila I. Horabin Methods in molecular biology ; 397., Totowa, USA, Humana Press (2007), 115-128 Ch. 10
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RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene to examine its involvement in biologicalRNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any processes. Many strategies exist to reduce the expression of a particular gene using RNAi. Some rely on delivering directly to cells the approximately 21-nucleotide long interfering double-stranded RNA (dsRNA) species that are central mediators of the silencing process. Others rely on the transgenic expression of longer dsRNA molecules, leaving it to the cellular machinery to process these hairpins into short active dsRNA. In this chapter, we describe a transgenic method to deplete a chosen protein from a specific Drosophila tissue following induction of long dsRNA. It was used to uncover the role of lipidic particles in Hedgehog signaling by silencing lipophorin in the fat body (1), and we routinely use it to deplete specific proteins from wing imaginal disc subdomains (2). The method, certainly not restricted to the study of Hedgehog signaling, allows fast and efficient construction of a plasmid incorporating various Drosophila genetic tools to allow heat-shock-induced expression of dsRNA at the desired time and in the desired tissue. For protocols involving injection of in vitro synthesized dsRNA in embryos to study Hedgehog signaling, see for example (3). For genomic screens to identify Hedgehog pathway components in tissue culture cells by transfection of small interfering RNAs, see refs. (4,5).

Yan Dong, Aliona Bogdanova, Bianca Habermann, Wolfgang Zachariae, Julie Ahringer
Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast.
BMC Dev Biol, 7 Art. No. 19 (2007)
Open Access PDF DOI

BACKGROUND: RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes. RESULTS: Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Delta mutant. CONCLUSION: These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.

Sabine Witzel, Vitaly Zimyanin, Filipa Carreira-Barbosa, Masazumi Tada, Carl-Philipp Heisenberg
Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane.
J Cell Biol, 175(5) 791-802 (2006)
PDF DOI

Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.

Otilia V Vieira, Katharina Gaus, Paul Verkade, Joachim Füllekrug, Winchil L. C. Vaz, Kai Simons
FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.
Proc Natl Acad Sci U.S.A., 103(49) 18556-18561 (2006)
PDF DOI

We have analyzed the role of the phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2), a component of the apical transport machinery, in cilium formation in polarized Madin-Darby canine kidney (MDCK) cells. We show that ciliogenesis is defective in FAPP2 knockdown cells. Furthermore, by using fluorescence recovery after photobleaching studies of domain connectivity and the generalized polarization spectra of Laurdan, we demonstrate that FAPP2 depletion impairs the formation of condensed apical membrane domains. Laurdan staining also revealed that the ciliary membrane has a highly condensed bilayer domain at its base that could function as a fence to separate the ciliary membrane from the surrounding apical membrane. These results indicate that the compartmentalization of the apical membrane in MDCK cells into the ciliary membrane and the surrounding membrane depends on the balance of raft and nonraft domains.

Pierre-Marie Dehé, Bernhard Dichtl, Daniel Schaft, Assen Roguev, Mercè Pamblanco, Régine Lebrun, Alfonso Rodríguez-Gil, Msau Mkandawire, Katharina Landsberg, Anna Shevchenko, Andrej Shevchenko, Lorena E Rosaleny, Vicente Tordera, Sebastián Chávez, A. Francis Stewart, Vincent Géli
Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation.
J Biol Chem, 281(46) 35404-35412 (2006)
PDF DOI

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Christian Bökel, Anja Schwabedissen, Eugeni V. Entchev, Olivier Renaud, Marcos González-Gaitán
Sara endosomes and the maintenance of Dpp signaling levels across mitosis.
Science, 314(5802) 1135-1139 (2006)
PDF DOI

During development, cells acquire positional information by reading the concentration of morphogens. In the developing fly wing, a gradient of the transforming growth factor-beta (TGF-beta)-type morphogen decapentaplegic (Dpp) is transduced into a gradient of concentration of the phosphorylated form of the R-Smad transcription factor Mad. The endosomal protein Sara (Smad anchor for receptor activation) recruits R-Smads for phosphorylation by the type I TGF-beta receptor. We found that Sara, Dpp, and its type I receptor Thickveins were targeted to a subpopulation of apical endosomes in the developing wing epithelial cells. During mitosis, the Sara endosomes and the receptors therein associated with the spindle machinery to segregate into the two daughter cells. Daughter cells thereby inherited equal amounts of signaling molecules and thus retained the Dpp signaling levels of the mother cell.

Tobias B Huber, Bernhard Schermer, Roman Ulrich Müller, Martin Höhne, Malte Bartram, Andrea Calixto, Henning Hagmann, Christian Reinhardt, Fabienne Koos, Karl Kunzelmann, Elena Shirokova, Dietmar Krautwurst, Christian Harteneck, Matias Simons, Hermann Pavenstädt, Dontscho Kerjaschki, Christoph Thiele, Gerd Walz, Martin Chalfie, Thomas Benzing
Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels.
Proc Natl Acad Sci U.S.A., 103(46) 17079-17086 (2006)
PDF DOI

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.

Agnes Toth-Petroczy, Agnes Szilagyi, Zsolt Ronai^#, Maria Sasvari-Szekely, András Guttman^#
Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by capillary gel electrophoresis.
J Chromatogr A, 1130(2) 201-205 (2006)
DOI

Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A capillary gel electrophoresis based genotype analysis method and an in-house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene.

David Stanĕk, Karla M. Neugebauer
The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze.
Chromosoma, 115(5) 343-354 (2006)
PDF DOI

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

Nayoung Suh, Britta Jedamzik, Christian R. Eckmann, Marvin Wickens, Judith Kimble
The GLD-2 poly(A) polymerase activates gld-1 mRNA in the Caenorhabditis elegans germ line.
Proc Natl Acad Sci U.S.A., 103(41) 15108-15112 (2006)
PDF DOI

mRNA regulation is crucial for many aspects of metazoan development and physiology, including regulation of stem cells and synaptic plasticity. In the nematode germ line, RNA regulators control stem cell maintenance, the sperm/oocyte decision, and progression through meiosis. Of particular importance to this work are three GLD (germ-line development) regulatory proteins, each of which promotes entry into the meiotic cell cycle: GLD-1 is a STAR/Quaking translational repressor, GLD-2 is a cytoplasmic poly(A) polymerase, and GLD-3 is a homolog of Bicaudal-C. Here we report that the gld-1 mRNA is a direct target of the GLD-2 poly(A) polymerase: polyadenylation of gld-1 mRNA depends on GLD-2, the abundance of GLD-1 protein is dependent on GLD-2, and the gld-1 mRNA coimmunoprecipitates with both GLD-2 and GLD-3 proteins. We suggest that the GLD-2 poly(A) polymerase enhances entry into the meiotic cell cycle at least in part by activating GLD-1 expression. The importance of this conclusion is twofold. First, the activation of gld-1 mRNA by GLD-2 identifies a positive regulatory step that reinforces the decision to enter the meiotic cell cycle. Second, gld-1 mRNA is initially repressed by FBF (for fem-3 binding factor) to maintain stem cells but then becomes activated by the GLD-2 poly(A) polymerase once stem cells begin to make the transition into the meiotic cell cycle. Therefore, a molecular switch regulates gld-1 mRNA activity to accomplish the transition from mitosis to meiosis.

Werner L. Straube, Elly M. Tanaka
Reversibility of the differentiated state: regeneration in amphibians.
Artificial organs, 30(10) 743-755 (2006)
PDF DOI

In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, tail, eye lens, or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, dermis, and cartilage. In the case of multinucleated skeletal muscle fibers, cell cycle reentry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo.

Jacob W. J. Kerssemakers, Jonathon Howard, Henry Hess, Stefan Diez
The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy.
Proc Natl Acad Sci U.S.A., 103(43) 15812-15817 (2006)
PDF DOI

Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor "heads"), a long coiled-coil domain (the "rod" or "stalk"), and two small globular "tail" domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 +/- 2 nm (mean +/- SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule ( approximately 60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport.

Christophe J. Echeverri, Philip A Beachy, Buzz Baum, Michael Boutros, Frank Buchholz, Sumit K Chanda, Julian Downward, Jan Ellenberg, Andrew G Fraser, Nir Hacohen, William C Hahn, Aimee L Jackson, Amy Kiger, Peter S Linsley, Lawrence Lum, Yong Ma, Bernard Mathey-Prévôt, David E Root, David M Sabatini, Jussi Taipale, Norbert Perrimon, René Bernards
Minimizing the risk of reporting false positives in large-scale RNAi screens.
Nat Methods, 3(10) 777-779 (2006)
PDF DOI

Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.

Martin Srayko^*, Eileen T. O'Toole^*, Anthony A. Hyman, Thomas Müller-Reichert
Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis.
Curr Biol, 16(19) 1944-1949 (2006)
PDF DOI

Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro. In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis. However, studies from plant cells, cultured neurons, and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanin's role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles. By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.

Silvia Cappello, Alessio Attardo, Xunwei Wu, Takuji Iwasato, Shigeyoshi Itohara, Michaela Wilsch-Bräuninger, Hanna M Eilken, Michael A Rieger, Timm T Schroeder, Wieland B. Huttner, Cord Brakebusch, Magdalena Götz
The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface.
Nat Neurosci, 9(9) 1099-1107 (2006)
PDF DOI

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

Cveta Tomova, Willie J C Geerts, Thomas Müller-Reichert, Rolf Entzeroth, Bruno M Humbel
New comprehension of the apicoplast of Sarcocystis by transmission electron tomography.
Biol Cell, 98(9) 535-545 (2006)
PDF DOI

BACKGROUND INFORMATION: Apicomplexan parasites (like Plasmodium, Toxoplasma, Eimeria and Sarcocystis) contain a distinctive organelle, the apicoplast, acquired by a secondary endosymbiotic process analogous to chloroplasts and mitochondria. The apicoplast is essential for long-term survival of the parasite. This prokaryotic origin implies that molecular and metabolic processes in the apicoplast differ from those of the eukaryotic host cells and therefore offer options for specific chemotherapeutic treatment. We studied the apicoplast in high-pressure frozen and freeze-substituted cysts of Sarcocystis sp. from roe deer (Capreolus capreolus) to get better insight in apicoplast morphology. RESULTS AND CONCLUSIONS: We observed that the apicoplast contains four continuous membranes. The two inner membranes have a circular shape with a constant distance from each other and large-sized protein complexes are located between them. The two outer membranes have irregular shapes. The periplastid membrane also contains large-sized protein complexes, while the outer membrane displays protuberances into the parasite cytoplasm. In addition, it is closely associated with the endoplasmic reticulum by 'contact sites'.

Alexis P. Rideau, Clare Gooding, Peter J. Simpson, Tom P. Monie, Mike Lorenz, Stefan Hüttelmaier, Robert H. Singer, Stephen Matthews, Stephen Curry, Christopher W. J. Smith
A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain.
Nat Struct Mol Biol, 13(9) 839-848 (2006)
PDF DOI

Polypyrimidine tract-binding protein (PTB) is a regulatory splicing repressor. Raver1 acts as a PTB corepressor for splicing of alpha-tropomyosin (Tpm1) exon 3. Here we define a minimal region of Raver1 that acts as a repressor domain when recruited to RNA. A conserved [S/G][I/L]LGxxP motif is essential for splicing repressor activity and sufficient for interaction with PTB. An adjacent proline-rich region is also essential for repressor activity but not for PTB interaction. NMR analysis shows that LLGxxP peptides interact with a hydrophobic groove on the dorsal surface of the RRM2 domain of PTB, which constitutes part of the minimal repressor region of PTB. The requirement for the PTB-Raver1 interaction that we have characterized may serve to bring the additional repressor regions of both proteins into a configuration that allows them to synergistically effect exon skipping.

Natalie Wielsch^*, Henrik Thomas^*, Vineeth Surendranath, Patrice Waridel, Ari Frank, Pavel Pevzner, Andrej Shevchenko
Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches.
J Proteome Res, 5(9) 2448-2456 (2006)
PDF DOI

Protein identifications with the borderline statistical confidence are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in the reliable and reproducible characterization of proteomes. Here, we present a method for rapid validation of borderline hits that circumvents the need in, often biased, manual inspection of raw MS/MS spectra. The approach takes advantage of the independent interpretation of corresponding MS/MS spectra by PepNovo de novo sequencing software followed by mass spectrometry-driven BLAST (MS BLAST) sequence-similarity database searches that utilize all partially inaccurate, degenerate and redundant candidate peptide sequences. In a case study involving the identification of more than 180 Caenorhabditis elegans proteins by nanoLC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, the approach enabled rapid assignment (confirmation or rejection) of more than 70% of Mascot hits of borderline statistical confidence.

Kurt I. Anderson, Jeremy Sanderson, Silke Gerwig, Jan Peychl
A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs.
Cytometry A, 69(8) 920-929 (2006)
PDF DOI

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572; Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging.

Gaspare Benenati
Osmotic balance and establishment of polarity in the C. elegans embryo require cytochrome P450 CYP31A
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Gerlinde Reim, Michael Brand
Maternal control of vertebrate dorsoventral axis formation and epiboly by the POU domain protein Spg/Pou2/Oct4.
Development, 133(14) 2757-2770 (2006)
PDF DOI

Dorsoventral (DV) axis formation of the vertebrate embryo is controlled by the maternal genome and is subsequently refined zygotically. In the zygote, repression of ventralizing Bmp activity on the dorsal side through chordin and noggin is crucial for establishment of a dorsally located organizer. This interplay generates a zygotic Bmp activity gradient that defines distinct positional values along the DV axis. The maternal processes that control expression of the zygotic genes implicated in DV patterning are largely unknown. spiel-ohne-grenzen (spg/pou2) is a maternally and zygotically expressed zebrafish gene that encodes the POU domain transcription factor Pou2, an ortholog of mammalian Oct4/Pou5f1. We show that embryos that are genetically depleted of both maternal and zygotic pou2 function (MZspg) exhibit extreme DV patterning defects and, independently, a blastoderm-specific arrest of epiboly. Dorsal tissues expand to the ventral side at the expense of ventrolateral tissue in MZspg embryos. Dorsally expressed Bmp-antagonists, such as Chd and Nog1, and Gsc are ectopically activated at ventral levels in MZspg. Lack of ventral specification is apparent very early, suggesting that maternal processes are affected in MZspg. Indeed, maternal pou2 function is necessary to initiate zygotic expression of ventrally expressed genes such as bmp2b and bmp4, and for proper activation of bmp7, vox, vent and eve1. A constitutively active Alk8-TGFbeta-receptor can ectopically induce bmp2b and bmp4 and rescues the dorsalization of MZspg. This indicates that pou2 acts upstream of Alk8, a maternally provided receptor implicated in the activation of zygotic bmp2b and bmp4 transcription. Consistent with this possibility, Bmp gene misexpression can rescue MZspg embryos, indicating that TGFbeta-mediated signal transduction itself is intact in absence of Pou2. Inhibition of Fgf signaling, another pathway with early dorsalizing activity, can also restore and even ventralize MZspg embryos. The requirement for pou2 to initiate bmp2b expression can therefore be bypassed by releasing the repressive function of Fgf signaling upon bmp2b transcription. In transplantation experiments, we find that dorsalized cells from prospective ventrolateral regions of MZspg embryos are non cell-autonomously respecified to a ventral fate within wild-type host embryos. Analysis of pou2 mRNA injected MZspg embryos shows that pou2 is required on the ventral side of cleavage stage embryos. Based on the maternal requirement for pou2 in ventral specification, we propose that ventral specification employs an active, pou2-dependent maternal induction step, rather than a default ventralizing program.

Heiner Grandel^*, Jan Kaslin^*, Julia Ganz, Isabell Wenzel, Michael Brand
Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate.
Dev Biol, 295(1) 263-277 (2006)
PDF DOI

Lifelong neurogenesis in vertebrates relies on stem cells producing proliferation zones that contain neuronal precursors with distinct fates. Proliferation zones in the adult zebrafish brain are located in distinct regions along its entire anterior-posterior axis. We show a previously unappreciated degree of conservation of brain proliferation patterns among teleosts, suggestive of a teleost ground plan. Pulse chase labeling of proliferating populations reveals a centrifugal movement of cells away from their places of birth into the surrounding mantle zone. We observe tangential migration of cells born in the ventral telencephalon, but only a minor rostral migratory stream to the olfactory bulb. In contrast, the lateral telencephalic area, a domain considered homologous to the mammalian dentate gyrus, shows production of interneurons and migration as in mammals. After a 46-day chase, newborn highly mobile cells have moved into nuclear areas surrounding the proliferation zones. They often show HuC/D immunoreactivity but importantly also more specific neuronal identities as indicated by immunoreactivity for tyrosine hydroxylase, serotonin and parvalbumin. Application of a second proliferation marker allows us to recognize label-retaining, actively cycling cells that remain in the proliferation zones. The latter population meets two key criteria of neural stem cells: label retention and self renewal.

Vesa M Olkkonen, Marie Johansson, Monika Suchanek, D Yan, Riikka Hynynen, C Ehnholm, M Jauhiainen, Christoph Thiele, Markku Lehto
The OSBP-related proteins (ORPs): global sterol sensors for co-ordination of cellular lipid metabolism, membrane trafficking and signalling processes?
Biochem Soc Trans, 34(Pt 3) 389-391 (2006)
PDF DOI

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

Maike Gravert
Novel regulators of trafficking in the yeast Golgi-endosomal systems
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Matthias Bochtler, Roman H Szczepanowski, Gintautas Tamulaitis, Saulius Grazulis, Honorata Czapinska, Elena Manakova, Virginijus Siksnys
Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease.
EMBO J, 25(10) 2219-2229 (2006)
PDF DOI

Restricion endonuclease Ecl18kI is specific for the sequence /CCNGG and cleaves it before the outer C to generate 5 nt 5'-overhangs. It has been suggested that Ecl18kI is evolutionarily related to NgoMIV, a 6-bp cutter that cleaves the sequence G/CCGGC and leaves 4 nt 5'-overhangs. Here, we report the crystal structure of the Ecl18kI-DNA complex at 1.7 A resolution and compare it with the known structure of the NgoMIV-DNA complex. We find that Ecl18kI flips both central nucleotides within the CCNGG sequence and buries the extruded bases in pockets within the protein. Nucleotide flipping disrupts Watson-Crick base pairing, induces a kink in the DNA and shifts the DNA register by 1 bp, making the distances between scissile phosphates in the Ecl18kI and NgoMIV cocrystal structures nearly identical. Therefore, the two enzymes can use a conserved DNA recognition module, yet recognize different sequences, and form superimposable dimers, yet generate different cleavage patterns. Hence, Ecl18kI is the first example of a restriction endonuclease that flips nucleotides to achieve specificity for its recognition site.

Massimo Galimberti, Iva M. Tolic-Norrelykke, Roberto Favillini, Raffaella Mercatelli, Francesco Annunziato, Lorenzo Cosmi, Francesco Liotta, Veronica Santarlasci, Enrico Maggi, Francesco S. Pavone
Hypergravity speeds up the development of T-lymphocyte motility.
Eur Biophys J, 35(5) 393-400 (2006)
PDF DOI

The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1-11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.

Karla M. Neugebauer
Please hold--the next available exon will be right with you.
Nat Struct Mol Biol, 13(5) 385-386 (2006)
PDF DOI

A major challenge in gene expression is to understand how precursor messenger RNA (pre-mRNA) processing events are integrated with transcription. A recent study suggests that distant exons in nascent RNA are held together during transcription, promoting accurate splicing independent of intron fate.

Jonne H. Helenius^*, Gary J. Brouhard^*, Yannis Kalaidzidis, Stefan Diez, Jonathon Howard
The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends.
Nature, 441(7089) 115-119 (2006)
PDF DOI

The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family. These proteins are crucial for the control of microtubule length in cell division, neuronal development and interphase microtubule dynamics. The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 microm2 s(-1). Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality" that has been proposed as the search strategy by which DNA enzymes find specific binding sites. We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.

Jun Yu, Sonia Bergaya, Takahisa Murata, Ilkay F. Alp, Michael P. Bauer, Michelle I Lin, Marek Drab, Teymuras V. Kurzchalia, Radu V Stan, William C Sessa
Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels.
J Clin Invest, 116(5) 1284-1291 (2006)
PDF DOI

Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels.

Anne-Kathrin Classen
Hexagonal packing of drosophila wing epithelial cells by the planar cell polarity pathway
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Catherine M Burgess, Dirk Jan Slotboom, Eric R Geertsma, Ria H Duurkens, B Poolman, Douwe van Sinderen
The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism.
J Bacteriol, 188(8) 2752-2760 (2006)
DOI

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

Karla M. Neugebauer
Keeping tabs on the women: life scientists in Europe.
PLoS Biol, 4(4) 97-97 (2006)
Open Access PDF DOI

Veronica Dudu, Thomas Bittig, Eugeni V. Entchev, Anna Kicheva, Frank Jülicher, Marcos González-Gaitán
Postsynaptic mad signaling at the Drosophila neuromuscular junction.
Curr Biol, 16(7) 625-635 (2006)
PDF DOI

BACKGROUND: Cell-to-cell communication at the synapse involves synaptic transmission as well as signaling mediated by growth factors, which provide developmental and plasticity cues. There is evidence that a retrograde, presynaptic transforming growth factor-beta (TGF-beta) signaling event regulates synapse development and function in Drosophila. RESULTS: Here we show that a postsynaptic TGF-beta signaling event occurs during larval development. The type I receptor Thick veins (Tkv) and the R-Smad transcription factor Mothers-against-dpp (Mad) are localized postsynaptically in the muscle. Furthermore, Mad phosphorylation occurs in regions facing the presynaptic active zones of neurotransmitter release within the postsynaptic subsynaptic reticulum (SSR). In order to monitor in real time the levels of TGF-beta signaling in the synapse during synaptic transmission, we have established a FRAP assay to measure Mad nuclear import/export in the muscle. We show that Mad nuclear trafficking depends on stimulation of the muscle. CONCLUSIONS: Our data suggest a mechanism linking synaptic transmission and postsynaptic TGF-beta signaling that may coordinate nerve-muscle development and function.

Caren Norden^*, Manuel Mendoza^*, Jeroen Dobbelaere, Chitra V Kotwaliwale, Sue Biggins, Yves Barral
The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage.
Cell, 125(1) 85-98 (2006)
PDF DOI

During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.

Tobias Langenberg^*, Tadeusz Dracz^*, Andrew C. Oates, Carl-Philipp Heisenberg, Michael Brand
Analysis and visualization of cell movement in the developing zebrafish brain.
Dev Dyn, 235(4) 928-933 (2006)
PDF DOI

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Axel Ring^*, Soazig Le Lay^*, Jürgen A. Pohl, Paul Verkade, Wolfgang Stremmel
Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.
Biochim Biophys Acta, 1761(4) 416-423 (2006)
PDF DOI

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

Steffen Scholpp, Olivia Wolf, Michael Brand, Andrew Lumsden
Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon.
Development, 133(5) 855-864 (2006)
PDF DOI

Midway between the anterior neural border and the midbrain-hindbrain boundary, two well-known local signalling centres in the early developing brain, is a further transverse boundary with putative signalling properties -- the zona limitans intrathalamica (ZLI). Here, we describe formation of the ZLI in zebrafish in relation to expression of sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), and to development of the forebrain regions that flank the ZLI: the prethalamus and thalamus. We find that enhanced Hh signalling increases the size of prethalamic and thalamic gene expression domains, whereas lack of Hh signalling leads to absence of these domains. In addition, we show that shh and twhh display both unique and redundant functions during diencephalic patterning. Genetic ablation of the basal plate shows that Hh expression in the ZLI alone is sufficient for diencephalic differentiation. Furthermore, acquisition of correct prethalamic and thalamic gene expression is dependent on direct Hh signalling. We conclude that proper maturation of the diencephalon requires ZLI-derived Hh signalling.

Ganka Nikolova, Normund Jabs, Irena Konstantinova, Anna Domogatskaya, Karl Tryggvason, Lydia Sorokin, Reinhard Fässler, Guoqiang Gu, Hans-Peter Gerber, Napoleone Ferrara, Douglas A Melton, Eckhard Lammert
The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation.
Dev Cell, 10(3) 397-405 (2006)
PDF DOI

Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required.

Angela Huebner, Philipp Mann, Elvira Rohde, Angela M Kaindl, Martin Witt, Paul Verkade, Sibylle Jakubiczka, Mario Menschikowski, Gisela Stoltenburg-Didinger, Katrin Koehler
Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome.
Mol Cell Biol, 26(5) 1879-1887 (2006)
PDF DOI

Triple A syndrome is a human autosomal recessive disorder characterized by adrenal insufficiency, achalasia, alacrima, and neurological abnormalities affecting the central, peripheral, and autonomic nervous systems. In humans, this disease is caused by mutations in the AAAS gene, which encodes ALADIN, a protein that belongs to the family of WD-repeat proteins and localizes to nuclear pore complexes. To analyze the function of the gene in the context of the whole organism and in an attempt to obtain an animal model for human triple A syndrome, we generated mice lacking a functional Aaas gene. The Aaas-/- animals were found to be externally indistinguishable from their wild-type littermates, although their body weight was on the average lower than that of wild-type mice. Histological analysis of various tissues failed to reveal any differences between Aaas-/- and wild-type mice. Aaas-/- mice exhibit unexpectedly mild abnormal behavior and only minor neurological deficits. Our data show that the lack of ALADIN in mice does not lead to a triple A syndrome-like disease. Thus, in mice either the function of ALADIN differs from that in humans, its loss can be readily compensated for, or additional factors, such as environmental conditions or genetic modifiers, contribute to the disease.

Lidia Sobkow, Hans-Henning Epperlein, Stephan Herklotz, Werner L. Straube, Elly M. Tanaka
A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration.
Dev Biol, 290(2) 386-397 (2006)
PDF DOI

The development of transgenesis in axolotls is crucial for studying development and regeneration as it would allow for long-term cell fate tracing as well as gene expression analysis. We demonstrate here that plasmid injection into the one-cell stage axolotl embryo generates mosaic transgenic animals that display germline transmission of the transgene. The inclusion of SceI meganuclease in the injections (Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., Joly, J.S., 2002. I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91-98) resulted in a higher percentage of F0 animals displaying strong expression throughout the body. This represents the first demonstration in the axolotl of germline transmission of a transgene. Using this technique we have generated a germline transgenic animal expressing GFP ubiquitously in all tissues examined. We have used this animal to study cell fate in the dorsal fin during development. We have uncovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled hosts. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity.

Christian Bökel, Sajith Dass, Michaela Wilsch-Bräuninger, Siegfried Roth
Drosophila Cornichon acts as cargo receptor for ER export of the TGFalpha-like growth factor Gurken.
Development, 133(3) 459-470 (2006)
PDF DOI

Drosophila Cornichon (Cni) is the founding member of a conserved protein family that also includes Erv14p, an integral component of the COPII-coated vesicles that mediate cargo export from the yeast endoplasmic reticulum (ER). During Drosophila oogenesis, Cni is required for transport of the TGFalpha growth factor Gurken (Grk) to the oocyte surface. Here, we show that Cni, but not the second Drosophila Cni homologue Cni-related (Cnir), binds to the extracellular domain of Grk, and propose that Cni acts as a cargo receptor recruiting Grk into COPII vesicles. Consequently, in the absence of Cni function, Grk fails to leave the oocyte ER. Proteolytic processing of Grk still occurs in cni mutant ovaries, demonstrating that release of the active growth factor from its transmembrane precursor occurs earlier during secretory transport than described for the other Drosophila TGFalpha homologues. Massive overexpression of Grk in a cni mutant background can overcome the requirement of Grk signalling for cni activity, confirming that cni is not essential for the production of the functional Grk ligand. However, the rescued egg chambers lack dorsoventral polarity. This demonstrates that the generation of temporally and spatially precisely coordinated Grk signals cannot be achieved by bulk flow secretion, but instead has to rely on fast and efficient ER export through cargo receptor-mediated recruitment of Grk into the secretory pathway.

Cerasela Z. Dinu
Leveraging the motor protein kinesin to manipulate DNA molecules in synthetic environment
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Klaus-Peter Knoch, Ronny Meisterfeld, Stephan Kersting, Hendrik Bergert, Anke Altkrüger, Carolin Wegbrod, Melanie Jäger, Hans-Detlev Saeger, Michele Solimena
cAMP-dependent phosphorylation of PTB1 promotes the expression of insulin secretory granule proteins in beta cells.
Cell Metab, 3(2) 123-134 (2006)
PDF DOI

Glucose stimulates the exocytosis of insulin secretory granules of pancreatic beta cells. Granule stores are quickly refilled by activation of posttranscriptional mechanisms that enhance the biosynthesis of granule components. Rapid replacement of granules is important to sustain insulin secretion, since new granules appear to be preferentially released. Posttranscriptional regulation of granule biogenesis includes the glucose-induced nucleocytoplasmic translocation of polypyrimidine tract binding protein 1 (PTB1), which binds mRNAs encoding granule proteins, and thus promotes their stabilization and translation. Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin gene expression and secretion by increasing cAMP levels in beta cells. Here, we show that elevation of cAMP levels causes the protein kinase A-dependent phosphorylation and nucleocytoplasmic translocation of PTB1, thereby preventing the rapid degradation of insulin mRNA and enhancing the expression of various granule proteins. Taken together, these findings identify PTB1 as a common downstream target of glucose and GLP-1 for the posttranscriptional upregulation of granule biogenesis.

Eric Marois, Ali Mahmoud, Suzanne Eaton
The endocytic pathway and formation of the Wingless morphogen gradient.
Development, 133(2) 307-317 (2006)
PDF DOI

Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. We have utilized short-term expression of dominant negative Rab proteins to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. Our results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As previously shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. We find that both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. We find that Dally-like is not required for Wingless spread when endocytosis is blocked, and propose that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient.

Veronique Dubreuil, Lilla M. Farkas, Federico Calegari, Yoichi Kosodo, Wieland B. Huttner
Neurogenesis in the central nervous system
In: Cell Signaling and Growth Factors in Development: Vol. 1. (Eds.) Klaus Unsicker, Kerstin Krieglstein, Hoboken, USA, Wiley (2006), 229-286 Ch. 7
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Michael Schroeder, Albert Burger, Patty Kostkova, Robert Stevens, Bianca Habermann, Rose Dieng-Kuntz
Sealife: a semantic grid browser for the life sciences applied to the study of infectious diseases.
Studies in health technology and informatics, 120 167-178 (2006)
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The objective of Sealife is the conception and realisation of a semantic Grid browser for the life sciences, which will link the existing Web to the currently emerging eScience infrastructure. The SeaLife Browser will allow users to automatically link a host of Web servers and Web/Grid services to the Web content he/she is visiting. This will be accomplished using eScience's growing number of Web/Grid Services and its XML-based standards and ontologies. The browser will identify terms in the pages being browsed through the background knowledge held in ontologies. Through the use of Semantic Hyperlinks, which link identified ontology terms to servers and services, the SeaLife Browser will offer a new dimension of context-based information integration. In this paper, we give an overview over the different components of the browser and their interplay. This SeaLife Browser will be demonstrated within three application scenarios in evidence-based medicine, literature & patent mining, and molecular biology, all relating to the study of infectious diseases. The three applications vertically integrate the molecule/cell, the tissue/organ and the patient/population level by covering the analysis of high-throughput screening data for endocytosis (the molecular entry pathway into the cell), the expression of proteins in the spatial context of tissue and organs, and a high-level library on infectious diseases designed for clinicians and their patients. For more information see http://www.biote.ctu-dresden.de/sealife.

Rafael A P Guércio, Anna Shevchenko, Andrej Shevchenko, Jorge L López-Lozano, Jaime Paba, Marcelo V. de Sousa, Carlos A. O. Ricart
Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox.
Proteome Sci, 4 11-11 (2006)
PDF DOI

BACKGROUND: Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults. RESULTS: Optimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins. CONCLUSION: The analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Tobias Oelschlägel
Meiosis-specific regulation of the anaphase-promoting complex
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Serena Bielli
Characterization of the Drosophila Egfl7/8 ortholog during oogenesis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

Anne-Kristin Heninger, Karol Kozak, Ralf Kittler, Jan Wagner, Annett Lohmann, Ina Poser, Hannes Grabner, Eberhard Krausz, Frank Buchholz
RNAi libraries for Funtional Genomics: Avoid the Difficulties of Predicting Efficient siRNAs and the Cost of Chemical Synthesis
Genetic Engineering News, 26(19) 22-23 (2006)
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Charles R. Bradshaw, Vineeth Surendranath, Bianca Habermann
ProFAT: a web-based tool for the functional annotation of protein sequences.
BMC Bioinformatics, 7 466-466 (2006)
PDF DOI

BACKGROUND: The functional annotation of proteins relies on published information concerning their close and remote homologues in sequence databases. Evidence for remote sequence similarity can be further strengthened by a similar biological background of the query sequence and identified database sequences. However, few tools exist so far, that provide a means to include functional information in sequence database searches. RESULTS: We present ProFAT, a web-based tool for the functional annotation of protein sequences based on remote sequence similarity. ProFAT combines sensitive sequence database search methods and a fold recognition algorithm with a simple text-mining approach. ProFAT extracts identified hits based on their biological background by keyword-mining of annotations, features and most importantly, literature associated with a sequence entry. A user-provided keyword list enables the user to specifically search for weak, but biologically relevant homologues of an input query. The ProFAT server has been evaluated using the complete set of proteins from three different domain families, including their weak relatives and could correctly identify between 90% and 100% of all domain family members studied in this context. ProFAT has furthermore been applied to a variety of proteins from different cellular contexts and we provide evidence on how ProFAT can help in functional prediction of proteins based on remotely conserved proteins. CONCLUSION: By employing sensitive database search programs as well as exploiting the functional information associated with database sequences, ProFAT can detect remote, but biologically relevant relationships between proteins and will assist researchers in the prediction of protein function based on remote homologies.

Ganka Nikolova
The vascular basement membrane : a niche for insuline gene expression and beta cell proliferation
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)
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Endocrine pancreatic beta cells depend on endothelial signals for their differentiation and

Doris Meder, Maria Joao Moreno, Paul Verkade, Winchil L. C. Vaz, Kai Simons
Phase coexistence and connectivity in the apical membrane of polarized epithelial cells.
Proc Natl Acad Sci U.S.A., 103(2) 329-334 (2006)
PDF DOI

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature ( approximately 25 degrees C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37 degrees C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12 degrees C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25 degrees C in which isolated domains of the nonraft phase are dispersed, whereas at 37 degrees C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.

Ivo F. Sbalzarini, J. Walther, M. Bergdorf, Simone E. Hieber, Evangelos M. Kotsalis, Petros Koumoutsakos
PPM – A highly efficient parallel particle–mesh library for the simulation of continuum systems
J Comput Phys, 215 566-588 (2006)
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Simon F. Tolić-Nørrelykke^*, Erik Schäffer^*, Jonathon Howard, Francesco S. Pavone, Frank Jülicher, Henrik Flyvbjerg
Calibration of optical tweezers with positional detection in the back focal plane
Rev Sci Instrum, 77(10) 1-11 (2006)
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We explain and demonstrate a new method of force and position calibrations for optical tweezers with back-focal-plane photodetection. The method combines power spectral measurements of thermal motion and the response to a sinusoidal motion of a translation stage. It consequently does not use the drag coefficient of the trapped object as an input. Thus, neither the viscosity, nor the size of the trapped object, nor its distance to nearby surfaces needs to be known. The method requires only a low level of instrumentation and can be applied in situ in all spatial dimensions. It is both accurate and precise: true values are returned, with small error bars. We tested this experimentally, near and far from surfaces in the lateral directions. Both position and force calibrations were accurate to within 3%. To calibrate, we moved the sample with a piezoelectric translation stage, but the laser beam could be moved instead, e.g., by acousto-optic deflectors. Near surfaces, this precision requires an improved formula for the hydrodynamical interaction between an infinite plane and a microsphere in nonconstant motion parallel to it. We give such a formula.

Dominik Schwudke, Jeffrey Oegema, Lyle Burton, Eugeni V. Entchev, J Thomas Hannich, Christer S. Ejsing, Teymuras V. Kurzchalia, Andrej Shevchenko
Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition.
Anal Chem, 78(2) 585-595 (2006)
PDF DOI

Data-dependent acquisition of MS/MS spectra from lipid precursors enables to emulate the simultaneous acquisition of an unlimited number of precursor and neutral loss scans in a single analysis. This approach takes full advantage of rich fragment patterns in tandem mass spectra of lipids and enables their profiling by complex (Boolean) scans, in which masses of several fragment ions are considered within a single logical framework. No separation of lipids is required, and the accuracy of identification and quantification is not compromised, compared to conventional precursor and neutral loss scanning.

Alessio Attardo
Novel in vivo imaging approaches to study embryonic and adult neurogenesis in the mouse
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2006)

F. U. Gast, P. S. Dittrich, Petra Schwille, M. Weigel, Michael Mertig, Jörg Opitz, Uta Queitsch, Stefan Diez, B. Lincoln, F. Wottawah, S. Schinkinger, Jochen Guck, J. Käs, J. Smolinski, K. Salchert, Carsten Werner, C. Duschl, M. S. Jäger, K. Uhlig, P. Geggier, S. Howitz
The microscopy cell (MicCell), a versatile modular flowthrough system for cell biology, biomaterial research, and nanotechnology
Microfluidics and Nanofluidics, 2(1) 21-26 (2006)
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We describe a novel microfluidic perfusion system for high-resolution microscopes. Its modular design allows pre-coating of the coverslip surface with reagents, biomolecules, or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special molding station, using masters made by photolithography and dry etching of silicon or by photoresist patterning on glass or silicon. This channel system can be reused while the coverslip is exchanged between experiments. As normal fluidic connectors are used, the link to external, computer-programmable syringe pumps is standardized and various fluidic channel networks can be used in the same setup. The system can house hydrogel microvalves and microelectrodes close to the imaging area to control the influx of reaction partners. We present a range of applications, including single-molecule analysis by fluorescence correlation spectroscopy (FCS), manipulation of single molecules for nanostructuring by hydrodynamic flow fields or the action of motor proteins, generation of concentration gradients, trapping and stretching of live cells using optical fibers precisely mounted in the PDMS layer, and the integration of microelectrodes for actuation and sensing.

Jessica Dessimoz, Robert Opoka, Jennifer J Kordich, Anne Grapin-Botton, James M Wells
FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo.
Mech Dev, 123(1) 42-55 (2006)
DOI

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.

Sandra Schubert
The role of β2-Syntrophin phosphorylation in secretory granule exocytosis
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Eric R Geertsma, Ria H Duurkens, B Poolman
The activity of the lactose transporter from Streptococcus thermophilus is increased by phosphorylated IIA and the action of beta-galactosidase.
Biochemistry, 44(48) 15889-15897 (2005)
DOI

The metabolism of lactose by Streptococcus thermophilus is highly regulated, allowing the bacterium to prefer lactose over glucose as main source of carbon and energy. In vitro analysis of the enzymes involved in transport and hydrolysis of lactose showed that the transport reaction benefits from the hydrolysis of lactose at the trans side of the membrane. Furthermore, the activity of LacS is modulated by PEP-dependent phosphorylation of the IIA domain via the general energy coupling proteins of the PTS, Enzyme I and HPr. To determine whether unphosphorylated LacS-IIA inhibited, or the phosphorylated form stimulated lactose counterflow, a LacS-IIA truncation mutant of LacS was constructed. Detailed analyses of transport in whole cells and in proteoliposomes indicated that unphosphorylated LacS-IIA does not functionally interact with the carrier domain. Instead, interaction of the phosphorylated form of LacS-IIA with the carrier stimulates lactose counterflow transport. The proposed mode of regulation thus proceeds via a mechanism opposite to the inducer exclusion type of regulation in gram-negative bacteria, where transporters are inhibited by binding of the unphosphorylated form of IIA(Glc).

Kai Simons, Carol Featherstone
The European research council on the brink.
Cell, 123(5) 747-750 (2005)
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Basic research in Europe is about to receive a shot in the arm with the creation of a European Research Council (ERC). This new agency will sustain fundamental investigation into all aspects of scientific knowledge and should drive up standards of scientific research across the continent.

Henrik Bringmann
Cytokinesis and the spindle midzone.
Cell Cycle, 4(12) 1709-1712 (2005)
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At the end of the cell cycle a cell physically divides into two daughter cells in a process called cytokinesis. Cytokinesis consists of at least four steps: (1) The position of the presumptive cytokinesis furrow is specified. (2) A contractile ring is formed. (3) The contractile ring contracts, resulting in furrow ingression. (4) Cytokinesis completes with sealing of the membranes. The mitotic spindle positions the cytokinesis furrow at the cell cortex midway along the longitudinal axis of the spindle, which is both the mid-point between the two asters and the location of the spindle midzone. The mitotic spindle emits two consecutive signals that position the furrow: Microtubule asters provide a first signal; the spindle midzone provides a second signal. Our results support the view that the spindle midzone is dispensable for completion of cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis, possibly because the aster- and midzone-positioned furrows compete for contractile elements.

Ricardo Zayas, Alvaro Hernández, Bianca Habermann, Yuying Wang, Joel M Stary, Phillip A Newmark
The planarian Schmidtea mediterranea as a model for epigenetic germ cell specification: analysis of ESTs from the hermaphroditic strain.
Proc Natl Acad Sci U.S.A., 102(51) 18491-18496 (2005)
PDF DOI

Freshwater planarians have prodigious regenerative abilities that enable them to form complete organisms from tiny body fragments. This plasticity is also exhibited by the planarian germ cell lineage. Unlike many model organisms in which germ cells are specified by localized determinants, planarian germ cells appear to be specified epigenetically, arising postembryonically from stem cells. The planarian Schmidtea mediterranea is well suited for investigating the mechanisms underlying epigenetic germ cell specification. Two strains of S. mediterranea exist: a hermaphroditic strain that reproduces sexually and an asexual strain that reproduces by means of transverse fission. To date, expressed sequence tags (ESTs) have been generated only from the asexual strain. To develop molecular reagents for studying epigenetic germ cell specification, we have sequenced 27,161 ESTs from two developmental stages of the hermaphroditic strain of S. mediterranea; this collection of ESTs represents approximately 10,000 unique transcripts. blast analysis of the assembled ESTs showed that 66% share similarity to sequences in public databases. We annotated the assembled ESTs using Gene Ontology terms as well as conserved protein domains and organized them in a relational database. To validate experimentally the Gene Ontology annotations, we used whole-mount in situ hybridization to examine the expression patterns of transcripts assigned to the biological process "reproduction." Of the 53 genes in this category, 87% were expressed in the reproductive organs. In addition to its utility for studying germ cell development, this EST collection will be an important resource for annotating the planarian genome and studying this animal's amazing regenerative abilities.

Anne-Kathrin Classen, Kurt I. Anderson, Eric Marois, Suzanne Eaton
Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway.
Dev Cell, 9(6) 805-817 (2005)
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The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. Here, we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. We propose that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems.

Takehiko Itoh, Kai S. Erdmann, Aurelien Roux, Bianca Habermann, Hauke Werner, Pietro De Camilli
Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.
Dev Cell, 9(6) 791-804 (2005)
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Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.

Wieland B. Huttner, Yoichi Kosodo
Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system.
Curr Opin Cell Biol, 17(6) 648-657 (2005)
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The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.

Verena Arndt, Christina Daniel, Wolfgang Nastainczyk, Simon Alberti, Jörg Höhfeld
BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP.
Mol Biol Cell, 16(12) 5891-5900 (2005)
DOI

Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.

Yanmei Liu, Anna Shevchenko, Andrej Shevchenko, Arnold J Berk
Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex.
J Virol, 79(22) 14004-14016 (2005)
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Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.

Alexander Picker, Michael Brand
Fgf signals from a novel signaling center determine axial patterning of the prospective neural retina.
Development, 132(22) 4951-4962 (2005)
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Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. We find that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina.

Sergey G Odintsov, Izabela Sabala, Gleb Bourenkov, Vladimir Rybin, Matthias Bochtler
Substrate access to the active sites in aminopeptidase T, a representative of a new metallopeptidase clan.
J Mol Biol, 354(2) 403-412 (2005)
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Aminopeptidase T (AmpT) from Thermus thermophilus is a metalloexopeptidase with no similarity to prototypical metallopeptidases with an HExxH or HxxEH motif. The crystal structure of the Staphylococcus aureus homologue of AmpT, which is known as aminopeptidase S (AmpS), has been reported recently. This structure revealed a dimeric protein with a very unusual, elongated shape and a large internal cavity. The active sites were found on the inner walls of the cavity and were entirely shielded from the environment, which suggested either that the dimer in the crystals was not physiologically relevant, or that an inactive conformation had been crystallized. Here, we show by gel-filtration and analytical ultracentrifugation that AmpT, like AmpS, forms dimers in solution, and we present the structure of AmpT in a crystal form with five protomers in the asymmetric unit. The five protomers take conformations that range from fully closed, as in the AmpS structure, to nearly open, so that the active site is almost directly accessible. The different conformations indicate flexibility between the AmpT N and C-domains, and explain how AmpT can be active, although the unusual AmpS dimerization mode applies to AmpT as well.

Saulius Grazulis, Elena Manakova, Manfred Roessle, Matthias Bochtler, Giedre Tamulaitiene, Robert Huber, Virginijus Siksnys
Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease.
Proc Natl Acad Sci U.S.A., 102(44) 15797-15802 (2005)
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Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-A resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a beta-barrel-like structure very similar to the effector DNA-binding domain of the Mg(2+)-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A psi-blast search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.

Magdalena Götz, Wieland B. Huttner
The cell biology of neurogenesis.
Nat Rev Mol Cell Biol, 6(10) 777-788 (2005)
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During the development of the mammalian central nervous system, neural stem cells and their derivative progenitor cells generate neurons by asymmetric and symmetric divisions. The proliferation versus differentiation of these cells and the type of division are closely linked to their epithelial characteristics, notably, their apical-basal polarity and cell-cycle length. Here, we discuss how these features change during development from neuroepithelial to radial glial cells, and how this transition affects cell fate and neurogenesis.

Ina Weisswange, Till Bretschneider, Kurt I. Anderson
The leading edge is a lipid diffusion barrier.
J Cell Sci, 118(Pt 19) 4375-4380 (2005)
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Actin polymerization drives many cellular events, including endocytosis, pathogen rocketing, and cell spreading. Force generation and polymerization regulation are intimately linked where an actin meshwork attaches to, and pushes against, an interface. We reasoned that interaction with actin filament plus-ends might stabilize the position of components within the plasma membrane at the leading edge, thereby slowing the diffusion of lipids within the bilayer where filament growth occurs. To test this hypothesis we focally labeled the outer membrane leaflet of migrating keratocytes and compared the initial diffusion of carbocyanine dyes in the dorsal and ventral lamellipodium membranes using sequential TIRF and epi-fluorescent imaging. Global diffusion analysis shows that lateral mobility of lipids in the outer membrane leaflet is blocked at the leading edge during protrusion. Cytochalasin treatment abolished this diffusion barrier, but we found no evidence to support the involvement of membrane microdomains. Our results demonstrate the immobilization of membrane components at the leading edge, and suggest that interaction between actin filaments and the plasma membrane is mediated by densely packed molecular complexes. We propose that actin polymerization traps regulatory proteins at the leading edge in a positive-feedback loop.

Mihail Sarov, A Francis Stewart
The best control for the specificity of RNAi
Trends Biotechnol, 23(9) 446-448 (2005)
PDF DOI

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

Eugene W Myers
The fragment assembly string graph.
Bioinformatics, 21 Suppl 2 79-85 (2005)
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We present a concept and formalism, the string graph, which represents all that is inferable about a DNA sequence from a collection of shotgun sequencing reads collected from it. We give time and space efficient algorithms for constructing a string graph given the collection of overlaps between the reads and, in particular, present a novel linear expected time algorithm for transitive reduction in this context. The result demonstrates that the decomposition of reads into kmers employed in the de Bruijn graph approach described earlier is not essential, and exposes its close connection to the unitig approach we developed at Celera. This paper is a preliminary piece giving the basic algorithm and results that demonstrate the efficiency and scalability of the method. These ideas are being used to build a next-generation whole genome assembler called BOA (Berkeley Open Assembler) that will easily scale to mammalian genomes.

Gregory Emery, Andrea Hutterer, Daniela Berdnik, Bernd Mayer, Frederik Wirtz-Peitz, Marcos González-Gaitán, Jürgen A. Knoblich
Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system.
Cell, 122(5) 763-773 (2005)
PDF DOI

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.

Otilia V Vieira, Paul Verkade, Aki Manninen, Kai Simons
FAPP2 is involved in the transport of apical cargo in polarized MDCK cells.
J Cell Biol, 170(4) 521-526 (2005)
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Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin-Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

Hye-Won Shin, Mitsuko Hayashi, Savvas Christoforidis, Sandra Lacas-Gervais, Sebastian Hoepfner, Markus R Wenk, Jan Modregger, Sandrine Uttenweiler-Joseph, Matthias Wilm, Arne Nystuen, Wayne N Frankel, Michele Solimena, Pietro De Camilli, Marino Zerial
An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway.
J Cell Biol, 170(4) 607-618 (2005)
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Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Martin Srayko, Aynur Kaya, Joanne Stamford, Anthony A. Hyman
Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo.
Dev Cell, 9(2) 223-236 (2005)
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Microtubules (MTs) are dynamic polymers that undergo cell cycle and position-sensitive regulation of polymerization and depolymerization. Although many different factors that regulate MT dynamics have been described, to date there has been no systematic analysis of genes required for MT dynamics in a single system. Here, we use a transgenic EB1::GFP strain, which labels the growing plus ends of MTs, to analyze the growth rate, nucleation rate, and distribution of growing MTs in the Caenorhabditis elegans embryo. We also present the results from an RNAi screen of 40 genes previously implicated in MT-based processes. Our findings suggest that fast microtubule growth is dependent on the amount of free tubulin and the ZYG-9-TAC-1 complex. Robust MT nucleation by centrosomes requires AIR-1, SPD-2, SPD-5, and gamma-tubulin. However, we found that centrosomes do not nucleate MTs to saturation; rather, the depolymerizing kinesin-13 subfamily member KLP-7 is required to limit microtubule outgrowth from centrosomes.

Nurhan Ozlü, Martin Srayko, Kazuhisa Kinoshita, Bianca Habermann, Eileen T. O'Toole, Thomas Müller-Reichert, Natalie Schmalz, Arshad Desai, Anthony A. Hyman
An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles.
Dev Cell, 9(2) 237-248 (2005)
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In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.

Lars Demmel
Unraveling phosphatidylinositol 4-kinase function in the yeast Golgi-endosomal system
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Ingmar H. Riedel
Mechanics of the axoneme: self-organized beating patterns and vortex arrays of spermatozoa
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)
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Cilia and eukaryotic flagella are long, thin extensions of cells that contain a structure known as axoneme. The key components of the axoneme are microtubule filaments and the motor proteins dynein. These dynein motors force the microtubules to slide in an oscillatory fashion leading to a wave pattern along the axoneme. How these motors are coordinated and how this phenomenon can be described quantitatively is not understood. I therefore studied the waveforms of sperm tails that contain such an axoneme. I observed these waveforms under different conditions with a high-speed camera and developed an automated image analysis tool allowing the extraction of long time series of this waveform. In a subsequent Fourier analysis I increased the precision by obtaining an averaged waveform. I then compared the data to the predictions of a theoretical framework (Camalet, Julicher et al. 1999) and found that they do not agree. I suggested extending this theoretical framework by considering a visco-elastic element at the base of the axoneme, which leads to a satisfactory agreement. This project leaves open questions hence further work is discussed. As a side finding, I discovered a new phenomenon on how spermatozoa form dynamic vortex arrays. I described this pattern in detail and introduced a novel order parameter to quantify the order among many particles. I showed that the array only forms above a critical sperm density. I suggested a model to explain the origin of the pattern and showed by simulation that the model can account for the main features of the pattern. Finally I estimated the typical interaction force between beating axonemes to be 0.1 pN and drew conclusions about their collective action in general that might be relevant for sperm cooperation or metachronal waves of cilia.

Agathi Papanikolaou, Alexandra Papafotika, Carol Murphy, Thomais Papamarcaki, Orestes Tsolas, Marek Drab, Teymuras V. Kurzchalia, Michael Kasper, Savvas Christoforidis
Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39.
J Biol Chem, 280(28) 26406-26414 (2005)
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CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.

Eric R Geertsma, Ria H Duurkens, B Poolman
Functional interactions between the subunits of the lactose transporter from Streptococcus thermophilus.
J Mol Biol, 350(1) 102-111 (2005)
DOI

Although the quaternary state has been assessed in detail for only a few members of the major facilitator superfamily (MFS), it is clear that multiple oligomeric states are represented within the MFS. One of its members, the lactose transporter LacS from Streptococcus thermophilus assumes a dimeric structure in the membrane and in vitro analysis showed functional interactions between both subunits when proton motive force ((Delta)p)-driven transport was assayed. To study the interactions in further detail, a covalent dimer was constructed consisting of in tandem fused LacS subunits. These covalent dimers, composed of active and completely inactive subunits, were expressed in Escherichia coli, and initial rates of (Delta)p-driven lactose uptake and lactose counterflow were determined. We now show that also in vivo, both subunits interact functionally; that is, partial complementation of the inactive subunit was observed for both transport modes. Thus, both subunits interact functionally in (Delta)p-driven uptake and in counterflow transport. In addition, analysis of in tandem fused LacS subunits containing one regulatory LacS-IIA domain showed that regulation is primarily an intramolecular event.

Federico Calegari^*, Wulf Haubensak^*, Christiane Haffner, Wieland B. Huttner
Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development.
J Neurosci, 25(28) 6533-6538 (2005)
PDF DOI

During embryonic development of the mammalian brain, the average cell-cycle length of progenitor cells in the ventricular zone is known to increase. However, for any given region of the developing cortex and stage of neurogenesis, the length of the cell cycle is thought to be similar in the two coexisting subpopulations of progenitors [i.e., those undergoing (symmetric) proliferative divisions and those undergoing (either asymmetric or symmetric) neuron-generating divisions]. Using cumulative bromodeoxyuridine labeling of Tis21-green fluorescent protein knock-in mouse embryos, in which these two subpopulations of progenitors can be distinguished in vivo, we now show that at the onset as well as advanced stages of telencephalic neurogenesis, progenitors undergoing neuron-generating divisions are characterized by a significantly longer cell cycle than progenitors undergoing proliferative divisions. In addition, we find that the recently characterized neuronal progenitors dividing at the basal side of the ventricular zone and in the subventricular zone have a longer G(2) phase than those dividing at the ventricular surface. These findings are consistent with the hypothesis (Calegari and Huttner, 2003) that cell-cycle lengthening can causally contribute to neural progenitors switching from proliferative to neuron-generating divisions and may have important implications for the expansion of somatic stem cells in general.

Andrew C. Oates, Laurel A. Rohde, Robert K Ho
Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event.
Dev Biol, 283(1) 204-214 (2005)
PDF DOI

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.

Janina Görnemann, Kimberly M. Kotovic, Katja Hujer, Karla M. Neugebauer
Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex.
Mol Cell, 19(1) 53-63 (2005)
PDF DOI

Coupling between transcription and pre-mRNA splicing is a key regulatory mechanism in gene expression. Here, we investigate cotranscriptional spliceosome assembly in yeast, using in vivo crosslinking to determine the distribution of spliceosome components along intron-containing genes. Accumulation of the U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) and the 3' splice site binding factors Mud2p and BBP was detected in patterns indicative of progressive and complete spliceosome assembly; recruitment of the nineteen complex (NTC) component Prp19p suggests that splicing catalysis is also cotranscriptional. The separate dynamics of the U1, U2, and U5 snRNPs are consistent with stepwise recruitment of individual snRNPs rather than a preformed "penta-snRNP", as recently proposed. Finally, we show that the cap binding complex (CBC) is necessary, but not sufficient, for cotranscriptional spliceosome assembly. Thus, the demonstration of an essential link between CBC and spliceosome assembly in vivo indicates that 5' end capping couples pre-mRNA splicing to transcription.

Tobias Langenberg, Michael Brand
Lineage restriction maintains a stable organizer cell population at the zebrafish midbrain-hindbrain boundary.
Development, 132(14) 3209-3216 (2005)
PDF DOI

The vertebrate hindbrain is subdivided into segments, termed neuromeres, that are units of gene expression, cell differentiation and behavior. A key property of such segments is that cells show a restricted ability to mix across segment borders -- termed lineage restriction. In order to address segmentation in the midbrain-hindbrain boundary (mhb) region, we have analyzed single cell behavior in the living embryo by acquiring time-lapse movies of the developing mhb region in a transgenic zebrafish line. We traced the movement of hundreds of nuclei, and by matching their position with the expression of a midbrain marker, we demonstrate that midbrain and hindbrain cells arise from two distinct cell populations. Single cell labeling and analysis of the distribution of their progeny shows that lineage restriction is probably established during late gastrulation stages. Our findings suggest that segmentation as an organizing principle in early brain development can be extended to the mhb region. We argue that lineage restriction serves to constrain the position of the mhb organizer cell population.

Anne-Marie Marzesco, Peggy Janich, Michaela Wilsch-Bräuninger, Veronique Dubreuil, Katja Langenfeld, Denis Corbeil, Wieland B. Huttner
Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells.
J Cell Sci, 118(Pt 13) 2849-2858 (2005)
PDF DOI

Apical plasma membrane constituents of mammalian neural stem/progenitor cells have recently been implicated in maintaining their stem/progenitor cell state. Here, we report that in the developing embryonic mouse brain, the fluid in the lumen of the neural tube contains membrane particles carrying the stem cell marker prominin-1 (CD133), a pentaspan membrane protein found on membrane protrusions of the apical surface of neuroepithelial cells. Two size classes of prominin-1-containing membrane particles were observed in the ventricular fluid: approximately 600-nm particles, referred to as P2 particles, and 50-80-nm vesicles, referred to as P4 particles. The P2 and P4 particles appeared in the ventricular fluid at the very onset and during the early phase of neurogenesis, respectively. Concomitant with their appearance, the nature of the prominin-1-containing apical plasma membrane protrusions of neuroepithelial cells changed, in that microvilli were lost and large pleiomorphic protuberances appeared. P4 particles were found in various body fluids of adult humans, including saliva, seminal fluid and urine, and were released by the epithelial model cell line Caco-2 upon differentiation. Importantly, P4 particles were distinct from exosomes. Our results demonstrate the widespread occurrence of a novel class of extracellular membrane particles containing proteins characteristic of stem cells, and raise the possibility that the release of the corresponding membrane subdomains from the apical surface of neural progenitors and other epithelial cells may have a role in tissue development and maintenance. Moreover, the presence of prominin-1-containing membrane particles in human body fluids may provide the basis for a protein-based diagnosis of certain diseases.

Dong Ji Zhang, Qi Wang, Jie Wei, Gyulnar Baimukanova, Frank Buchholz, A. Francis Stewart, Xiaohong Mao, Nigel Killeen
Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene.
J Immunol, 174(11) 6725-6731 (2005)
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Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.

Audra Lee, Jessica D Kessler, Tracy-Ann Read, Constanze Kaiser, Denis Corbeil, Wieland B. Huttner, Jane E Johnson, Robert J Wechsler-Reya
Isolation of neural stem cells from the postnatal cerebellum.
Nat Neurosci, 8(6) 723-729 (2005)
PDF DOI

The cerebellum is critical for motor coordination and cognitive function and is the target of transformation in medulloblastoma, the most common malignant brain tumor in children. Although the development of granule cells, the most abundant neurons in the cerebellum, has been studied in detail, the origins of other cerebellar neurons and glia remain poorly understood. Here we show that the murine postnatal cerebellum contains multipotent neural stem cells (NSCs). These cells can be prospectively isolated based on their expression of the NSC marker prominin-1 (CD133) and their lack of markers of neuronal and glial lineages (lin-). Purified prominin+ lin- cells form self-renewing neurospheres and can differentiate into astrocytes, oligodendrocytes and neurons in vitro. Moreover, they can generate each of these lineages after transplantation into the cerebellum. Identification of cerebellar stem cells has important implications for the understanding of cerebellar development and the origins of medulloblastoma.

Cosima Luedeke, Stéphanie Buvelot Frei, Ivo F. Sbalzarini, Heinz Schwarz, Anne Spang, Yves Barral
Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth.
J Cell Biol, 169(6) 897-908 (2005)
PDF DOI

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

Amy Shaub Maddox, Bianca Habermann, Arshad Desai, Karen Oegema
Distinct roles for two C. elegans anillins in the gonad and early embryo.
Development, 132(12) 2837-2848 (2005)
PDF DOI

Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.

Kate Poole^*, Khaled Khairy^*, Jens Friedrichs^*, Clemens Franz, David A. Cisneros, Jonathon Howard, Daniel J. Müller
Molecular-scale topographic cues induce the orientation and directional movement of fibroblasts on two-dimensional collagen surfaces.
J Mol Biol, 349(2) 380-386 (2005)
PDF DOI

Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.

Thimo Kurz, Nurhan Ozlü, Fabian Rudolf, Sean M O'Rourke, Brian Luke, Kay Hofmann, Anthony A. Hyman, Bruce Bowerman, Matthias Peter
The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae.
Nature, 435(7046) 1257-1261 (2005)
PDF DOI

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Zuzanna Kroneková
Assembly of mitochondrial ubiquinol-cytochrome c oxidoreductase complex in qeast Saccharomyces cerevisiae : the role of Cbp3p and Cbp4p assembly factors
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Fabien Alpy, Vinoth K Latchumanan, Valérie Kedinger, Agnes Janoshazi, Christoph Thiele, Corinne Wendling, Marie-Christine Rio, Catherine Tomasetto
Functional characterization of the MENTAL domain.
J Biol Chem, 280(18) 17945-17952 (2005)
PDF DOI

Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH(2)-terminal) domain shared with an unique protein called MENTHO (MLN64 NH(2)-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).

M Kamran Azim, Walter Goehring, Hyun Kyu Song, Ravishankar Ramachandran, Matthias Bochtler, Peter Goettig
Characterization of the HslU chaperone affinity for HslV protease.
Protein Sci, 14(5) 1357-1362 (2005)
PDF DOI

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.

Sebastian Hoepfner, Fedor F. Severin, Alicia Cabezas, Bianca Habermann, Anja Runge, David J. Gillooly, Harald Stenmark, Marino Zerial
Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B.
Cell, 121(3) 437-450 (2005)
PDF DOI

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.

Stefan Diez, Günther Gerisch, Kurt I. Anderson, Annette Müller-Taubenberger, Till Bretschneider
Subsecond reorganization of the actin network in cell motility and chemotaxis.
Proc Natl Acad Sci U.S.A., 102(21) 7601-7606 (2005)
PDF DOI

Actin networks are continuously reorganized in cells that rapidly change their shape. Applying total internal reflection fluorescence microscopy at acquisition rates of 10-20 Hz, we measured an average growth rate of 3 microm.sec(-1) for filamentous actin structures throughout the entire substrate-attached cortex of Dictyostelium cells. New filaments often proceed along preexisting ones, resulting in bundle formation concurrent with filament growth. In cells that orientate in a gradient of chemoattractant, prominent assemblies of actin enriched in the Arp2/3 complex are inserted into the network, primarily at the base of filopods that point into the direction of the gradient. We propose that high turnover rates of actin filaments confer the plasticity to the cell cortex that is required for rapid accommodation to external stimuli.

Eugeni V. Entchev, Teymuras V. Kurzchalia
Requirement of sterols in the life cycle of the nematode Caenorhabditis elegans.
Semin Cell Dev Biol, 16(2) 175-182 (2005)
PDF DOI

The nematode Caenorhabditis elegans represents an excellent model for studying many aspects of sterol function on the level of a whole organism. Recent studies show that especially two processes in the life cycle of the worm, dauer larva formation and molting, depend on sterols. In both cases, cholesterol or its derivatives seem to act as hormones rather than being structural components of the membrane. Investigations on C. elegans could provide information on the etiology of human diseases that display defects in the transport or metabolism of sterols.

Gert Dandanell, Roman H Szczepanowski, Borys Kierdaszuk, David Shugar, Matthias Bochtler
Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene.
J Mol Biol, 348(1) 113-125 (2005)
PDF DOI

Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.

Doris Meder^#, Kai Simons^#
Ras on the roundabout.
Science, 307(5716) 1731-1733 (2005)
PDF DOI

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

Gabriela Kania, Denis Corbeil, Joerg Fuchs, Kirill V Tarasov, Przemyslaw Blyszczuk, Wieland B. Huttner, Kenneth R Boheler, Anna M Wobus
The somatic stem cell marker prominin-1/CD133 is expressed in embryonic stem cell-derived progenitors
Eur J Cell Biol, 84(Supplement 55) 76-76 (2005)

Anne Grapin-Botton
Ductal cells of the pancreas.
Int. J. Biochem. Cell Biol., 37(3) 504-510 (2005)
DOI

Ductal cells of the pancreas form the epithelial lining of the branched tubes that deliver enzymes produced by pancreatic acinar cells into the duodenum. In addition, these cells secrete bicarbonate that neutralizes stomach acidity. During development, epithelium of endodermal origin evaginates from the future duodenum area and invades the mesenchyme to form a complex branched network. All endocrine, acinar and ductal cells arise from common precursors in this epithelial structure. Adult ductal cells share some similarities with embryonic primitive ducts and may retain the ability to generate endocrine cells in the adult. Based on challenged pancreas regeneration experiments, the adult ductal cells have been proposed to be pancreatic stem cells but their role in normal endocrine cell turnover has recently been challenged. Manipulating their ability to give rise to endocrine cells may open new avenues in the treatment of diabetes and therefore they have recently been under scrutiny. In addition, in the main form of pancreatic cancer, pancreas adenocarcinoma, tumor cells share similarities with ductal cells. The secrets of an appropriate therapy for this deadly cancer may thus reside in the biology of ductal cells.

Kirsten Bacia, Petra Schwille, Teymuras V. Kurzchalia
Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes.
Proc Natl Acad Sci U.S.A., 102(9) 3272-3277 (2005)
PDF DOI

The existence of lipid rafts in biological membranes in vivo is still debated. In contrast, the formation of domains in model systems has been well documented. In giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol, a clear separation of liquid-disordered and sphingomyelin-enriched, liquid-ordered phases could be observed. This phase separation can lead to the fission of the liquid-ordered phase from the vesicle. Here we show that in cholesterol-containing GUVs, the phase separation can involve dynamic redistribution of lipids from one phase into another as a result of a cross-linking perturbation. We found that the molecular structure of a sterol used for the preparation of GUVs determines (i) its ability to induce phase separation and (ii) the curvature (positive or negative) of the formed liquid-ordered phase. As a consequence, the latter can pinch off to the outside or inside of the vesicle. Remarkably, some mixtures of sterols induce liquid-ordered domains exhibiting both positive and negative curvature, which can lead to a new type of budding behavior in GUVs. Our findings could have implications for the role of sterols in various cell-biological processes such as budding of secretory vesicles, endocytosis, or formation of multivesicular bodies.

Jie Shen, Christian Dahmann
The role of Dpp signaling in maintaining the Drosophila anteroposterior compartment boundary.
Dev Biol, 279(1) 31-43 (2005)
PDF DOI

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it was not known whether and in which cells Dpp signal transduction was required for maintaining the A/P boundary. Here, we have investigated the role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary by clonal analysis. We show that a transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, we find that Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, we provide evidence that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. We propose that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary.

Juan-Antonio Montero, Lara Carvalho, Michaela Wilsch-Bräuninger, Beate Kilian, Chigdem Mustafa, Carl-Philipp Heisenberg
Shield formation at the onset of zebrafish gastrulation.
Development, 132(6) 1187-1198 (2005)
PDF DOI

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.

Tim L. Noetzel, David N. Drechsel, Anthony A. Hyman, Kazuhisa Kinoshita
A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1.
Philos Trans R Soc Lond B Biol Sci, 360(1455) 591-594 (2005)
PDF DOI

During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics.

Tobias Oelschlaegel^*, Martin Schwickart^*, Joao Matos, Aliona Bogdanova, Alain Camasses, Jan Havlis, Andrej Shevchenko, Wolfgang Zachariae
The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1.
Cell, 120(6) 773-788 (2005)
PDF DOI

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.

Muriel Rhinn, Klaus Lun, Marta Luz, Michaela Werner, Michael Brand
Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling.
Development, 132(6) 1261-1272 (2005)
PDF DOI

The organizing center located at the midbrain-hindbrain boundary (MHB) patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates showed that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, we find that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. We demonstrate that positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm, including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. Our findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organizer.

Elisa B Vervoort, Jelle B Bultema, Gea K Schuurman-Wolters, Eric R Geertsma, Jaap Broos, B Poolman
The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane.
J Mol Biol, 346(3) 733-743 (2005)
DOI

The mannitol permease (EII(Mtl)) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII(Mtl) contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII(Mtl), all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII(Mtl) undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.

Veronica Dudu
TGF-β signaling at the cellular junctions
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Martin Schwickart
Molecular analysis of novel subunits of the anaphase-promoting complex (APC/C) in yeast
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Karla M. Neugebauer, Kimberly M. Kotovic, Jennifer A. Geiger, David Stanĕk
Pre-mRNA Processing in the Nuclear Landscape
In: Visions of the Cell Nucleus. (Eds.) Stephan Diekmann, Peter Hemmerich, Stevenson Ranch, USA, American Scientific Publishers (2005), 106-119 Ch. 7
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All eukaryotic protein-coding genes are transcribed by RNA polymerase II (pol II), and each mRNA is the product not only of transcription but a variety of pre-mRNA processing events. In humans, every pre-mRNA acquires a methyl-guanosine cap at its 5` end, and nearly every transcript is internally spliced and polyadenylated at its 3` end. The purpose of this chapter is to place these three major pre-mRNA processing steps within the context of the three dimensional space of the cell nucleus. Because capping, splicing and polyadenylation at least begin during RNA synthesis, these reactions occur largely at sites of gene transcription, which are distributed throughout the nucleus and not localized to particular domains or substructures. Splicing and polyadenylation often continue post-transcriptionally, most likely in the interchromatin space. In addition, pre-mRNA processing factors are components of a number of subnuclear structures, such as Cajal Bodies and Cleavage Bodies, suggesting that some functions related to pre-mRNA processing are compartmentalized within the nuclear landscape.

Andriy Kovalchuk
Molecular analysis of the LTR Retrotransposon Ylt1 from the genome of dimorphic fungus yarrowia lipolytica
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Anna Shevchenko^*, Mirta M. L. de Sousa^*, Patrice Waridel, Silvia Tolfo Bittencourt, Marcelo V. de Sousa, Andrej Shevchenko
Sequence similarity-based proteomics in insects: characterization of the larvae venom of the Brazilian moth Cerodirphia speciosa.
J Proteome Res, 4(3) 862-869 (2005)
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Using a combination of tandem mass spectrometric sequencing and sequence similarity searches, we characterized the larvae venom of the moth Cerodirphia speciosa, which belongs to the Saturniidae family of the Lepidoptera order. Despite the paucity of available database sequence resources, the approach enabled us to identify 48 out of 58 attempted spots on its two-dimensional gel electrophoresis map, which represented 37 unique proteins, whereas it was only possible to identify 13 proteins by conventional non-error tolerant database searching methods. The majority of cross-species hits were made to proteins from the phylogenetically related Lepidoptera organism, the silk worm Bombyx mori. The protein composition of the venom suggested that envenoming by C. speciosa toxins might proceed through the contact with its hemolymph, similarly to another toxic Lepidoptera organism, Lonomia obliqua.

Esther Schnapp
Studying the Molecular Mechanisms for Generating Progenitor Cells during Tail Regeneration in Ambystoma mexicanum
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2005)

Andrei I. Pozniakovsky^*, Dmitry A. Knorre^*, Olga V. Markova^*, Anthony A. Hyman, Vladimir P. Skulachev, Fedor F. Severin
Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast.
J Cell Biol, 168(2) 257-269 (2005)
PDF DOI

Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.

Anne Grapin-Botton
Antero-posterior patterning of the vertebrate digestive tract: 40 years after Nicole Le Douarin's PhD thesis.
Int. J. Dev. Biol., 49(2-3) 335-347 (2005)
DOI

This review is dedicated to the work on chick digestive tract organogenesis that Nicole Le Douarin performed as a PhD student under the direction of Etienne Wolf. I discuss how she laid the grounds for future work by establishing fate maps at somitic stages, by describing morphogenetic movements between germ layers and by pointing to signaling events between endoderm and mesoderm. Her inspiring work was extended by others, in particular at the molecular level, leading to a better understanding of antero-posterior patterning in the digestive tract. Antero-posterior patterning of endoderm is initiated at gastrulation when future anterior and posterior endoderm ingress at different times and accordingly express different genes. Plasticity is however maintained at somite stages and even later, when organ primordia can be delineated. There is a cross-talk between endoderm and mesoderm and the two layers exchange instructive signals that induce specific antero-posterior identities as well as permissive signals required for organogenesis from previously patterned fields. Recent experiments suggest that several signaling molecules involved in neural tube antero-posterior patterning are also instrumental in the digestive tract including retinoic acid and FGF4.

Doris Meder, Anna Shevchenko, Kai Simons, Joachim Füllekrug
Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells.
J Cell Biol, 168(2) 303-313 (2005)
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Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

Sebastian Schuck, Kai Simons
Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane.
J Cell Sci, 117(Pt 25) 5955-5964 (2004)
PDF DOI

Polarized cells establish and maintain functionally distinct surface domains by an elaborate sorting process, which ensures accurate delivery of biosynthetic cargo to different parts of the plasma membrane. This is particularly evident in polarized epithelial cells, which have been used as a model system for studies of sorting mechanisms. The clustering of lipid rafts through the oligomerization of raft components could be utilized for segregating apical from basolateral cargo and for the generation of intracellular transport carriers. Besides functioning in polarized sorting in differentiated cells, raft clustering might also play an important role in the biogenesis of apical membrane domains during development.

Florian Raible, Michael Brand
Divide et Impera--the midbrain-hindbrain boundary and its organizer.
Trends Neurosci, 27(12) 727-734 (2004)
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The midbrain-hindbrain organizer (MHO) is a signalling centre that orchestrates development of the mesencephalic and anterior metencephalic primordia. In recent years, details have been revealed about the molecular nature of these signals, their transmission and reception, and the regulatory processes associated with MHO function. This article reviews recent progress in understanding the genetic and molecular components of the MHO, and how they synergize to control brain development.

P Huw Williams, Anja Hagemann, Marcos González-Gaitán, James C Smith
Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo.
Curr Biol, 14(21) 1916-1923 (2004)
PDF DOI

One way in which cells acquire positional information during embryonic development is by measuring the local concentration of a signaling factor, or morphogen, that is secreted by an organizing center . The ways in which morphogen gradients are established, particularly in vertebrates, remain obscure, although various suggestions have been made for the mechanisms by which signaling molecules traverse fields of cells. These include simple diffusion, "cytonemes", filopodia, "argosomes", and "transcytosis". In this study, we use a functional EGFP-tagged ligand to visualize long-range signaling in the Xenopus embryo in real time. Our results show that the TGF-beta family member Xnr2 is secreted efficiently from embryonic cells, and a new method of tissue recombination allows us to investigate the way in which the morphogen traverses multiple cell diameters. This reveals that Xnr2 exerts long-range effects by diffusing rapidly through the extracellular milieu of nonexpressing cells. No evidence has been obtained for long-range signaling through cytonemes, filopodia, argosomes, or transcytosis. In demonstrating that long-range signaling in the early Xenopus embryo occurs by diffusion rather than by these alternative routes, our results suggest that different morphogens in different developmental contexts use different means of transport.

Claudia Esser, Simon Alberti, Jörg Höhfeld
Cooperation of molecular chaperones with the ubiquitin/proteasome system.
Biochim Biophys Acta, 1695(1-3) 171-188 (2004)
DOI

Molecular chaperones and energy-dependent proteases have long been viewed as opposing forces that control protein biogenesis. Molecular chaperones are specialized in protein folding, whereas energy-dependent proteases such as the proteasome mediate efficient protein degradation. Recent data, however, suggest that molecular chaperones directly cooperate with the ubiquitin/proteasome system during protein quality control in eukaryotic cells. Modulating the intracellular balance of protein folding and protein degradation may open new strategies for the treatment of human diseases that involve chaperone pathways such as cancer and diverse amyloid diseases.

Sebastian Schuck
Protein sorting to the apical membrane of epithelial cells
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2004)

Tobias Langenberg
Neuromeric organization of the midbrain-hindbrain boundary region in zebrafish
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2004)

Karsten Kruse^*, Periklis Pantazis^*, Tobias Bollenbach, Frank Jülicher, Marcos González-Gaitán
Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model.
Development, 131(19) 4843-4856 (2004)
PDF DOI

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. Here, we perform a theoretical study of a model for morphogen spreading based on extracellular diffusion, which takes into account receptor binding and trafficking. We compare profiles of ligand and surface receptors obtained in this model with experimental data. To this end, we monitored directly the pool of surface receptors and extracellular Dpp with specific antibodies. We conclude that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement.

Simon Alberti, Karsten Böhse, Verena Arndt, Anton Schmitz, Jörg Höhfeld
The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator.
Mol Biol Cell, 15(9) 4003-4010 (2004)
DOI

The CHIP ubiquitin ligase turns molecular chaperones into protein degradation factors. CHIP associates with the chaperones Hsc70 and Hsp90 during the regulation of signaling pathways and during protein quality control, and directs chaperone-bound clients to the proteasome for degradation. Obviously, this destructive activity should be carefully controlled. Here, we identify the cochaperone HspBP1 as an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms of the cystic fibrosis transmembrane conductance regulator (CFTR) and stimulates CFTR maturation. Our data reveal a novel regulatory mechanism that determines folding and degradation activities of molecular chaperones.

Christian R. Eckmann, Sarah L Crittenden, Nayoung Suh, Judith Kimble
GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans.
Genetics, 168(1) 147-160 (2004)
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Germ cells can divide mitotically to replenish germline tissue or meiotically to produce gametes. In this article, we report that GLD-3, a Caenorhabditis elegans Bicaudal-C homolog, promotes the transition from mitosis to meiosis together with the GLD-2 poly(A) polymerase. GLD-3 binds GLD-2 via a small N-terminal region present in both GLD-3S and GLD-3L isoforms, and GLD-2 and GLD-3 can be co-immunoprecipitated from worm extracts. The FBF repressor binds specifically to elements in the gld-3S 3'-UTR, and FBF regulates gld-3 expression. Furthermore, FBF acts largely upstream of gld-3 in the mitosis/meiosis decision. By contrast, GLD-3 acts upstream of FBF in the sperm/oocyte decision, and GLD-3 protein can antagonize FBF binding to RNA regulatory elements. To address the relative importance of these two regulatory mechanisms in the mitosis/meiosis and sperm/oocyte decisions, we isolated a deletion mutant, gld-3(q741), that removes the FBF-binding site from GLD-3L, but leaves the GLD-2-binding site intact. Animals homozygous for gld-3(q741) enter meiosis, but are feminized. Therefore, GLD-3 promotes meiosis primarily via its interaction with GLD-2, and it promotes spermatogenesis primarily via its interaction with FBF.

Sandra Lacas-Gervais, Jun Guo, Nicola Strenzke, Eric Scarfone, Melanie Kolpe, Monika Jahkel, Pietro De Camilli, Tobias Moser, Matthew N Rasband, Michele Solimena
BetaIVSigma1 spectrin stabilizes the nodes of Ranvier and axon initial segments.
J Cell Biol, 166(7) 983-990 (2004)
PDF DOI

Saltatory electric conduction requires clustered voltage-gated sodium channels (VGSCs) at axon initial segments (AIS) and nodes of Ranvier (NR). A dense membrane undercoat is present at these sites, which is thought to be key for the focal accumulation of channels. Here, we prove that betaIVSigma1 spectrin, the only betaIV spectrin with an actin-binding domain, is an essential component of this coat. Specifically, betaIVSigma1 coexists with betaIVSigma6 at both AIS and NR, being the predominant spectrin at AIS. Removal of betaIVSigma1 alone causes the disappearance of the nodal coat, an increased diameter of the NR, and the presence of dilations filled with organelles. Moreover, in myelinated cochlear afferent fibers, VGSC and ankyrin G clusters appear fragmented. These ultrastructural changes can explain the motor and auditory neuropathies present in betaIVSigma1 -/- mice and point to the betaIVSigma1 spectrin isoform as a master-stabilizing factor of AIS/NR membranes.

Adam J. Liska^*, Andrei V. Popov^*, Shamil Sunyaev, Peg Coughlin, Bianca Habermann, Anna Shevchenko, Peer Bork, Eric Karsenti, Andrej Shevchenko
Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome.
Proteomics, 4(9) 2707-2721 (2004)
PDF DOI

The application of functional proteomics to important model organisms with unsequenced genomes is restricted because of the limited ability to identify proteins by conventional mass spectrometry (MS) methods. Here we applied MS and sequence-similarity database searching strategies to characterize the Xenopus laevis microtubule-associated proteome. We identified over 40 unique, and many novel, microtubule-bound proteins, as well as two macromolecular protein complexes involved in protein translation. This finding was corroborated by electron microscopy showing the presence of ribosomes on spindles assembled from frog egg extracts. Taken together, these results suggest that protein translation occurs on the spindle during meiosis in the Xenopus oocyte. These findings were made possible due to the application of sequence-similarity methods, which extended mass spectrometric protein identification capabilities by 2-fold compared to conventional methods.

Carsten Schnatwinkel, Savvas Christoforidis, Margaret R Lindsay, Sandrine Uttenweiler-Joseph, Matthias Wilm, Robert G. Parton, Marino Zerial
The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.
PLoS Biol, 2(9) 261-261 (2004)
Open Access PDF DOI

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

Jie Shen
The role of Decapentaplegic (Dpp) in Drosophila wing development
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2004)

Yang Yang, Sandra Lacas-Gervais, D Kent Morest, Michele Solimena, Matthew N Rasband
BetaIV spectrins are essential for membrane stability and the molecular organization of nodes of Ranvier.
J Neurosci, 24(33) 7230-7240 (2004)
PDF DOI

High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on betaIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv(3J)"quivering" mice, whose betaIV spectrins have a truncated proline-rich "specific" domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv(3J) mice, which lack betaIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv(3J) nodes, some with detectable betaIV spectrins, are less affected. In contrast, a larger truncation of betaIV spectrins in qv(4J) mice, deleting the SD, PH, and ankyrinG binding domains, causes betaIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt betaIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.

Gijs van Haaften, Nadine Vastenhouw, Ellen A A Nollen, Ronald H A Plasterk, Marcel Tijsterman
Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality.
Proc Natl Acad Sci U.S.A., 101(35) 12992-12996 (2004)
DOI

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect the germ line against DNA double-strand breaks. Besides known DNA-repair proteins such as the C. elegans orthologs of TopBP1, RPA2, and RAD51, eight genes previously unassociated with a double-strand-break response were identified. Knockdown of these genes increased sensitivity to ionizing radiation and camptothecin and resulted in increased chromosomal nondisjunction. All genes have human orthologs that may play a role in human carcinogenesis.

Adam J. Liska
The morality of problem selection in proteomics.
Proteomics, 4(7) 1929-1931 (2004)
PDF DOI

The emerging power of new technologies in proteomics and the biological sciences to alter the human condition demands that scientists hold a new perspective on the social responsibilities of their research. Ethical theory can help scientists recognize not only those research projects that are harmful, but also those research paths that can create the greatest improvements in human health on a global scale. Whereas individual choices are important for the direction of scientific research, these choices may have limited social effects if they are not coordinated with larger institutional and inter-institutional structures. The perspective presented here calls for the Human Proteome Organization to recognize the ten most ethically significant proteomes to be characterized, with the hopes of rallying support and directing the research efforts of scientists in the proteomics community toward these goals.

Ravi Sawhney, Jonathon Howard
Molecular dissection of the fibroblast-traction machinery.
Cell Motil Cytoskeleton, 58(3) 175-185 (2004)
PDF DOI

Motile fibroblasts generate forces that can be expressed as cell migration or as traction, the drawing-in of extracellular matrix. Traction by cultured fibroblasts can induce a rapid concerted reorganization of collagen gel, creating a pattern of collagen alignment similar to that seen in tendons and ligaments. In such fibrous connective tissues, after pattern morphogenesis is complete, ongoing traction may be responsible for the maintenance of proper form and function. The molecules that generate and transmit forces have been catalogued; however, how these nanometer-scale molecules contribute to millimeter-scale patterns has not been directly tested. Here, we placed pairs of explants of human periodontal ligament fibroblasts in collagen gels, where ligament-like straps of anisotropic collagen formed on the axes between them. We scrutinized the traction apparatus using electron microscopy, video microscopy, and computer-based pattern analysis, augmented with pharmacologic inhibitors of cytoskeletal function. Patterning was marked by the co-alignment of collagen, fibroblasts, and their actin cytoskeletons, all parallel to the axis between explants. The pattern was diminished by depolymerizing actin filaments or by blocking myosin activity, but was accentuated by depolymerizing microtubules. The plasma membrane also seems to contribute to the traction force. These molecular components combine to exert a sub-maximal traction force on the matrix, suggesting that the force may be regulated to ensure tissue tensional homeostasis.

Nadine Vastenhouw, Ronald H A Plasterk
RNAi protects the Caenorhabditis elegans germline against transposition.
Trends Genet, 20(7) 314-319 (2004)
DOI

Gregor Wollensak, Michaela Wilsch, Eberhard Spoerl, Theo Seiler
Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA.
Cornea, 23(5) 503-507 (2004)
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OBJECTIVE: Collagen crosslinking of the cornea has been developed recently as a quasiconservative treatment of keratoconus. Biomechanical in vitro measurements have demonstrated a significant increase in biomechanical stiffness of the crosslinked cornea. The aim of the present study was to evaluate the effect of this new procedure on the collagen fiber diameter of the rabbit cornea. METHODS: The corneas of the right eyes of 10 New Zealand White albino rabbits were crosslinked by application of the photosensitizer riboflavin and exposure to UVA light (370 nm, 3 mW/cm2) for 30 minutes. The left fellow control eyes were either left untreated (rabbits 1-4), deepithelialized (rabbits 5-7), or deepithelialized and treated with riboflavin/dextran solution (rabbits 8-10) to exclude an influence of epithelial debridement or hydration changes on the fiber diameter. On ultrathin sections of samples from the anterior and posterior cornea, the collagen fiber diameter was measured semiautomatically with the help of morphometric computer software. RESULTS: In the anterior stroma, the collagen fiber diameter in the treated corneas was significantly increased by 12.2% (3.96 nm), and in the posterior stroma by 4.6% (1.63 nm), compared with the control fellow eyes. In the crosslinked eyes, the collagen fiber diameter was also significantly increased by, on average, 9.3% (3.1 nm) in the anterior compared with the posterior stroma within the same eye. CONCLUSIONS: Collagen crosslinking using riboflavin and UVA leads to a significant increase in corneal collagen diameter. This alteration is the morphologic correlate of the crosslinking process leading to an increase in biomechanical stability. The crosslinking effect is strongest in the anterior half of the stroma because of the rapid decrease in UVA irradiance across the corneal stroma as a result of riboflavin-enhanced UVA absorption.

Andreas Henschel, Frank Buchholz, Bianca Habermann
DEQOR: a web-based tool for the design and quality control of siRNAs.
Nucleic Acids Res, 32(Web Server issue) 113-120 (2004)
PDF DOI

RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNA-mediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at http://cluster-1.mpi-cbg.de/Deqor/deqor.html.

Srinath Sampath, Ryoma Ohi, Oliver Leismann, Adrian Salic, Andrei I. Pozniakovsky, Hironori Funabiki
The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly.
Cell, 118(2) 187-202 (2004)
PDF DOI

In cells lacking centrosomes, such as those found in female meiosis, chromosomes must nucleate and stabilize microtubules in order to form a bipolar spindle. Here we report the identification of Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex containing Incenp, Survivin, and the kinase Aurora B, and demonstrate that this complex is required for chromatin-induced microtubule stabilization and spindle formation. The failure of microtubule stabilization caused by depletion of the chromosomal passenger complex was rescued by codepletion of the microtubule-depolymerizing kinesin MCAK, whose activity is negatively regulated by Aurora B. By contrast, we present evidence that the Ran-GTP pathway of chromatin-induced microtubule nucleation does not require the chromosomal passenger complex, indicating that the mechanisms of microtubule assembly by these two pathways are distinct. We propose that the chromosomal passenger complex regulates local MCAK activity to permit spindle formation via stabilization of chromatin-associated microtubules.

Steffen Scholpp^*, Casper Groth^*, Claudia Lohs, Michael Lardelli, Michael Brand
Zebrafish fgfr1 is a member of the fgf8 synexpression group and is required for fgf8 signalling at the midbrain-hindbrain boundary.
Dev Genes Evol, 214(6) 285-295 (2004)
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FGFR1 is an important signalling molecule during embryogenesis and in adulthood. FGFR1 mutations in human may lead to developmental defects and pathological conditions, including cancer and Alzheimer's disease. Here, we describe cloning and expression analysis of the zebrafish fibroblast growth factor receptor 1 ( fgfr1). Initially, fgfr1 is expressed in the adaxial mesoderm with transcripts distinctly localised to the anterior portion of each half-somite. Hereupon, fgfr1 is also strongly expressed in the otic vesicles, branchial arches and the brain, especially at the midbrain-hindbrain boundary (MHB). The expression patterns of fgfr1 and fgf8 are strikingly similar and knock-down of fgfr1 phenocopies many aspects observed in the fgf8 mutant acerebellar, suggesting that Fgf8 exerts its function mainly by binding to FgfR1.

Yoichi Kosodo^*, Katja Röper^*, Wulf Haubensak, Anne-Marie Marzesco, Denis Corbeil, Wieland B. Huttner
Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.
EMBO J, 23(11) 2314-2324 (2004)
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At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.

Kate Poole, Doris Meder, Kai Simons, Daniel J. Müller
The effect of raft lipid depletion on microvilli formation in MDCK cells, visualized by atomic force microscopy.
FEBS Lett, 565(1-3) 53-58 (2004)
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We have investigated whether raft lipids of Madin-Darby canine kidney (MDCK) cells play any role in microvilli maintenance using a combination of atomic force microscopy (AFM) and laser scanning confocal microscopy. MDCK cells were treated to reduce the amount of sphingolipids, cholesterol, or both and subsequently imaged, in buffer solution, using AFM. It was observed that inhibition of either sphingolipid or cholesterol biosynthesis led to a reduction in the number of microvilli on the surface of MDCK cells. However, this effect was not uniform across the monolayer, with some cells resembling those in untreated controls. The subsequent extraction of cholesterol from cells grown in the presence of inhibitors led to a further reduction in microvilli on the surface of the cells and, in some cases, resulted in monolayers devoid of full length microvilli. Significantly, smaller spikes were observed on the surface of the smoother cells.

Hae Yong Yoo, Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko, William G Dunphy
Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase.
Cell, 117(5) 575-588 (2004)
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The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.

Laurence Pelletier, Nurhan Ozlü, Eva Hannak, Carrie R. Cowan, Bianca Habermann, Martine Ruer, Thomas Müller-Reichert, Anthony A. Hyman
The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication.
Curr Biol, 14(10) 863-873 (2004)
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BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.

Mihai D Morariu, Erik Schäffer, Ullrich Steiner
Molecular forces caused by the confinement of thermal noise.
Phys Rev Lett, 92(15) 156102-156102 (2004)
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We have investigated spontaneous surface instabilities of very thin polymer films. Film stability and the wavelength of the dominating unstable mode were found to depend sensitively on the media adjacent to the film. Our experimental results cannot be explained by van der Waals interactions alone. To account for the presence of an additional destabilizing force, we propose that the geometrical confinement of thermally excited acoustic waves gives rise to a force that is strong enough to destabilize thin films. This thermoacoustic effect is of similar magnitude as van der Waals forces.

Martin Schwickart, Jan Havlis, Bianca Habermann, Aliona Bogdanova, Alain Camasses, Tobias Oelschlaegel, Andrej Shevchenko, Wolfgang Zachariae
Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27.
Mol Cell Biol, 24(8) 3562-3576 (2004)
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The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

Flaviana Gentile^*, Gaetano Calì^*, Chiara Zurzolo, Annunziata Corteggio, Patrizia Rosa, Federico Calegari, Andrea Levi, Roberta Possenti, Claudia Puri, Carlo Tacchetti, Lucio Nitsch
The neuroendocrine protein VGF is sorted into dense-core granules and is secreted apically by polarized rat thyroid epithelial cells.
Exp Cell Res, 295(1) 269-280 (2004)
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We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route.

Wulf Haubensak, Alessio Attardo, Winfried Denk, Wieland B. Huttner
Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis.
Proc Natl Acad Sci U.S.A., 101(9) 3196-3201 (2004)
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Neurons of the mammalian CNS are thought to originate from progenitors dividing at the apical surface of the neuroepithelium. Here we use mouse embryos expressing GFP from the Tis21 locus, a gene expressed throughout the neural tube in most, if not all, neuron-generating progenitors, to specifically reveal the cell divisions that produce CNS neurons. In addition to the apical, asymmetric divisions of neuroepithelial (NE) cells that generate another NE cell and a neuron, we find, from the onset of neurogenesis, a second population of progenitors that divide in the basal region of the neuroepithelium and generate two neurons. Basal progenitors are most frequent in the telencephalon, where they outnumber the apically dividing neuron-generating NE cells. Our observations reconcile previous data on the origin and lineage of CNS neurons and show that basal, rather than apical, progenitors are the major source of the neurons of the mammalian neocortex.

Jonathon Howard, Susanne Bechstedt
Hypothesis: a helix of ankyrin repeats of the NOMPC-TRP ion channel is the gating spring of mechanoreceptors.
Curr Biol, 14(6) 224-226 (2004)
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Evdokia Pasheva, Mihail Sarov, Kiril Bidjekov, Iva Ugrinova, Bettina Sarg, Herbert Lindner, Iliya G Pashev
In vitro acetylation of HMGB-1 and -2 proteins by CBP: the role of the acidic tail
Biochemistry, 43(10) 2935-2940 (2004)
DOI

Histone acetyltransferases CBP, PCAF, and Tip60 have been tested for their ability to in vitro acetylate HMGB-1 and -2 proteins and their truncated forms lacking the C-terminal tail. It was found that these proteins were substrates for CBP only. Analyses of modified proteins by electrophoresis, amino acid sequencing, and mass spectrometry showed that full-length HMGB-1 and -2 were monoacetylated at Lys2. Removal of the C terminus resulted in (i) an increased incorporation of radiolabeled acetate within the proteins to a level close to that observed with histones H3/H4 and (ii) creation of a novel target site at Lys81. Acetylated and nonmodified HMGB-1 and -2 protein lacking the acidic tail were compared relative to their binding affinity to distorted DNA and the ability to bend linear DNA. Both proteins showed similar affinities to cisplatin-damaged DNA; the acetylated protein, however, was 3-fold more effective in inducing ligase-mediated circularization of a 111-bp DNA fragment. The alterations in the acetylation pattern of HMGB-1 and -2 upon removal of the C-terminal tail are regarded as a means by which the acidic domain modulates some properties of these proteins.

Bianca Habermann, Jeffrey Oegema, Shamil Sunyaev, Andrej Shevchenko
The power and the limitations of cross-species protein identification by mass spectrometry-driven sequence similarity searches.
Mol Cell Proteomics, 3(3) 238-249 (2004)
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Mass spectrometry-driven BLAST (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins available in a database. MS BLAST utilizes redundant, degenerate, and partially inaccurate peptide sequence data obtained by de novo interpretation of tandem mass spectra and has become a powerful tool in functional proteomic research. Using computational modeling, we evaluated the potential of MS BLAST for proteome-wide identification of unknown proteins. We determined how the success rate of protein identification depends on the full-length sequence identity between the queried protein and its closest homologue in a database. We also estimated phylogenetic distances between organisms under study and related reference organisms with completely sequenced genomes that allow substantial coverage of unknown proteomes.

Assen Roguev, Anna Shevchenko, Daniel Schaft, Henrik Thomas, A. Francis Stewart, Andrej Shevchenko
A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Mol Cell Proteomics, 3(2) 125-132 (2004)
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The sequential application of protein tagging, affinity purification, and mass spectrometry enables highly accurate charting of proteomic environments by the characterization of stable protein assemblies and the identification of subunits that are shared between two or more protein complexes, termed here "proteomic hyperlinks." We have charted the proteomic environments surrounding the histone methyltransferase, Set1, in both yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Although the composition of these nonessential Set1 complexes is remarkably conserved, they differ with respect to their hyperlinks to their proteomic environments. We speculate that conservation of the core components of protein assemblies and variability of hyperlinks represents a general principle in the molecular organization of eukaryotic proteomes.

Muriel Rhinn, Klaus Lun, Michaela Werner, Angela Simeone, Michael Brand
Isolation and expression of the homeobox gene Gbx1 during mouse development.
Dev Dyn, 229(2) 334-339 (2004)
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In zebrafish, gbx1 and otx2 are among the earliest genes expressed in the neuroectoderm, dividing it into an anterior and a posterior domain with a common border that marks the midbrain-hindbrain boundary (MHB) primordium. Here, we describe the sequence and expression pattern of Gbx1 in mouse. The first transcripts are found at embryonic day 7.75 in the hindbrain. Later on, expression of Gbx1 is detectable in the hindbrain (rhombomeres 2 to 7), spinal cord, optic vesicles, and in the ventral telencephalon. In mouse, Gbx1 expression is not observed at the MHB as is the case during early zebrafish development. We suggest that an evolutionary switch occurred: in mouse Gbx2 is involved in the early specification of the MHB primordium, whereas in zebrafish, gbx1 is required instead of gbx2.

Doris Meder
Organization and formation of the apical membrane of epithelial cells
Ph.D. Thesis, Technische Universität Dresden, Dresden, Germany (2004)

Gerlinde Reim, Takamasa Mizoguchi, Didier Y.R. Stainier, Yutaka Kikuchi, Michael Brand
The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova.
Dev Cell, 6(1) 91-101 (2004)
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The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.

V J P Robert, Nadine Vastenhouw, Ronald H A Plasterk
RNA interference, transposon silencing, and cosuppression in the Caenorhabditis elegans germ line: similarities and differences.
Cold Spring Harb Symp Quant Biol, 69 397-402 (2004)
DOI

Till Bretschneider, Stefan Diez, Kurt I. Anderson, John Heuser, Margaret Clarke, Annette Müller-Taubenberger, Jana Köhler, Günther Gerisch
Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells.
Curr Biol, 14(1) 1-10 (2004)
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BACKGROUND: In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS: Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS: The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.

Wolfgang Zachariae
Destruction with a box: substrate recognition by the anaphase-promoting complex.
Mol Cell, 13(1) 2-3 (2004)
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Destruction boxes mark cyclin B and other proteins degraded in mitosis for ubiquitination by the anaphase-promoting complex (APC/C). In a paper in this issue of Molecular Cell, Yamano et al. show that destruction boxes directly bind to the APC/C in a cell cycle-regulated manner. Interestingly, this interaction does not require APC/C activators of the Cdc20 family, which were thought to be essential for recruiting substrates to the APC/C.

Bruce T Schaar, Kazuhisa Kinoshita, Susan K McConnell
Doublecortin microtubule affinity is regulated by a balance of kinase and phosphatase activity at the leading edge of migrating neurons.
Neuron, 41(2) 203-213 (2004)
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Doublecortin (Dcx) is a microtubule-associated protein that is mutated in X-linked lissencephaly (X-LIS), a neuronal migration disorder associated with epilepsy and mental retardation. Although Dcx can bind ubiquitously to microtubules in nonneuronal cells, Dcx is highly enriched in the leading processes of migrating neurons and the growth cone region of differentiating neurons. We present evidence that Dcx/microtubule interactions are negatively controlled by Protein Kinase A (PKA) and the MARK/PAR-1 family of protein kinases. In addition to a consensus MARK site, we identified a serine within a novel sequence that is crucial for the PKA- and MARK-dependent regulation of Dcx's microtubule binding activity in vitro. This serine is mutated in two families affected by X-LIS. Immunostaining neurons with an antibody that recognizes phosphorylated substrates of MARK supports the conclusion that Dcx localization and function are regulated at the leading edge of migrating cells by a balance of kinase and phosphatase activity.

Mark Lommel, Michel Bagnat, Sabine Strahl
Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants.
Mol Cell Biol, 24(1) 46-57 (2004)

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.

Henrik Thomas, Jan Havlis, Jan Peychl, Andrej Shevchenko
Dried-droplet probe preparation on AnchorChip targets for navigating the acquisition of matrix-assisted laser desorption/ionization time-of-flight spectra by fluorescence of matrix/analyte crystals.
Rapid Commun Mass Spectrom, 18(9) 923-930 (2004)
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We have developed a dried-droplet probe preparation method for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which uses AnchorChip targets and alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Upon drying of a matrix and analyte mixture on the AnchorChip, salts and low molecular weight contaminants were pooled at the hydrophilic metal anchor, whereas 10-50 microm matrix/peptide crystals firmly adhered at the surface of a hydrophobic polymer and the entire target could be subsequently washed by submerging it in 5% formic acid for 2-3 min. Epifluorescence microscopy suggested that peptides were completely co-localized with CHCA crystals at the AnchorChip surface. Fluorescent images of the probes were of good contrast and were background-free, compared with images taken by a video camera built into the ion source. CHCA/peptide crystals were easy to recognize at the surface and peptide mass maps were acquired from them without further adjustment of the position of the laser beam. These crystals were remarkably stable towards the laser depletion and almost no matrix-related ions were typically observed in the low m/z region of peptide mass maps. The sensitivity of the peptide mass mapping was at the low-femtomole level.

Sophie Quintin, Paul E Mains, Andrea Zinke, Anthony A. Hyman
The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo.
EMBO Rep, 4(12) 1175-1181 (2003)
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The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.

József Jászai, Frank Reifers, Alexander Picker, Tobias Langenberg, Michael Brand
Isthmus-to-midbrain transformation in the absence of midbrain-hindbrain organizer activity.
Development, 130(26) 6611-6623 (2003)
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In zebrafish acerebellar (ace) embryos, because of a point mutation in fgf8, the isthmic constriction containing the midbrain-hindbrain boundary (MHB) organizer fails to form. The mutants lack cerebellar development by morphological criteria, and they appear to have an enlarged tectum, showing no obvious reduction in the tissue mass at the dorsal mesencephalic/metencephalic alar plate. To reveal the molecular identity of the tissues located at equivalent rostrocaudal positions along the neuraxis as the isthmic and cerebellar primordia in wild-types, we undertook a detailed analysis of ace embryos. In ace mutants, the appearance of forebrain and midbrain specific marker genes (otx2, dmbx1, wnt4) in the caudal tectal enlargement reveals a marked rostralized gene expression profile during early somitogenesis, followed by the lack of early and late cerebellar-specific gene expression (zath1/atoh1, gap43, tag1/cntn2, neurod, zebrin II). The Locus coeruleus (LC) derived from rostral rhombomere 1 is also absent in the mutants. A new interface between otx2 and epha4a suggests that the rostralization stops at the caudal part of rhombomere 1. The mesencephalic basal plate is also affected in the mutant embryos, as indicated by the caudal expansion of the diencephalic expression domains of epha4a, zash1b/ashb, gap43 and tag1/cntn2, and by the dramatic reduction of twhh expression. No marked differences are seen in cell proliferation and apoptotic patterns around the time the rostralization of gene expression becomes evident in the mutants. Therefore, locally distinct cell proliferation and cell death is unlikely to be the cause of the fate alteration of the isthmic and cerebellar primordia in the mutants. Dil cell-lineage labeling of isthmic primordial cells reveals that cells, at the location equivalent of the wild-type MHB, give rise to caudal tectum in ace embryos. This suggests that a caudalto-rostral transformation leads to the tectal expansion in the mutants. Fgf8-coated beads are able to rescue morphological MHB formation, and elicit the normal molecular identity of the isthmic and cerebellar primordium in ace embryos. Taken together, our analysis reveals that cells of the isthmic and cerebellar primordia acquire a more rostral, tectal identity in the absence of the functional MHB organizer signal Fgf8.

Suzanne Eaton
Cell biology of planar polarity transmission in the Drosophila wing.
Mech Dev, 120(11) 1257-1264 (2003)
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The coordination of epithelial planar polarization is a critical step in the formation of well-ordered tissues. The process has been extensively studied in Drosophila, where genetic analysis has identified a set of "tissue polarity" genes that serve to coordinate planar polarity of cells in the developing wings, bristles and eyes. In the last several years, it has emerged that six of these genes encode junctional proteins. In the wing epithelium, these proteins undergo a polarized redistribution, forming separate proximal and distal cortical domains within each cell. The mechanisms that mediate cortical polarization and cue its direction have been the subject of intense investigation. Cuing the orientation of cortical polarization appears to depend on the atypical Cadherins Fat and Dachsous, although these proteins do not become polarized themselves, nor do they colocalize with components of polarized cortical domains. Interestingly, these Cadherins also act at earlier developmental stages to polarize tissue growth along the proximal-distal axis and it will be interesting to see whether these processes are mechanistically related. Once the axis of polarization is determined, cortical polarity seems to be propagated, at least locally, by a cascade of direct cell-cell interactions mediated by the proximal and distal domains. The cell biological mechanisms leading to polarization are still unclear, but the process depends on the control of Protein Phosphatase 2A activity by its regulatory subunit, Widerborst. Interestingly, Widerborst is found on a planar web of microtubules with connections to apical junctions, suggesting that these microtubules may have an important function in polarizing the cortex.

Steffen Scholpp, Michael Brand
Integrity of the midbrain region is required to maintain the diencephalic-mesencephalic boundary in zebrafish no isthmus/pax2.1 mutants.
Dev Dyn, 228(3) 313-322 (2003)
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Initial anterior-posterior patterning of the neural tube into forebrain, midbrain, and hindbrain primordia occurs already during gastrulation, in response to signals patterning the gastrula embryo. After the initial establishment, further development within each brain part is thought to proceed largely independently of the others. However, mechanisms should exist that ensure proper delineation of brain subdivisions also at later stages; such mechanisms are, however, poorly understood. In zebrafish no isthmus mutant embryos, inactivation of the pax2.1 gene leads to a failure of the midbrain and isthmus primordium to develop normally from the gastrula stage onward (Lun and Brand [1998] Development 125:3049-3062). Here, we report that, after the initially correct establishment during gastrulation stages, the neighbouring forebrain primordium and, partially, the hindbrain primordium expand into the misspecified midbrain territory in no isthmus mutant embryos. The expansion is particularly evident for the posterior part of the diencephalon and less so for the first rhombomeric segment, the territories immediately abutting the midbrain/isthmus primordium. The nucleus of the posterior commissure is expanded in size, and marker genes of the forebrain and rhombomere 1 expand progressively into the misspecified midbrain primordium, eventually resulting in respecification of the midbrain primordium. We therefore suggest that the genetic program controlled by Pax2.1 is not only involved in initiating but also in maintaining the identity of midbrain and isthmus cells to prevent them from assuming a forebrain or hindbrain fate.

Florian Ulrich^*, Miguel L Concha^*, Paul J Heid, Ed Voss, Sabine Witzel, Henry Roehl, Masazumi Tada, Stephen W. Wilson, Richard J. Adams, David R Soll, Carl-Philipp Heisenberg
Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation.
Development, 130(22) 5375-5384 (2003)
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During vertebrate gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. In zebrafish, silberblick (slb)/wnt11 regulates normal gastrulation movements by activating a signalling pathway similar to the Frizzled-signalling pathway, which establishes epithelial planar cell polarity (PCP) in Drosophila. However, the cellular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still unclear. Using high-resolution two-photon confocal imaging followed by computer-assisted reconstruction and motion analysis, we have analysed the movement and morphology of individual cells in three dimensions during the course of gastrulation. We show that in slb-mutant embryos, hypoblast cells within the forming germ ring have slower, less directed migratory movements at the onset of gastrulation. These aberrant cell movements are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. We conclude that slb/wnt11-mediated orientation of cellular processes plays a role in facilitating and stabilising movements of hypoblast cells in the germ ring, thereby pointing at a novel function of the slb/wnt11 signalling pathway for the regulation of migratory cell movements at early stages of gastrulation.

Eileen T. O'Toole, Kent McDonald, Jana Mäntler, J. Richard McIntosh, Anthony A. Hyman, Thomas Müller-Reichert
Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans.
J Cell Biol, 163(3) 451-456 (2003)
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During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.

Eric R Geertsma, Ria H Duurkens, B Poolman
Identification of the dimer interface of the lactose transport protein from Streptococcus thermophilus.
J Mol Biol, 332(5) 1165-1174 (2003)
DOI

The lactose transporter from Streptococcus thermophilus catalyses the symport of galactosides and protons. The carrier domain of the protein harbours the contact sites for dimerization, and the individual subunits in the dimer interact functionally during the transport reaction. As a first step towards the elucidation of the mechanism behind the cooperation between the subunits, regions involved in the dimer interface were determined by oxidative and chemical cross-linking of 12 cysteine substitution mutants. Four positions in the protein were found to be susceptible to intermolecular cross-linking. To ensure that the observed cross-links were not the result of randomly colliding particles, the cross-linking was studied in samples in which either the concentration of LacS in the membrane was varied or the oligomeric state was manipulated. These experiments showed that the cross-links were formed specifically within the dimer. The four regions of the protein located at the dimer interface are close to the extracellular ends of transmembrane segments V and VIII and the intracellular ends of transmembrane segments VI and VII.

Arshad Desai, Sonja Rybina, Thomas Müller-Reichert, Andrej Shevchenko, Anna Shevchenko, Anthony A. Hyman, Karen Oegema
KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans.
Genes Dev, 17(19) 2421-2435 (2003)
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Segregation of the replicated genome during cell division requires kinetochores, mechanochemical organelles that assemble on mitotic chromosomes to connect them to spindle microtubules. CENP-A, a histone H3 variant, and CENP-C, a conserved structural protein, form the DNA-proximal foundation for kinetochore assembly. Using RNA interference-based genomics in Caenorhabditis elegans, we identified KNL-1, a novel kinetochore protein whose depletion, like that of CeCENP-A or CeCENP-C, leads to a "kinetochore-null" phenotype. KNL-1 is downstream of CeCENP-A and CeCENP-C in a linear assembly hierarchy. In embryonic extracts, KNL-1 exhibits substoichiometric interactions with CeCENP-C and forms a near-stoichiometric complex with CeNDC-80 and HIM-10, the C. elegans homologs of Ndc80p/HEC1p and Nuf2p-two widely conserved outer kinetochore components. However, CeNDC-80 and HIM-10 are not functionally equivalent to KNL-1 because their inhibition, although preventing formation of a mechanically stable kinetochore-microtubule interface and causing chromosome missegregation, does not result in a kinetochore-null phenotype. The greater functional importance of KNL-1 may be due to its requirement for targeting multiple components of the outer kinetochore, including CeNDC-80 and HIM-10. Thus, KNL-1 plays a central role in translating the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a functional microtubule-binding interface.

Renata Filipek^*, Malgorzata Rzychon^*, Aneta Oleksy, Milosz Gruca, Adam Dubin, Jan Potempa, Matthias Bochtler
The Staphostatin-staphopain complex: a forward binding inhibitor in complex with its target cysteine protease.
J Biol Chem, 278(42) 40959-40966 (2003)
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Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Our recent crystal structure of staphostatin B has shown that this inhibitor forms a mixed, eight-stranded beta-barrel with statistically significant similarity to lipocalins, but not to cystatins. We now present the 1.8-A crystal structure of staphostatin B in complex with an inactive mutant of its target protease. The complex is held together through extensive interactions and buries a total surface area of 2300 A2. Unexpectedly for a cysteine protease inhibitor, staphostatin B binds to staphopain B in an almost substrate-like manner. The inhibitor polypeptide chain runs through the protease active site cleft in the forward direction, with residues IG-TS in P2 to P2' positions. Both in the free and complexed forms, the P1 glycine residue of the inhibitor is in a main chain conformation only accessible to glycines. Mutations in this residue lead to a loss of affinity of the inhibitor for protease and convert the inhibitor into a substrate.

Steffen Scholpp, Claudia Lohs, Michael Brand
Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon.
Development, 130(20) 4881-4893 (2003)
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Specification of the forebrain, midbrain and hindbrain primordia occurs during gastrulation in response to signals that pattern the gastrula embryo. Following establishment of the primordia, each brain part is thought to develop largely independently from the others under the influence of local organizing centers like the midbrain-hindbrain boundary (MHB, or isthmic) organizer. Mechanisms that maintain the integrity of brain subdivisions at later stages are not yet known. To examine such mechanisms in the anterior neural tube, we have studied the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB). We show that maintenance of the DMB requires both the presence of a specified midbrain and a functional MHB organizer. Expression of pax6.1, a key regulator of forebrain development, is posteriorly suppressed by the Engrailed proteins, Eng2 and Eng3. Mis-expression of eng3 in the forebrain primordium causes downregulation of pax6.1, and forebrain cells correspondingly change their fate and acquire midbrain identity. Conversely, in embryos lacking both eng2 and eng3, the DMB shifts caudally into the midbrain territory. However, a patch of midbrain tissue remains between the forebrain and the hindbrain primordia in such embryos. This suggests that an additional factor maintains midbrain cell fate. We find that Fgf8 is a candidate for this signal, as it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB anteriorly independently of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, we show that cells in the presumptive midbrain neural plate require an Fgf signal to keep them from following a forebrain fate. Combined loss of both Eng2/Eng3 and Fgf8 leads to complete loss of midbrain identity, resulting in fusion of the forebrain and the hindbrain primordia. Thus, Eng2/Eng3 and Fgf8 are necessary to maintain midbrain identity in the neural plate and thereby position the DMB. This provides an example of a mechanism needed to maintain the subdivision of the anterior neural plate into forebrain and midbrain.

Gregor Wollensak, Eberhard Spoerl, Michaela Wilsch, Theo Seiler
Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit.
J Cataract Refract Surg, 29(9) 1786-1790 (2003)
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PURPOSE: To evaluate the possible cytotoxic effect of combined riboflavin-ultraviolet-A (UVA) treatment on the corneal endothelium. SETTING: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. METHODS: The right eyes of 34 New Zealand White rabbits were treated with riboflavin and various endothelial UVA doses ranging from 0.16 to 0.9 J/cm2 (0.09 to 0.5 mW/cm2, 370 nm) and postoperative enucleation times of 4 hours and 24 hours. The endothelial cells were evaluated in histological sections. The terminal deoxynulceotidyl transferase deoxy-UTP-nick-end labeling (TUNEL) technique and transmission electron microscopy were used to detect apoptosis. RESULTS: There was no endothelial damage in the 6 rabbit eyes enucleated at 4 hours. In those enucleated at 24 hours, there was significant necrosis and apoptosis of endothelial cells in the corneas treated with an endothelial dose of > or =0.65 J/cm2 (0.36 mW/cm2), which is about twice the endothelial UVA dose used in the treatment of keratoconus patients. CONCLUSIONS: In rabbit corneas with a corneal thickness less than 400 microm, the endothelial UVA dose reached a cytotoxic level of > or =0.65 J/cm2 (0.36 mW/cm2) using the standard surface UVA dose of 5.4 J/cm2 (3 mW/cm2). Pachymetry should be routinely performed before riboflavin-UVA treatment; in thinner corneas, irradiation should not be done because of the cytotoxic risk to the endothelium.

Tiina Lehto, Marta Miaczynska, Marino Zerial, Daniel J. Müller, Fedor F. Severin
Observing the growth of individual actin filaments in cell extracts by time-lapse atomic force microscopy.
FEBS Lett, 551(1-3) 25-28 (2003)
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High-resolution atomic force microscopy (AFM) was applied to directly observe the dynamic assembly of single actin filaments in HeLa cell extracts in vitro. The F-actin filaments established a dynamic network and formed different types of junctions and branches. The connections of this network were X-, Y- or T-shaped. It was found that the actin filaments were densely covered by endosomes and vesicles from the cell extract, which are thought to stabilize their structures. Using time-lapse AFM, the growth, shrinkage, branching and the interaction of actin filaments with endosomes could be characterized. Our results indicate that the majority of F-actin filaments are static in HeLa extract and that only a minor fraction of filaments undergo dynamic changes. Furthermore, the AFM imaging approach not only provides unique insights into the assembly and dynamics of actin networks; it also builds an avenue to study in vitro assays of complex biological systems.

Michele Solimena, Hans Hermann Gerdes
Secretory granules: and the last shall be first...
Trends Cell Biol, 13(8) 399-402 (2003)
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By tagging secretory granules with the fluorescent protein dsRed-E5, which changes its emission from green to red over time, Duncan et al. analysed the age-dependent distribution of secretory vesicles within chromaffin cells. This elegant study illustrates as never before how age is a critical factor that segregates granules with respect to their localization and mobility and the probability of them undergoing exocytosis in response to different stimuli.

Ralf Kittler, Frank Buchholz
RNA interference: gene silencing in the fast lane.
Semin Cancer Biol, 13(4) 259-265 (2003)
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Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. RNA interference (RNAi) is becoming the method of choice for gene function analysis in cells and whole organisms. Here we review the approaches available to perform RNAi experiments in mammalian cells and in mice. We discuss usage of RNAi in cancer research and as a possible therapeutic tool for cancer treatment.

Hein Sprong, Sophie Degroote, Tommy Nilsson, Masao Kawakita, Nobuhiro Ishida, Peter van der Sluijs, Gerrit van Meer
Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.
Mol Biol Cell, 14(8) 3482-3493 (2003)
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UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Kimberly M. Kotovic, Daniel Lockshon, Lamia Boric, Karla M. Neugebauer
Cotranscriptional recruitment of the U1 snRNP to intron-containing genes in yeast.
Mol Cell Biol, 23(16) 5768-5779 (2003)
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Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5' splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

Alain Camasses, Aliona Bogdanova, Andrej Shevchenko, Wolfgang Zachariae
The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20.
Mol Cell, 12(1) 87-100 (2003)
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The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.

Stephan W. Grill, Jonathon Howard, Erik Schäffer, Ernst H K Stelzer, Anthony A. Hyman
The distribution of active force generators controls mitotic spindle position.
Science, 301(5632) 518-521 (2003)
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During unequal cell divisions a mitotic spindle is eccentrically positioned before cell cleavage. To determine the basis of the net force imbalance that causes spindle displacement in one-cell Caenorhabditis elegans embryos, we fragmented centrosomes with an ultraviolet laser. Analysis of the mean and variance of fragment speeds suggests that the force imbalance is due to a larger number of force generators pulling on astral microtubules of the posterior aster relative to the anterior aster. Moreover, activation of heterotrimeric guanine nucleotide- binding protein (Gprotein) alpha subunits is required to generate these astral forces.

H Lucius, Tim Friedrichson, Teymuras V. Kurzchalia, G R Lewin
Identification of caveolae-like structures on the surface of intact cells using scanning force microscopy.
J Membr Biol, 194(2) 97-108 (2003)
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Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl-beta-cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.

Folker Spitzenberger, Massimo Pietropaolo, Paul Verkade, Bianca Habermann, Sandra Lacas-Gervais, Hassan Mziaut, Susan Pietropaolo, Michele Solimena
Islet cell autoantigen of 69 kDa is an arfaptin-related protein associated with the Golgi complex of insulinoma INS-1 cells.
J Biol Chem, 278(28) 26166-26173 (2003)
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Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Involvement of ICA69 in neurosecretion has been suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene ric-19 in C. elegans. In this study, we have further investigated the localization of ICA69 in neurons and insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas it did not co-localize with markers of synaptic vesicles/synaptic-like microvesicles. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules. The association of ICA69 with these organelles was confirmed by immunoelectron microscopy. Virtually no ICA69 immunogold labeling was observed on secretory granules near the plasma membrane, suggesting that ICA69 dissociates from secretory granule membranes during their maturation. In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules. ICA69 is therefore a novel arfaptin-related protein that is likely to play a role in membrane trafficking at the Golgi complex and immature secretory granules in neurosecretory cells.

Tanja Wucherpfennig, Michaela Wilsch-Bräuninger, Marcos González-Gaitán
Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release.
J Cell Biol, 161(3) 609-624 (2003)
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During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, we monitored an endosomal compartment in Drosophila neuromuscular synapses. We showed that in conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and only recovers from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. We demonstrate that the small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance.

Daniel Schaft^*, Assen Roguev^*, Kimberly M. Kotovic, Anna Shevchenko, Mihail Sarov, Andrej Shevchenko, Karla M. Neugebauer, A. Francis Stewart
The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation.
Nucleic Acids Res, 31(10) 2475-2482 (2003)
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Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor. Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation. First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2. Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II. Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3. Fourth, in the absence of ssn3, mRNA production from gal1 required SET2. Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription. Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions. Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation.

Jonathon Howard, Anthony A. Hyman
Dynamics and mechanics of the microtubule plus end.
Nature, 422(6933) 753-758 (2003)
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An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around inside cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how does this coupling regulate the stability and distribution of the microtubules? It is now clear that there are at least three properties of a microtubule end: it has alternate structures; it has a biochemical transition defined by GTP hydrolysis; and it forms a distinct target for the binding of specific proteins. These different properties can be unified by thinking of the microtubule as a molecular machine, which switches between growing and shrinking modes. Each mode is associated with a specific end structure on which end-binding proteins can assemble to modulate dynamics and couple the dynamic properties of microtubules to the movement of cellular structures.

Thomas Müller-Reichert, Ingrid Sassoon, Eileen T. O'Toole, Maryse Romao, Anthony J. Ashford, Anthony A. Hyman, Claude Antony
Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles.
Chromosoma, 111(7) 417-428 (2003)
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Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions. We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase. By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally. About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules. Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p. Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle.

Mark van Breugel, David N. Drechsel, Anthony A. Hyman
Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.
J Cell Biol, 161(2) 359-369 (2003)
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The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

Beate Kilian, Hannu Mansukoski, Filipa Carreira Barbosa, Florian Ulrich, Masazumi Tada, Carl-Philipp Heisenberg
The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation.
Mech Dev, 120(4) 467-476 (2003)
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Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.

Mark D Adams, Granger G Sutton, Hamilton O Smith, Eugene W Myers, J C Venter
The independence of our genome assemblies.
Proc Natl Acad Sci U.S.A., 100(6) 3025-3026 (2003)
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Marcos González-Gaitán
Signal dispersal and transduction through the endocytic pathway.
Nat Rev Mol Cell Biol, 4(3) 213-224 (2003)
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During cell signalling, information that is encoded by ligands travels from one place, the source, to another, the target, where signals are transduced by receptors. Evidence has emerged recently that uncovers a role for the endocytic pathway in the secretion of ligands at the source, their dispersion through developing target tissues and the transduction of the signals from endocytic compartments. As a result, endosomes have become the focus of attention in cell-cell communication studies.

Christine A. Fargeas, Mareike Florek, Wieland B. Huttner, Denis Corbeil
Characterization of prominin-2, a new member of the prominin family of pentaspan membrane glycoproteins.
J Biol Chem, 278(10) 8586-8596 (2003)
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Prominin/CD133 is a 115/120-kDa integral membrane glycoprotein specifically associated with plasma membrane protrusions in epithelial and non-epithelial cells including neuroepithelial and hematopoietic stem cells. Here we report the identification as well as molecular and cell biological characterization of mouse, rat, and human prominin-2, a 112-kDa glycoprotein structurally related to prominin (referred to as prominin-1). Although the amino acid identity between prominin-2 and prominin-1 is low (<30%), their genomic organization is strikingly similar, suggesting an early gene duplication event. Like prominin-1, prominin-2 exhibits a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Upon its ectopic expression in Chinese hamster ovary cells as a green fluorescent protein fusion chimera, prominin-2 was also found to be associated with plasma membrane protrusions, as revealed by its co-localization with prominin-1, suggesting a related role. Consistent with this, prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract as well as in various other epithelial tissues. However, in contrast to prominin-1, prominin-2 was not detected in the eye, which perhaps explains why a loss-of function mutation in the human prominin-1 gene causes retinal degeneration but no other obvious pathological signs. Finally, we present evidence for the existence of a family of pentaspan membrane proteins, the prominins, which are conserved in evolution.

A Bernardo Carvalho, Maria D Vibranovski, Joseph W Carlson, Susan E Celniker, Roger A Hoskins, Gerald M Rubin, Granger G Sutton, Mark D Adams, Eugene W Myers, Andrew Clark
Y chromosome and other heterochromatic sequences of the Drosophila melanogaster genome: how far can we go?
Genetica, 117(2-3) 227-237 (2003)
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Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.

Shamil Sunyaev, Adam J. Liska, Alexander Golod, Anna Shevchenko, Andrej Shevchenko
MultiTag: multiple error-tolerant sequence tag search for the sequence-similarity identification of proteins by mass spectrometry.
Anal Chem, 75(6) 1307-1315 (2003)
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The characterization of proteomes by mass spectrometry is largely limited to organisms with sequenced genomes. To identify proteins from organisms with unsequenced genomes, database sequences from related species must be employed for sequence-similarity protein identifications. Peptide sequence tags (Mann, 1994) have been used successfully for the identification of proteins in sequence databases using partially interpreted tandem mass spectra of tryptic peptides. We have extended the ability of sequence tag searching to the identification of proteins whose sequences are yet unknown but are homologous to known database entries. The MultiTag method presented here assigns statistical significance to matches of multiple error-tolerant sequence tags to a database entry and ranks alignments by their significance. The MultiTag approach has the distinct advantage over other sequence-similarity approaches of being able to perform sequence-similarity identifications using only very short (2-4) amino acid residue stretches of peptide sequences, rather than complete peptide sequences deduced by de novo interpretation of tandem mass spectra. This feature facilitates the identification of low abundance proteins, since noisy and low-intensity tandem mass spectra can be utilized.

Assen Roguev^*, Daniel Schaft^*, Anna Shevchenko, Rein Aasland, Andrej Shevchenko, A. Francis Stewart
High conservation of the Set1/Rad6 axis of histone 3 lysine 4 methylation in budding and fission yeasts.
J Biol Chem, 278(10) 8487-8493 (2003)
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Histone 3 lysine 4 (H3 Lys(4)) methylation in Saccharomyces cerevisiae is mediated by the Set1 complex (Set1C) and is dependent upon ubiquitinylation of H2B by Rad6. Mutually exclusive methylation of H3 at Lys(4) or Lys(9) is central to chromatin regulation; however, S. cerevisiae lacks Lys(9) methylation. Furthermore, a different H3 Lys(4) methylase, Set 7/9, has been identified in mammals, thereby questioning the relevance of the S. cerevisiae findings for eukaryotes in general. We report that the majority of Lys(4) methylation in Schizosaccharomyces pombe, like in S. cerevisiae, is mediated by Set1C and is Rad6-dependent. S. pombe Set1C mediates H3 Lys(4) methylation in vitro and contains the same eight subunits found in S. cerevisiae, including the homologue of the Drosophila trithorax Group protein, Ash2. Three additional features of S. pombe Set1C each involve PHD fingers. Notably, the Spp1 subunit is dispensable for H3 Lys(4) methylation in budding yeast but required in fission yeast, and Sp_Set1C has a novel proteomic hyperlink to a new complex that includes the homologue of another trithorax Group protein, Lid (little imaginal discs). Thus, we infer that Set1C is highly conserved in eukaryotes but observe that its links to the proteome are not.

Andrew W Hunter, Michael Caplow, David L. Coy, William O Hancock, Stefan Diez, Linda Wordeman, Jonathon Howard
The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends.
Mol Cell, 11(2) 445-457 (2003)
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MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.

Jonathon Howard
A molecular view of the cell
Nature, 421(1) 115-115 (2003)
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Rafael Mattera, Cecilia N Arighi, Robert Lodge, Marino Zerial, Juan S Bonifacino
Divalent interaction of the GGAs with the Rabaptin-5-Rabex-5 complex.
EMBO J, 22(1) 78-88 (2003)
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Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5-Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439-443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the gamma1- and gamma2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA-Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)-Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/gamma-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

Hyun Kyu Song, Matthias Bochtler, M Kamran Azim, Claudia Hartmann, Robert Huber, Ravishankar Ramachandran
Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV.
Biophys Chem, 100(1-3) 437-452 (2003)
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Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 degrees C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-A resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.

Bruce D. Wyse, Ian A. Prior, Hongwei Qian, Isabel C. Morrow, Susan J. Nixon, Cornelia Muncke, Teymuras V. Kurzchalia, Walter G. Thomas, Robert G. Parton, John F. Hancock
Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane
J Biol Chem, 278(26) 23738-23746 (2003)
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The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterolindependent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R aveolin complexes are readily detectable e cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.

John Clemmens, Henry Hess, Jonathon Howard, Viola Vogel
Analysis of Microtubule Guidance in Open Microfabricated Channels Coated with the Motor Protein Kinesin
Langmuir, 19(5) 1738-1744 (2003)
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Motor proteins such as kinesin are used in cells to transport intracellular cargo along defined filament paths. The constituents of the transport system (i.e., motor proteins and filaments) can be reconstructed on synthetic surfaces to serve as molecular shuttles that actively transport cargo. Microfabricated open channels have been used in the past to guide microtubules propelled by kinesin adsorbed to the surface. In an effort to better understand the mechanism by which guidance occurs, we present a quantitative measure of the transport efficiency of microtubules along straight polyurethane channels 1 m deep. Our analysis shows that the probability of a microtubule remaining in a channel drops exponentially as a function of its travel distance and that this characteristic decay distance varies with channel width. We identify microtubule-sidewall collisions as the key event for microtubule escape from a channel and thus the determinant of guiding efficiency. To better understand the relationship between collisions and travel distance, we determine the effect of channel width on (1) the microtubule approach angle to the sidewall, (2) the collision outcome (guided or escaped), and (3) the distance traveled between collisions. Also discussed is how the physical properties of microtubules or actin filaments (i.e., length, bending stiffness, persistence length) and changes in channel geometry (width, height) can describe measured parameters.

Robert Ehehalt, Patrick Keller, Christian Haass, Christoph Thiele, Kai Simons
Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.
J Cell Biol, 160(1) 113-123 (2003)
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Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.

Adam J. Liska, Andrej Shevchenko
Expanding the organismal scope of proteomics: cross-species protein identification by mass spectrometry and its implications.
Proteomics, 3(1) 19-28 (2003)
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Due to the limited applicability of conventional protein identification methods to the proteomes of organisms with unsequenced genomes, researchers have developed approaches to identify proteins using mass spectrometry and sequence similarity database searches. Both the integration of mass spectrometry with bioinformatics and genomic sequencing drive the expanding organismal scope of proteomics.

Simon Alberti, Jens Demand, Claudia Esser, Niels Emmerich, Hansjorg Schild, Jorg Hohfeld
Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome.
J Biol Chem, 277(48) 45920-45927 (2002)
DOI

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.

Claire E. Walczak, Eugene C Gan, Arshad Desai, Timothy J. Mitchison, Susan L Kline-Smith
The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly.
Curr Biol, 12(21) 1885-1889 (2002)
DOI

Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.

Robert A Holt, G Mani Subramanian, Aaron L. Halpern, Granger G Sutton, Rosane Charlab, D R Nusskern, Patrick Wincker, Andrew Clark, José M C Ribeiro, Ron Wides, Steven L Salzberg, Brendan Loftus, Mark D Yandell, William H Majoros, Douglas B Rusch, Zhongwu Lai, Cheryl L Kraft, Josep F Abril, Veronique Anthouard, Peter Arensburger, Peter W Atkinson, Holly Baden, Veronique de Berardinis, Danita Baldwin, Vladimir Benes, Jim Biedler, Claudia Blass, R A Bolanos, Didier Boscus, Mary Barnstead, Shuang Cai, Angela Center, Kabir Chaturverdi, George K Christophides, Mathew A Chrystal, Michele Clamp, Anibal Cravchik, Val Curwen, Ali Dana, A L Delcher, I M Dew, Cheryl A Evans, M J Flanigan, Anne Grundschober-Freimoser, Lisa Friedli, Zhiping Gu, Ping Guan, Roderic Guigo, Maureen E Hillenmeyer, Susanne L Hladun, James R Hogan, Young S Hong, Jeffrey Hoover, Olivier Jaillon, Zhaoxi Ke, Chinnappa Kodira, Elena Kokoza, Anastasios Koutsos, Ivica Letunic, Alex Levitsky, Yong Liang, Jhy-Jhu Lin, Neil F Lobo, John R Lopez, Joel A Malek, Tina C McIntosh, Stephan Meister, Jason Miller, C M Mobarry, Emmanuel Mongin, Sean D Murphy, David A O'Brochta, Cynthia Pfannkoch, Rong Qi, Megan A Regier, K A Remington, Hongguang Shao, Maria V Sharakhova, Cynthia D Sitter, Jyoti Shetty, Thomas J Smith, Renee Strong, Jingtao Sun, Dana Thomasova, Lucas Q Ton, Pantelis Topalis, Zhijian Tu, Maria F Unger, Brian Walenz, Aihui Wang, Jian Wang, Mei Wang, Xuelan Wang, Kerry J Woodford, Jennifer R Wortman, Martin Wu, Alison Yao, Evgeny M Zdobnov, Hongyu Zhang, Qi Zhao, Senming Zhao, Shiaoping C Zhu, Igor Zhimulev, Mario Coluzzi, Alessandra della Torre, Charles W Roth, Christos Louis, Francis Kalush, Richard J Mural, Eugene W Myers, Mark D Adams, Hamilton O Smith, Samuel Broder, Melissa Gardner, Claire M Fraser, Ewan Birney, Peer Bork, Paul T Brey, J Craig Venter, Jean Weissenbach, Fotis C Kafatos, Frank H Collins, Stephen L Hoffman
The genome sequence of the malaria mosquito Anopheles gambiae.
Science, 298(5591) 129-149 (2002)
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Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

Karla M. Neugebauer
On the importance of being co-transcriptional.
J Cell Sci, 115(Pt 20) 3865-3871 (2002)
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Intense research in recent years has shown that many pre-mRNA processing events are co-transcriptional or at least begin during RNA synthesis by RNA polymerase II (Pol II). But is it important that pre-mRNA processing occurs co-transcriptionally? Whereas Pol II directs 5' capping of mRNA by binding to and recruiting all three capping activities to transcription units, co-transcriptional splicing is not obligatory. In some cases, such as alternative splicing, splicing may occur post-transcriptionally owing to the slower kinetics of splicing unfavorable introns. Despite recent models in which splicing factors are bound directly to the C-terminal domain (CTD) of Pol II, little evidence supports that view. Instead, interactions between snRNPs and transcription elongation factors provide the strongest molecular evidence for a physical link between transcription and splicing. Transcription termination depends on polyadenylation signals, but, like splicing, polyadenylation per se probably begins co-transcriptionally and continues post-transcriptionally. Nascent RNA plays an important role in determining which transcripts are polyadenylated and which alternative terminal exon is used. A recent addition to co-transcriptional RNA processing is a possible RNA surveillance step prior to release of the mRNP from the transcription unit, which appears to coordinate nuclear transport with mRNA processing and may be mediated by components of the nuclear exosome.

Arshad Desai
The (Theoretical) Yin and Yang of Spindle Mechanics.
Dev Cell, 3(4) 465-467 (2002)

Antiparallel overlap of microtubules is central to the morphogenesis of bipolar mitotic spindles. How does this overlap arise, and how is it maintained? A recent theoretical study uses computer simulations to investigate whether motor protein complexes can achieve this task. The "virtual" results reveal that a mixed polarity motor complex is needed to do the job.

Daniel H. Huson, Knut Reinert, Eugene W Myers
The greedy path-merging algorithm for contig scaffolding
Journal of the ACM (JACM), 49(5) 603-615 (2002)
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Josephine N Harada, Anna Shevchenko, Andrej Shevchenko, David C. Pallas, Arnold J Berk
Analysis of the adenovirus E1B-55K-anchored proteome reveals its link to ubiquitination machinery.
J Virol, 76(18) 9194-9206 (2002)
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During the early phase of infection, the E1B-55K protein of adenovirus type 5 (Ad5) counters the E1A-induced stabilization of p53, whereas in the late phase, E1B-55K modulates the preferential nucleocytoplasmic transport and translation of the late viral mRNAs. The mechanism(s) by which E1B-55K performs these functions has not yet been clearly elucidated. In this study, we have taken a proteomics-based approach to identify and characterize novel E1B-55K-associated proteins. A multiprotein E1B-55K-containing complex was immunopurified from Ad5-infected HeLa cells and found to contain E4-orf6, as well as several cellular factors previously implicated in the ubiquitin-proteasome-mediated destruction of proteins, including Cullin-5, Rbx1/ROC1/Hrt1, and Elongins B and C. We further demonstrate that a complex containing these as well as other proteins is capable of directing the polyubiquitination of p53 in vitro. These ubiquitin ligase components were found in a high-molecular-mass complex of 800 to 900 kDa. We propose that these newly identified binding partners (Cullin-5, Elongins B and C, and Rbx1) complex with E1B-55K and E4-orf6 during Ad infection to form part of an E3 ubiquitin ligase that targets specific protein substrates for degradation. We further suggest that E1B-55K functions as the principal substrate recognition component of this SCF-type ubiquitin ligase, whereas E4-orf6 may serve to nucleate the assembly of the complex. Lastly, we describe the identification and characterization of two novel E1B-55K interacting factors, importin-alpha 1 and pp32, that may also participate in the functions previously ascribed to E1B-55K and E4-orf6.

Andrei V. Popov, Fedor F. Severin, Eric Karsenti
XMAP215 is required for the microtubule-nucleating activity of centrosomes.
Curr Biol, 12(15) 1326-1330 (2002)
DOI

Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin.

Jeffrey A Bailey, Zhiping Gu, Royden A Clark, Knut Reinert, Rhea V Samonte, Stuart Schwartz, Mark D Adams, Eugene W Myers, Peter W Li, Evan E Eichler
Recent segmental duplications in the human genome.
Science, 297(5583) 1003-1007 (2002)
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Primate-specific segmental duplications are considered important in human disease and evolution. The inability to distinguish between allelic and duplication sequence overlap has hampered their characterization as well as assembly and annotation of our genome. We developed a method whereby each public sequence is analyzed at the clone level for overrepresentation within a whole-genome shotgun sequence. This test has the ability to detect duplications larger than 15 kilobases irrespective of copy number, location, or high sequence similarity. We mapped 169 large regions flanked by highly similar duplications. Twenty-four of these hot spots of genomic instability have been associated with genetic disease. Our analysis indicates a highly nonrandom chromosomal and genic distribution of recent segmental duplications, with a likely role in expanding protein diversity.

Thomas Wendl, Klaus Lun, Marina Mione, Jack Favor, Michael Brand, Stephen W. Wilson, Klaus B Rohr
pax2.1 is required for the development of thyroid follicles in zebrafish.
Development, 129(15) 3751-3760 (2002)
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The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

Daniela Berdnik^*, Tibor Török^*, Marcos González-Gaitán, Jürgen A. Knoblich
The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.
Dev Cell, 3(2) 221-231 (2002)
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During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch.

K A Johansson, Anne Grapin-Botton
Development and diseases of the pancreas.
Clin. Genet., 62(1) 14-23 (2002)
DOI

The pancreas is a vital gland of exocrine and endocrine function. It is the target of two main affections: diabetes and pancreatic cancer. We describe the tissue interactions, signaling pathways and intracellular targets that are involved in the emergence of the pancreas primordium and its proliferation, morphogenesis and differentiation. It appears that several genes of developmental relevance have an adult function and are involved in pancreas affections. Embryological experimentation in animals contributed to provide candidate genes for human disease and holds promise for future treatments.

James T Murray, Christina Panaretou, Harald Stenmark, Marta Miaczynska, Jonathan M Backer
Role of Rab5 in the recruitment of hVps34/p150 to the early endosome.
Traffic, 3(6) 416-427 (2002)
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PI 3-kinases are important regulators of endocytic trafficking. We have previously proposed a model in which the Rab5 GTPase recruits EEA1 to the early endosome both directly, by binding to EEA1, and indirectly, through the recruitment of the p150/hVps34 PI 3-kinase and the production of PI[3]P in the endosomal membrane. In this study we have examined this model in vivo. We find that both endogenous hVps34 and p150 are targeted to enlarged endosomal structures in cells expressing constitutively activated Rab5, where they are significantly colocalized with EEA1. Recombinant fragments of p150 disrupt the endosomal localization of EEA1, showing that p150 is required for EEA1 targeting. We further analyzed the mechanism of GTP-dependent Rab5-p150 binding, and showed the p150 HEAT and WD40 domains are required for binding, whereas deletion of the protein kinase domain increases binding to Rab5. Overexpression of constitutively active Rab5 caused a redistribution of epitope-tagged hVps34 and p150 to Rab5-positive endosomes. However, subcellular fractionation showed that this was not due to a significant recruitment of hVps34 or p150 from the cytosolic to the particulate fraction. These data suggest that the binding of Rab5 to the HEAT/WD40 domains of p150 is important in regulating the localization of hVps34/p150. However, Rab5 does not appear to act by directly recruiting p150/hVps34 complexes from the cytosol to the endosomal membrane.

Kazuhisa Kinoshita, Bianca Habermann, Anthony A. Hyman
XMAP215: a key component of the dynamic microtubule cytoskeleton.
Trends Cell Biol, 12(6) 267-273 (2002)
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Microtubules are essential for various cellular processes including cell division and intracellular organization. Their function depends on their ability to rearrange their distribution at different times and places. Microtubules are dynamic polymers and their behaviour is described as dynamic instability. Rearrangement of the microtubule cytoskeleton is made possible by proteins that modulate the parameters of dynamic instability. Studies using Xenopus egg extracts led to identification of a microtubule-associated protein called XMAP215 as a major regulator of physiological microtubule dynamics. XMAP215 belongs to an evolutionarily conserved protein family present in organisms ranging from yeast to mammals. Together with members of the Kin I family of kinesins, XMAP215 and its orthologues form an essential circuit for generating dynamic microtubules in vivo.

Heiner Grandel, Klaus Lun, Gerd-Jörg Rauch, Muriel Rhinn, Tatjana Piotrowski, Corinne Houart, Paolo Sordino, Axel M Küchler, Stefan Schulte-Merker, Robert Geisler, Nigel Holder, Stephen W. Wilson, Michael Brand
Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud.
Development, 129(12) 2851-2865 (2002)
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A number of studies have suggested that retinoic acid (RA) is an important signal for patterning the hindbrain, the branchial arches and the limb bud. Retinoic acid is thought to act on the posterior hindbrain and the limb buds at somitogenesis stages in chick and mouse embryos. Here we report a much earlier requirement for RA signalling during pre-segmentation stages for proper development of these structures in zebrafish. We present evidence that a RA signal is necessary during pre-segmentation stages for proper expression of the spinal cord markers hoxb5a and hoxb6b, suggesting an influence of RA on anteroposterior patterning of the neural plate posterior to the hindbrain. We report the identification and expression pattern of the zebrafish retinaldehyde dehydrogenase2 (raldh2/aldh1a2) gene. Raldh2 synthesises retinoic acid (RA) from its immediate precursor retinal. It is expressed in a highly ordered spatial and temporal fashion during gastrulation in the involuting mesoderm and during later embryogenesis in paraxial mesoderm, branchial arches, eyes and fin buds, suggesting the involvement of RA at different times of development in different functional contexts. Mapping of the raldh2 gene reveals close linkage to no-fin (nof), a newly discovered mutant lacking pectoral fins and cartilaginous gill arches. Cloning and functional tests of the wild-type and nof alleles of raldh2 reveal that nof is a raldh2 mutant. By treating nof mutants with RA during different time windows and by making use of a retinoic acid receptor antagonist, we show that RA signalling during pre-segmentation stages is necessary for anteroposterior patterning in the CNS and for fin induction to occur.

Joan Cerdà, Christine Gründ, Werner W Franke, Michael Brand
Molecular characterization of Calymmin, a novel notochord sheath-associated extracellular matrix protein in the zebrafish embryo.
Dev Dyn, 224(2) 200-209 (2002)
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During the screening of a zebrafish postsomitogenesis embryo cDNA library, we have identified a cDNA corresponding to a novel type of protein localized to the notochordal sheath-associated extracellular matrix (ECM) of the embryo. The 4.049-kb mRNA encodes a predicted polypeptide of 1,207 amino acids (122 kDa, pI 10.50) with a potential signal peptide of 20 amino acids. After the signal peptide, the mature protein consists of 1,187 amino acids (119 kDa, pI 10.46), for which the name "Calymmin" (from Greek chialphalambdanumumualpha, to envelop, to cover) is proposed. The Calymmin mRNA is highly and transiently expressed by the notochord cells of the embryo from the 10- to 12-somite stage to the pharyngula period (13 and 24 hours postfertilization, respectively), and light and electron microscopical immunolocalization analysis revealed that the protein was specifically localized within a granular and filamentous layer of the ECM compartment surrounding the notochord. In zebrafish no tail mutants (ntl(tc41)), in which the notochord precursor cells are present but fail to differentiate, the Calymmin protein was not detected, confirming the notochord origin of Calymmin. These results indicate that Calymmin is a novel constitutive protein of the ECM compartment associated to the perinotochordal sheath in the zebrafish embryo, which is specifically expressed by the differentiating notochord cells.

Ravi Sawhney, Jonathon Howard
Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels.
J Cell Biol, 157(6) 1083-1091 (2002)
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Aclassic model for tissue morphogenesis is the formation of ligament-like straps between explants of fibroblasts placed in collagen gels. The patterns arise from mechanical forces exerted by cells on their substrates (Harris et al., 1981). However, where do such straps come from, and how are slow local movements of cells transduced into dramatic long-distance redistributions of collagen? We embedded primary mouse skin and human periodontal ligament fibroblasts in collagen gels and measured the time course of patterning by using a novel computer algorithm to calculate anisotropy, and by tracking glass beads dispersed in the gel. As fibroblasts began to spread into their immediate environments, a coordinated rearrangement of collagen commenced throughout the gel, producing a strap on a time scale of minutes. Killing of cells afterwards resulted in a partial relaxation of the matrix strain. Surprisingly, relatively small movements of collagen molecules on the tensile axis between two pulling explants induced a much larger concomitant compression of the gel perpendicular to the axis, organizing and aligning fibers into a strap. We propose that this amplification is due to the geometry of the collagen matrix, and that analogous amplified movements may drive morphological changes in other biological meshes, both outside and inside the cell.

Darinel Ortiz^*, Martina Medkova^*, Christiane Walch-Solimena, Peter Novick
Ypt32 recruits the Sec4p guanine nucleotide exchange factor, Sec2p, to secretory vesicles; evidence for a Rab cascade in yeast.
J Cell Biol, 157(6) 1005-1015 (2002)
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SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.

Susanne Kaitna, Heinke Schnabel, Ralf Schnabel, Anthony A. Hyman, Michael Glotzer
A ubiquitin C-terminal hydrolase is required to maintain osmotic balance and execute actin-dependent processes in the early C. elegans embryo.
J Cell Sci, 115(Pt 11) 2293-2302 (2002)
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In the early Caenorhabditis elegans embryo, establishment of cell polarity and cytokinesis are both dependent upon reorganization of the actin cytoskeleton. Mutations in the cyk-3 gene cause maternal effect embryonic lethality. Embryos produced by homozygous cyk-3 mutant animals become multinucleate. We have further analyzed the cyk-3 mutant phenotype and have found that cyk-3 mutant embryos fail to properly polarize the actin cytoskeleton and fail to segregate germline determinants. In addition, they fail to assemble an intact cleavage furrow. However, we have found that cyk-3 mutant embryos are intrinsically defective in osmotic regulation and that the cytokinesis defects can be partially rescued by providing osmotic support. The cyk-3 gene has been identified and found to encode a ubiquitin C-terminal hydrolase that is active against model substrates. These data indicate that the deubiquitination of certain substrates by CYK-3 is crucial for cellular osmoregulation. Defects in osmoregulation appear to indirectly affect actin-dependent processes.

Ravishankar Ramachandran, Claudia Hartmann, Hyun Kyu Song, Robert Huber, Matthias Bochtler
Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY).
Proc Natl Acad Sci U.S.A., 99(11) 7396-7401 (2002)
PDF DOI

HslVU is a bacterial homolog of the proteasome, where HslV is the protease that is activated by HslU, an ATPase and chaperone. Structures of singly and doubly capped HslVU particles have been reported, and different binding modes have been observed. Even among HslVU structures with I-domains distal to HslV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HslVU structure, insertion of the C termini of HslU into pockets in HslV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HslV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HslV with 10(-5) M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HslU. Enhanced HslV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HslV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HslV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with I-domains distal to HslV captures the active species and point to significant differences in the activation mechanism of HslV, ClpP, and the proteasome.

Eva Hannak^*, Karen Oegema^*, Matthew Kirkham^*, Pierre Gönczy, Bianca Habermann, Anthony A. Hyman
The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent.
J Cell Biol, 157(4) 591-602 (2002)
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gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

Richard J Mural, Mark D Adams, Eugene W Myers, Hamilton O Smith, George L Gabor Miklos, Ron Wides, Aaron L. Halpern, Peter W Li, Granger G Sutton, Joe Nadeau, Steven L Salzberg, Robert A Holt, Chinnappa Kodira, Fu Lu, Lin Chen, Zuoming Deng, Carlos C Evangelista, Weiniu Gan, Thomas J Heiman, Jiayin Li, Zhenya Li, Gennady V Merkulov, Natalia V Milshina, Ashwinikumar K Naik, Rong Qi, Bixiong Chris Shue, Aihui Wang, Jian Wang, Xin Wang, Xianghe Yan, Jane Ye, Shibu Yooseph, Qi Zhao, Liansheng Zheng, Shiaoping C Zhu, Kendra Biddick, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Daniel H. Huson, S A Kravitz, Jason R Miller, C M Mobarry, Knut Reinert, K A Remington, Qinyu Zhang, Xiangqun H Zheng, D R Nusskern, Zhongwu Lai, Yiding Lei, Wenyan Zhong, Alison Yao, Ping Guan, Rui-Ru Ji, Zhiping Gu, Zhen-Yuan Wang, Fei Zhong, Chunlin Xiao, Chia-Chien Chiang, Mark D Yandell, Jennifer R Wortman, Peter G Amanatides, Susanne L Hladun, Eric C Pratts, Jeffery E Johnson, Kristina L Dodson, Kerry J Woodford, Cheryl A Evans, Barry Gropman, Douglas B Rusch, Eli Venter, Mei Wang, Thomas J Smith, Jarrett T Houck, Donald E Tompkins, Charles Haynes, Debbie Jacob, Soo H Chin, David R Allen, Carl E Dahlke, Robert Sanders, Kelvin Li, Xiangjun Liu, Alex Levitsky, William H Majoros, Quan Chen, Ashley C Xia, John R Lopez, Michael T Donnelly, Matthew H Newman, Anna Glodek, Cheryl L Kraft, Marc Nodell, Feroze Ali, Hui-Jin An, Danita Baldwin-Pitts, Karen Y Beeson, Shuang Cai, Mark Carnes, Amy Carver, Parris M Caulk, Angela Center, Yen-Hui Chen, Ming-Lai Cheng, My D Coyne, Michelle Crowder, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Lisa Doup, Patrick Dullaghan, Steven Ferriera, Carl R Fosler, Harold C Gire, Andres Gluecksmann, Jeannine D Gocayne, Jonathan Gray, Brit Hart, Jason Haynes, Jeffrey Hoover, Tim Howland, Chinyere Ibegwam, Mena Jalali, David Johns, Leslie Kline, Daniel S Ma, Steven MacCawley, Anand Magoon, Felecia Mann, David May, Tina C McIntosh, Somil Mehta, Linda Moy, Mee C Moy, Brian J Murphy, Sean D Murphy, Keith A Nelson, Zubeda Nuri, Kimberly A Parker, Alexandre C Prudhomme, Vinita N Puri, Hina Qureshi, John C Raley, Matthew S Reardon, Megan A Regier, Yu-Hui C Rogers, Deanna L Romblad, Jakob Schutz, John L Scott, Richard Scott, Cynthia D Sitter, Michella Smallwood, Andrew Sprague, Erin Stewart, Renee Strong, Ellen Suh, Karena Sylvester, Reginald Thomas, Ni Ni Tint, Christopher Tsonis, Gary Wang, George Wang, Monica S Williams, Sherita M Williams, Sandra M Windsor, Keriellen Wolfe, Mitchell M Wu, Jayshree Zaveri, Kabir Chaturvedi, Andrei E Gabrielian, Zhaoxi Ke, Jingtao Sun, Gangadharan Subramanian, J Craig Venter, Cynthia Pfannkoch, Mary Barnstead, Lisa D Stephenson
A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome.
Science, 296(5573) 1661-1671 (2002)
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The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

Eugene W Myers, Granger G Sutton, Hamilton O Smith, Mark D Adams, J Craig Venter
On the sequencing and assembly of the human genome.
Proc Natl Acad Sci U.S.A., 99(7) 4145-4146 (2002)
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Robert Ehehalt, Beate Michel, Davide De Pietri Tonelli, Daniele Zacchetti, Kai Simons, Patrick Keller
Splice variants of the beta-site APP-cleaving enzyme BACE1 in human brain and pancreas.
Biochem Biophys Res Commun, 293(1) 30-37 (2002)
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BACE is the beta-secretase responsible for the first step in amyloidogenic processing of the amyloid precursor protein APP. We have identified two BACE isoforms, BACE1B and BACE1C, lacking 25 and 44 amino acids, respectively. Whereas the BACE1B transcript is present in human pancreas and brain, the BACE1C transcript is found in pancreas only. In transfected cells both BACE1A, which encodes the originally described full-length BACE1 protein and the close homolog BACE2 localized mainly to post-Golgi membranes. In contrast, the two shorter isoforms were found in the endoplasmic reticulum only, and they did not display beta-secretase activity. Using RNase protection we in addition show that the major pancreatic transcript is BACE1A. This suggests that the known absence of beta-secretase activity in the pancreas is not due to a missing BACE1A transcript.

József Jászai, Michael Brand
Cloning and expression of Ventrhoid, a novel vertebrate homologue of the Drosophila EGF pathway gene rhomboid.
Mech Dev, 113(1) 73-77 (2002)
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In Drosophila melanogaster, the seven-pass transmembrane protein Rhomboid (Rho) is a crucial positive modulator of EGF signaling playing a substantial role in patterning of the ventral neuroectoderm and fate specification of neuroblasts. Here, we describe the cloning and expression pattern of Ventrhoid (Vrho), the novel evolutionarily conserved vertebrate cDNA related to fruit fly rho. Most importantly, like rho in Drosophila, Vrho is also expressed in a spatially restricted manner. Vrho expression is most prominent along the developing ventral neural tube, and is also detectable in the ventral forebrain, prospective diencephalon, otic vesicles, mandibular arches, cranial sensory placodes, last formed pair of somites and hindgut in midgestational mouse embryos.

Antoine Guichet, Tanja Wucherpfennig, Veronica Dudu, Sylvain Etter, Michaela Wilsch-Bräuninger, Andrea Hellwig, Marcos González-Gaitán, Wieland B. Huttner, Anne A Schmidt
Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction.
EMBO J, 21(7) 1661-1672 (2002)
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We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.

Yuri Voziyanov, A F Stewart, Makkuni Jayaram
A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity.
Nucleic Acids Res, 30(7) 1656-1663 (2002)
Open Access DOI

We have developed a dual reporter screen in Escherichia coli for identifying variants of the Flp site-specific recombinase that have acquired reactivity at an altered target site (mFRT). In one reporter, the lacZalpha gene segment is flanked by mFRTs in direct orientation. In the other, the red fluorescence protein (RFP) gene is flanked by the native FRTs. Hence, the color of a colony on an X-gal indicator plate indicates the recombination potential of the variant Flp protein expressed in it: blue if no recombination or only FRT recombination occurs, red if only mFRT recombination occurs and white if both FRT and mFRT recombinations occur. The scheme was validated by identification and in vivo characterization of Flp variants that show either relaxed specificity (active on FRT and mFRT) or moderately shifted specificity toward mFRT. We find that alteration of Lys-82 to Met, Thr, Arg or His enables the corresponding Flp variants to recombine FRT sites as well as altered FRT sites containing a substitution of G-C by C-G at position 1 of the Flp binding element (mFRT11). In contrast, wild-type Flp has no detectable activity on mFRT11. When Lys-82 is replaced by Tyr, the resulting Flp variant shows a small but reproducible preference for mFRT11 over FRT. However, this preference for mFRT11 is nearly lost when Tyr-82 is substituted by Phe.

Heike B Eberle, Ramon L Serrano, Joachim Füllekrug, Andreas Schlosser, Wolf D Lehmann, Friedrich Lottspeich, Dora Kaloyanova, Felix T. Wieland, J Bernd Helms
Identification and characterization of a novel human plant pathogenesis-related protein that localizes to lipid-enriched microdomains in the Golgi complex.
J Cell Sci, 115(Pt 4) 827-838 (2002)

Group 1 of plant pathogenesis-related proteins (PR-1) and a variety of related mammalian proteins constitute a superfamily of proteins that share structural similarities. Little is known about their function, but all the family members identified to date are co-translationally translocated to the lumen of the endoplasmic reticulum and are secreted as soluble proteins or are targeted to vacuoles. Here we report the identification of a novel family member that localizes to the cytosolic site of the endomembrane system in mammalian cells. After detergent solubilization of isolated Golgi membranes, a 17 kDa protein was found associated with a low-density detergent-insoluble fraction. The amino-acid sequence, determined by microsequencing and molecular cloning, revealed a significant homology with the superfamily of PR-1 proteins. Golgi-associated PR-1 protein (GAPR-1) showed a brefeldin-A-sensitive Golgi localization in immunofluorescence. Interestingly, the protein remained associated with the microdomain fraction in the presence of Brefeldin A. By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1. Tissue screening revealed that GAPR-1 is not detectably expressed in liver, heart or adrenal glands. High expression was found in monocytes, leukocytes, lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.

Shawn Burgess^*, Gerlinde Reim^*, Wenbiao Chen, Nancy Hopkins, Michael Brand
The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis.
Development, 129(4) 905-916 (2002)
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In early embryonic development, the brain is divided into three main regions along the anteroposterior axis: the forebrain, midbrain and hindbrain. Through retroviral insertional mutagenesis and chemical mutagenesis experiments in zebrafish, we have isolated mutations that cause abnormal hindbrain organization and a failure of the midbrain-hindbrain boundary (MHB) to form, a region that acts as an organizer for the adjacent brain regions. The mutations fail to complement the spiel-ohne-grenzen (spg) mutation, which causes a similar phenotype, but for which the affected gene is unknown. We show through genetic mapping, cloning of the proviral insertion site and allele sequencing that spg mutations disrupt pou2, a gene encoding the Pou2 transcription factor. Based on chromosomal synteny, phylogenetic sequence comparison, and expression and functional data, we suggest that pou2 is the zebrafish ortholog of mouse Oct3/Oct4 and human POU5F1. For the mammalian genes, a function in brain development has so far not been described. In the absence of functional pou2, expression of markers for the midbrain, MHB and the hindbrain primordium (pax2.1, wnt1, krox20) are severely reduced, correlating with the neuroectoderm-specific expression phase of pou2. Injection of pou2 mRNA restores these defects in spg mutant embryos, but does not activate these markers ectopically, demonstrating a permissive role for pou2. Injections of pou2-morpholinos phenocopy the spg phenotype at low concentration, further proving that spg encodes pou2. Two observations suggest that pou2 has an additional earlier function: higher pou2-morpholino concentrations specifically cause a pre-gastrula arrest of cell division and morphogenesis, and expression of pou2 mRNA itself is reduced in spg-homozygous embryos at this stage. These experiments suggest two roles for pou2. Initially, Pou2 functions during early proliferation and morphogenesis of the blastomeres, similar to Oct3/4 in mammals during formation of the inner cell mass. During zebrafish brain formation, Pou2 then functions a second time to activate gene expression in the midbrain and hindbrain primordium, which is reflected at later stages in the specific lack in spg embryos of the MHB and associated defects in the mid- and hindbrain.

Stefano de Renzis, Birte Sönnichsen, Marino Zerial
Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes.
Nat Cell Biol, 4(2) 124-133 (2002)
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The three GTPases Rab5, Rab4 and Rab11 regulate sequential transport steps along the endocytic/recycling pathway, and occupy distinct membrane domains on early and recycling endosomes. To address the mechanisms that regulate communication between such domains, we searched for proteins that interact with both Rab5 and Rab4. Here, we report that Rabenosyn-5, a previously identified Rab5 effector, also binds to Rab4. Rabenosyn-5 overexpression increased the association between Rab5 and Rab4 endosomal domains and decreased the fraction of Rab4- and Rab11-positive structures. This redistribution was accompanied by a faster rate of transferrin recycling from early endosomes to the cell surface and reduced transport to Rab11-containing perinuclear recycling endosomes. These effects depend on the ability of Rabenosyn-5 to interact with Rab4. We propose that divalent Rab effectors regulate protein sorting and recycling by connecting Rab domains on early endosomes.

Ivo F. Sbalzarini
Diffusion in the Endoplasmic Reticulum
Diploma Thesis,ETH Zurich, Department of Computer Science, Zurich, Switzerland (2002)
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Marta Miaczynska, Marino Zerial
Mosaic organization of the endocytic pathway.
Exp Cell Res, 272(1) 8-14 (2002)
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Stephen W. Wilson, Michael Brand, Judith S Eisen
Patterning the zebrafish central nervous system.
Results Probl Cell Differ, 40 181-215 (2002)
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We have described the formation of the zebrafish central nervous system. The spinal cord has the simplest organization and was considered first, followed by the forebrain, midbrain and hindbrain. We have discussed many studies that have revealed the molecular mechanisms, including extrinsic signals and intrinsic responses to them, underlying the establishment of nervous system regions and the wide diversity of neuronal cell types of which they are comprised. Wherever possible, we have tried to compare what has been learned from zebrafish with what is known in other vertebrate species. The simplicity of the developing nervous system makes zebrafish embryos particularly amenable to studies of nervous system development. Thus, many aspects of nervous system patterning that were unknown from other vertebrates have been revealed by studies in zebrafish. However, the relationship between embryonic and adult nervous system morphology is still not entirely clear and remains an important avenue for further studies.

Dimitrios Fotiadis, Simon Scheuring, Shirley A Müller, Andreas Engel, Daniel J. Müller
Imaging and manipulation of biological structures with the AFM.
Micron, 33(4) 385-397 (2002)
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Many biologists have dreamt of physically touching and manipulating the biomolecules they were investigating. With the invention of the atomic force microscope (AFM), this dream has come true. Here, recent applications of the AFM to image and to manipulate biological systems at the nanometer scale are reviewed. Macromolecular biological assemblies as well as individual biomolecules can be subjected to controlled nanomanipulation. Examples of AFM application in imaging and nanomanipulation include the extraction of chromosomal DNA for genetic analysis, the disruption of antibody--antigen bonds, the dissection of biological membranes, the nanodissection of protein complexes, and the controlled modulation of protein conformations. Also reviewed is the novel combination of single molecule imaging and force spectroscopy which allows biomolecules to be imaged, and inter- and intramolecular forces to be measured. Future application of these nanotechniques will reveal new information on the structure, function and assembly of biomolecules.

D R Shimshek, J Kim, M R Hübner, D J Spergel, Frank Buchholz, E Casanova, A. Francis Stewart, P H Seeburg, R. Sprengel
Codon-improved Cre recombinase (iCre) expression in the mouse.
Genesis, 32(1) 19-26 (2002)
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By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Raymond J. Deshaies, Jae Hong Seol, W Hayes McDonald, Gregory Cope, Svetlana Lyapina, Andrej Shevchenko, Anna Shevchenko, Rati Verma, John R. Yates
Charting the protein complexome in yeast by mass spectrometry.
Mol Cell Proteomics, 1(1) 3-10 (2002)
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It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.

Susan E Celniker, David A Wheeler, Brent Kronmiller, Joseph W Carlson, Aaron L. Halpern, Sandeep Patel, Mark Adams, Mark Champe, Shannon P Dugan, Erwin Frise, Ann Hodgson, Reed A George, Roger A Hoskins, Todd R Laverty, Donna M Muzny, Catherine R Nelson, Joanne M Pacleb, Soo Park, Barret D Pfeiffer, Stephen Richards, Erica J Sodergren, Robert R Svirskas, Paul E Tabor, Kenneth Wan, Mark Stapleton, Granger G Sutton, Craig Venter, George Weinstock, Steven E Scherer, Eugene W Myers, Richard A Gibbs, Gerald M Rubin
Finishing a whole-genome shotgun: release 3 of the Drosophila melanogaster euchromatic genome sequence.
Genome Biol, 3(12) Art. No. research0079.1–0079.14 (2002)
Open Access PDF

The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.

Benjamin P Berman, Pavel Tomancák, Yutaka Nibu, Barret D Pfeiffer, Susan E Celniker, Michael Levine, Gerald M Rubin, Michael B Eisen
Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome.
Proc Natl Acad Sci U.S.A., 99(2) 757-762 (2002)
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A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt), and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.

Assen Roguev, Daniel Schaft, A Shevchenko, W W M Pim Pijnappel, Matthias Wilm, Rein Aasland, A F Stewart
The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.
EMBO J, 20(24) 7137-7148 (2001)
DOI

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.

André Bachmann, M Schneider, Eva Theilenberg, Ferdi Grawe, Elisabeth Knust
Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity.
Nature, 414(6864) 638-643 (2001)
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The polarized architecture of epithelial cells depends on the highly stereotypic distribution of cellular junctions and other membrane-associated protein complexes. In epithelial cells of the Drosophila embryo, three distinct domains subdivide the lateral plasma membrane. The most apical one comprises the subapical complex (SAC). It is followed by the zonula adherens (ZA) and, further basally, by the septate junction. A core component of the SAC is the transmembrane protein Crumbs, the cytoplasmic domain of which recruits the PDZ-protein Discs Lost into the complex. Cells lacking crumbs or the functionally related gene stardust fail to organize a continuous ZA and to maintain cell polarity. Here we show that stardust provides an essential component of the SAC. Stardust proteins colocalize with Crumbs and bind to the carboxy-terminal amino acids of its cytoplasmic tail. We introduce two different Stardust proteins here: one MAGUK protein, characterized by a PDZ domain, an SH3 domain and a guanylate kinase domain; and a second isoform comprising only the guanylate kinase domain. The Stardust proteins represent versatile candidates as structural and possibly regulatory constituents of the SAC, a crucial element in the control of epithelial cell polarity.

Alexandra C. Moreno-Borchart, Katrin Strasser, Martin G. Finkbeiner, Anna Shevchenko, Andrej Shevchenko, Michael Knop
Prospore membrane formation linked to the leading edge protein (LEP) coat assembly.
EMBO J, 20(24) 6946-6957 (2001)
PDF DOI

In yeast, the differentiation process at the end of meiosis generates four daughter cells inside the boundaries of the mother cell. A meiosis-specific plaque (MP) at the spindle pole bodies (SPBs) serves as the starting site for the formation of the prospore membranes (PSMs) that are destined to encapsulate the post-meiotic nuclei. Here we report the identification of Ady3p and Ssp1p, which are functional components of the leading edge protein (LEP) coat, that covers the ring-shaped opening of the PSMs. Ssp1p is required for the assembly of the LEP coat, which consists of at least three proteins (Ssp1p, Ady3p and Don1p). The assembly of the LEP coat starts with the formation of cytosolic precursors, which then bind in an Ady3p-dependent manner to the SPBs. Subsequent processes at the SPBs leading to functional LEP coats require Ssp1p and the MP components. During growth of the PSMs, the LEP coat functions in formation of the cup-shaped membrane structure that is indispensable for the regulated cellularization of the cytoplasm around the post-meiotic nuclei.

Thomas Meier, Ulrich Matthey, Fabienne Henzen, Peter Dimroth, Daniel J. Müller
The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids
FEBS Lett, 505(5) 353-356 (2001)
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The isolated rotor cylinder of the ATP synthase from Ilyobacter tartaricus was reconstituted into two-dimensional crystalline arrays. Atomic force microscopy imaging indicated a central cavity on one side of the rotor and a central plug protruding from the other side. Upon incubation with phospholipase C, the plug disappeared, but the appearance of the surrounding c subunit oligomer was not affected. This indicates that the plug consists of phospholipids. As the detergent-purified c cylinder is completely devoid of phospholipids, these are incorporated into the central hole from one side of the cylinder during the reconstitution procedure.

W W M Pim Pijnappel, Daniel Schaft, Assen Roguev, A Shevchenko, H Tekotte, Matthias Wilm, Guillaume Rigaut, Bertrand Séraphin, Rein Aasland, A F Stewart
The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.
Genes Dev, 15(22) 2991-3004 (2001)
DOI

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Fedor F. Severin, Anthony A. Hyman, Simonetta Piatti
Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.
J Cell Biol, 155(5) 711-718 (2001)
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At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

Katherine S. Long, Tommy Cedervall, Christiane Walch-Solimena, Dennis A. Noe, Michael J. Huddleston, Roland S. Annan, Sandra L. Wolin
Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.
RNA, 7(11) 1589-1602 (2001)
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An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Joanna Rowe, Federico Calegari, Elena Taverna, Renato Longhi, Patrizia Rosa
Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins.
J Cell Sci, 114(Pt 18) 3323-3332 (2001)
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SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.

Valentina Greco, Michael Hannus, Suzanne Eaton
Argosomes: a potential vehicle for the spread of morphogens through epithelia.
Cell, 106(5) 633-645 (2001)
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The formation of morphogen gradients is essential for tissue patterning. Morphogens are released from producing cells and spread through adjacent tissue; paradoxically, however, many morphogens, including Wingless, associate tightly with the cell membrane. Here, we describe a novel cell biological mechanism that disperses membrane fragments over large distances through the Drosophila imaginal disc epithelium. We call these membrane exovesicles argosomes. Argosomes are derived from basolateral membranes and are produced by many different regions of the disc. They travel through adjacent tissue where they are found predominantly in endosomes. Wingless protein colocalizes with argosomes derived from Wingless-producing cells. The properties of argosomes are consistent with their being a vehicle for the spread of Wingless protein.

Matthias Bochtler, Hyun Kyu Song, Claudia Hartmann, Ravishankar Ramachandran, Robert Huber
The quaternary arrangement of HslU and HslV in a cocrystal: a response to Wang, Yale.
J Struct Biol, 135(3) 281-293 (2001)
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Protease HslV and ATPase HslU form an ATP-dependent protease in bacteria. We have previously determined the structure of the components of this protease. In the case of HslU, the structure was derived from HslU-HslV cocrystals, combining phase information from MAD and the previously determined HslV model. Whereas the structures of the components were confirmed in detail by later structures, the quaternary arrangement of HslV and HslU was not reproduced in later crystal forms. In a recent communication to this journal, Wang attempted a reinterpretation of our original data to account for this difference. In response, we demonstrate that difference Pattersons, difference Fouriers, molecular replacement calculations, R factors, and omit maps all support our original analysis and prove that the suggested reinterpretation is false by these criteria. In particular, we show that our crystals are essentially untwinned and that only the originally reported quaternary arrangement of HslV and HslU particles is consistent with the experimental data. We finally demonstrate that Wang's newly introduced R(tpart) method to predict translational corrections for a subset of the unit cell contents is systematically flawed.

Anna Shevchenko, Andrej Shevchenko
Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry.
Anal Biochem, 296(2) 279-283 (2001)
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NOTES & TIPS Evaluation of the Efficiency of In-Gel Digestion of Proteins by Peptide Isotopic Labeling and MALDI Mass Spectrometry Anna Shevchenko and Andrej Shevchenko 1 MPI of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany; and European Molecular Biology Laboratory (EMBL), 69012 Heidelberg, Germany Received December 21, 2000; published online August 15, 2001 Visualization of proteins separated by one-dimensional or two-dimensional polyacrylamide gel electrophoresis, in-gel digestion of excised protein bands (spots), followed by identification of proteins by mass spectrometry underpin many proteome characterization strategies (reviewed in (1 4)). A multitude of protocols for staining of polyacrylamide gels (reviewed in (5)) and of enzymatic in-gel digestion of proteins (reviewed in (1, 6)) at the low picomole femtomole level has been reported. A combination of protein visualization and in-gel digestion methods designed for proteomic application is usually evaluated according to two major criteria. First, the lowest limit of reliable visualization of protein spot (band) is determined (5, 7, 8). Second, the sequence coverage of MALDI peptide mass fingerprints acquired from in-gel digests of the visualized protein spots and the sensitivity of mass spectrometric detection are evaluated (8 10). However, the latter criteria are particularly difficult to apply. The intensity of peptide signals detected in complex mixtures by MALDI MS strongly depends on the employed sample handling and probe preparation routines. Furthermore, unavoidable micro-heterogeneity of MALDI probes and inevitable presence of the residual amount of dyes, salts, and detergents result in significant shot-to-shot variation of peptide and matrix signals, requires consistent tuning of the laser fluence (11) and, consequently, does not allow quantitative comparison of spectra acquired in the separate experiments. Without direct measurement of the yield of peptide digestion products it is difficult to provide consistent evaluation of the efficiency of in-gel digestion protocols. Sequencing of proteins from silver-stained gels may serve as an example. Successful identification of silver stained proteins and high sequence coverage of MALDI peptide mass maps at the low femtomole level was reported (12), and almost identical peptide mass fingerprints were observed in the digests of silver and Coomassie stained protein bands (13). However, other authors reported substantially lower sequence coverage of peptide mass maps acquired from the digests of silver-stained proteins (8, 10, 14) that, however, could be improved by destaining of the bands prior to in-gel trypsinolysis (15, 16). We therefore set out to develop a method for direct and quantitative evaluation of the efficiency of in-gel cleavage of proteins in order to outline a rational procedure for comparison of in-gel digestion efficiency. The yield of digestion products was determined by MALDI MS using 18 O-isotopically labeled peptides as the internal standards (17, 18). We further examined whether conventional methods of staining of polyacrylamide gels (Coomassie staining, silver staining, and zinc-imidazole staining) might affect the in-gel digestion efficiency. Materials and Methods Materials and reagents. All major chemicals were purchased from Sigma (Sigma Chemicals, St. Louis, MO) and were of analytical grade. H 2 18 O (Cambridge Isotopic Laboratories, MA) was purified by microdistillation as described (19). Gel electrophoresis and visualization of protein bands. The aliquots containing 1 pmol of bovine serum albumin (Sigma Chemicals) in Laemmli buffer were loaded onto separate lanes of a one-dimensional polyacrylamide gel. Immediately after electrophoresis, the gel was cut. Separate parts of the gel slab each containing three lanes with the BSA standards were stained by various methods. Coomassie staining and silver staining were performed as described (13). Zinc imidazole staining (negative staining) was performed according to (20). In a separate experiment the gel was stained with silver. Four BSA bands were excised from the gel and then destained with potassium ferricyanide and sodium thiosulfate as described (15). Stained and Preliminary results were reported at the 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, 2000. 1 Corresponding author: E-mail: shevchenko@mpi-cbg.de. Analytical Biochemistry 296, 279 283 (2001) 279 doi:10.1006/abio.2001.5321 0003-2697/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved. All articles available online at http://www.idealibrary.com on destained bands were further processed in parallel using conventional recipe. In-gel digestion, preparation of sample probes and MALDI analysis. To prepare a standard mixture of 18 O-labeled peptides, a solution of 0.14 pmol/ L BSA in 25 mM ammonium bicarbonate buffer in H 2 18 O was digested overnight at 37 C; enzyme:substrate ratio 1:10 (w/w). Protein bands (three for each method of staining) were in parallel in-gel digested with trypsin (unmodified, sequencing grade, Roche Diagnostics GmbH, Germany) as described (13, 19). Gel pieces were extracted with 5% formic acid and acetonitrile and the extracts were dried down in a vacuum concentrator. Tryptic peptides were redissolved in 10 L of 10% formic acid. A 2- L aliquot was withdrawn and mixed with 1 L of an 18 O-labeled mixture of peptides (internal standard) prepared as described above. Four 0.5- L aliquots of every mixed sample were analyzed in parallel by MALDI MS as described in (13, 21) on a modified REFLEX III mass spectrometer (Bruker Daltonics, Germany). The determined relative concentrations of peptides were averaged. The relative standard deviation of the concentrations in all series of measurements was better than 20%. Results and Discussion Quantification of peptides in in-gel tryptic digests. Upon digesting of a protein in the buffer, which contains H 2 18 O, tryptic peptides incorporate one or two 18 O-atoms into their C-terminal carboxyl groups (22). Comparison of the peptide mass maps acquired from the digests of various standard proteins revealed that peptides, which contain arginine residue at their Cterminus incorporate two 18 O atoms (2 18 O peptides) mostly, whereas peptides having C-terminal lysine residue incorporate one 18 O atom (1 18 O peptides) (Fig. 1). Incubation of 2 18 O peptides in 10% formic acid in H 2 16 O at room temperature resulted in a mixture of unlabeled, 1 18 O and 2 18 O forms. However, this process was slow and required several days before substantial alteration of the isotopic profile was detected (data not shown). Isotopically labeled peptides produced by digesting of a protein in H 2 18 O could be employed as internal standards for quantitative measurements by MALDI MS. A standard mixture of 18 O-labeled peptides was prepared by digesting BSA with trypsin in solution in the buffer containing H 2 18 O. Very similar profiles of tryptic peptides were detected in MALDI peptide FIG. 1. A part of the spectrum of the tryptic digest of BSA in the buffer containing H 2 18 O. Peaks in the spectrum are designated with corresponding peptide sequences and m/z calculated for the unlabeled monoisotopic ions. Blowouts demonstrate isotopic profiles typical for the peptide ions having arginine or lysine residues at their C-termini. The positions of the corresponding monoisotopic unlabeled ions are designated with unfilled arrows. 280 NOTES & TIPS maps of in-solution digests and of in-gel digests, although the relative intensity of peptide peaks was altered. Equal volume of the mixture of 18 O-labeled peptides was spiked into the aliquots withdrawn from the experimental in-gel digests that were performed in H 2 16 O and the samples were analyzed by MALDI MS. Relative concentration of digestion products was calculated as a ratio of the intensity of the monoisotopic peak of the unlabeled peptide and the intensity of the monoisotopic peak of the corresponding 2 18 O peptide standard (Fig. 2). Linearity of the calibration curve was tested by analyzing the series of samples obtained by successive diluting of the aliquot withdrawn from the in-gel digest of 1 pmol of BSA. The relative concentrations calculated for various peptides were found linear over 1:5 dilution range and were affected by chemical noise at larger dilution ratios (data not shown). The effect of gel staining on the recovery of tryptic peptides. This was examined by analyzing in-gel digests of the bands containing 1 pmol of BSA, which were stained with Coomassie, silver, and zinc-imidazole. A similar profile of tryptic peptides was detected TABLE 1 Relative Concentration of Tryptic Peptides of BSA in In-Gel Digests of Bands Stained by Various Methods Staining method Relative concentration a (%) m/z 927.49 m/z 1439.81 m/z 1479.80 m/z 1567.74 m/z 1639.94 Silver, with reduction and alkylation 100 100 100 100 100 Coomassie 105 136 74 95 121 Zn/Imidazole 117 61 74 79 100 a Relative concentrations of peptides were normalized to the concentrations in the digests of silver-stained bands. FIG. 2. Calculation of the relative concentration of peptides. A blowout of the isotopic cluster of the peptide peak with m/z 1439.93. The monoisotopic peak of the unlabeled peptide is designated with a filled arrow. The peak of the isotopicaly labeled peptide, which incorporated two 18 O atoms (2 18 O) was used as an internal standard. The relative concentration of the unlabeled peptide (R c ) was calculated as R c I p /I st , where I p stands for the intensity of the peptide peak and I st stands for the intensity of the peak of the standard. 281 NOTES & TIPS in each of those samples (data not shown) and relative concentrations determined for five most intense peptide ions were compared. We observed slight variation of the relative concentration of individual peptides. However, no one method of staining provided significantly better recovery of peptides compared to other methods in the test (Table 1). We further tested whether the recovery of peptides from silver stained gels could be improved by destaining of protein bands prior to in-gel digestion (15). To this end we compared the relative concentrations of peptides in the in-gel digests of the destained bands and of the bands treated according to the conventional protocol. We observed no significant increase in the number of detected peptide peaks as well as in the yield of peptides if destaining of bands was applied (Table 2). Similar conclusion was reached by Moertz et al. (12) on the basis of MALDI analysis of a large number of automatically processed samples. Reduction and alkylation steps did not influence the recovery of peptides, which do not contain cysteine residues and therefore for the purpose of protein identification those steps could, in principle, be omitted (23). Notably cysteine-containing peptides were missing if reduction and alkylation steps were skipped (Fig. 3). We therefore concluded that at the level of 1 pmol of protein starting material the method of protein visualization FIG. 3. Comparison of the peptide maps of the silver stained bands processed using destaining, reduction, and alkylation (the upper spectrum) and using only destaining (the lower spectrum). Peaks are designated with corresponding m/z; peptide sequences are presented in Fig. 1. Three intense peptide peaks (designated with unfilled arrows) having matching cysteine-containig peptides from BSA were additionally detected after reduction and alkylation. Corresponding peptide sequences are: m/z 1419.66 SLHTLFGDELCK; m/z 1539.79 LCVLHEKTPVSEK; m/z 1880.91 RPCFSALTPDETYVPK. C stands for cysteine-S-acetamide residue. TABLE 2 Relative Concentration of Peptides Recovered from Silver-Stained Gels Sample preparation method Relative concentration a (%) m/z 927.49 m/z 1439.81 m/z 1479.80 m/z 1567.74 m/z 1639.94 With destaining, reduction and alkylation 100 100 100 100 100 With destaining only 95 110 93 97 98 With reduction and alkylation 115 104 102 83 94 Untreated b 5 12 14 6 19 a Relative concentrations of peptides were normalized to the concentrations in the digests of destained, reduced and alkylated bands. b Predigestion washing, destaining, reduction and alkylation were skipped. 282 NOTES & TIPS does not have any noticeable impact on the recovery of tryptic peptides. We also observed that relative concentration of all peptides in the digests of silver-stained bands, which were directly treated with trypsin (i.e. washing steps as well as destaining, reduction and alkylation were omitted), was dramatically lower. Nevertheless, the number of detected peptide was always sufficient for unambiguous identification of BSA upon searching a database. We therefore speculate that the sequence coverage of MALDI peptide maps alone does not constitute an adequate measure of the digestion efficiency and should be complemented by direct quantification of peptide products. Thus we have demonstrated that application of isotopically labeled peptide standards and MALDI MS enabled direct and quantitative evaluation of the efficiency of in-gel digestion. The method paves the way for further optimization of sample processing routines, thus improving sensitivity and throughput of the characterization of proteomes by mass spectrometry. Acknowledgments. The authors are grateful for members of Protein and Peptide Group for experimental support and useful discussions. REFERENCES 1. Lahm, H. W., and Langen, H. (2000) Electrophoresis 21, 2105 2114. 2. Pandey, A., and Mann, M. (2000) Nature 405, 837 846. 3. Andersen, J. S., and Mann, M. (2000) FEBS Lett. 480, 25 31. 4. Anderson, N. L., Matheson, A. D., and Steiner, S. (2000) Curr. Opin. Biotechnol. 11, 408 412. 5. Rabilloud, T. (2000) Anal Chem. 72, 48A 55A. 6. Patterson, S. D., and Aebersold, R. (1995) Electrophoresis 16, 791 814. 7. Rabilloud, T. (1990) Electrophoresis 11, 785 794. 8. Lopez, M. F., Berggren, K., Chernokalskaya, E., Lazarev, A., Robinson, M., and Patton, W. F. (2000) Electrophoresis 21, 3673 3683. 9. Yan, J. X., Wait, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H., and Dunn, M. J. (2000) Electrophoresis 21, 3666 3672. 10. Lauber, W. M., Carrol, J. A., Dunfield, D. R., Kiesel, J. R., Radabaugh, M. R., and Malone, J. P. (2001) Electrophoresis 22, 906 918. 11. Jensen, O. N., Mortensen, P., Vorm, O., and Mann, M. (1997) Anal. Chem. 69, 1706 1714. 12. Moertz, E., Krogh, T. N., Vorum, H., and Go rg, A. (2000) Proceedings, 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach CA, pp. 1115 1116. 13. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850 858. 14. Scheler, C., Lamer, S., Pan, Z., Li, X. P., Salnikow, J., and Jungblut, P. (1998) Electrophoresis 19, 918 27. 15. Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Electrophoresis 20, 601 605. 16. Sumner, L. W., White, S., Wolf-Sumner, B., and Asirvatham, V. S. (2001) Abstracts 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago IL. 17. Shevchenko, A., Wilm, M., and Shevchenko, A. (2000) Proceedings, 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach CA, pp. 859 860. 18. Mirgorodskaya, O. A., Kozmin, Y. P., Titov, M. I., Korner, R., Sonksen, C. P., and Roepstorff, P. (2000) Rapid Commun. Mass Spectrom. 14, 1226 1232. 19. Shevchenko, A., Chernushevich, I., Wilm, M., and Mann, M. (2000) in Protein in Peptide Analysis (Chapman, J. R., Ed.), Vol. 146, pp. 1 16, Humana Press, Totowa, NJ. 20. Fernandez-Patron, C., Calero, M., Collazo, P. R., Garcia, J. R., Madrazo, J., Musacchio, A., Soriano, F., Estrada, R., Frank, R., and Castellanos-Serra, L. R. (1995) Anal. Biochem. 224, 203 211. 21. Jensen, O. N., Podtelejnikov, A., and Mann, M. (1996) Rapid Commun. Mass Spectrom. 10, 1371 1378. 22. Schno lzer, M., Jedrzejewski, P., and Lehmann, W. D. (1996) Electrophoresis 17, 945 953. 23. Borchers, C., Peter, J. F., Hall, M. C., Kunkel, T. A., and Tomer, K. B. (2000) Anal. Chem. 72, 1163 1168. Reutilization of Immunoblots after Chemiluminescent Detection Scott H. Kaufmann Division of Oncology Research, Mayo Clinic, and Department of Molecular Pharmacology, Mayo Graduate School, Rochester, Minnesota 55905 Received March 9, 2001; published online August 16, 2001 Immunoblotting is widely utilized to evaluate the presence of antigens of interest in various biological samples, monitor antigen purification, assess epitope retention after antigen degradation, or assay for the presence of antibodies of a particular specificity in biological fluids [reviewed in Refs. (1 4)]. Under certain circumstances, e.g., if a blot suggests an unexpected difference in antigen expression between two samples or the antigens being analyzed are derived from a precious source, it can be important to sequentially probe the same blot for the presence of multiple antigens. The present study demonstrates that treatment with sodium azide after detection of bound HRP 1 -coupled secondary antibodies results in inhibition of the reporter group, thereby facilitating sequential probing of blots if reagents raised in multiple species are available. A number of approaches have been previously proposed for the detection of multiple antigens on immu-1 Abbreviations used: HRP, horseradish peroxidase; PBS, calciumand magnesium-free phosphate-buffered saline. 283 NOTES & TIPS Analytical Biochemistry 296, 283 286 (2001) doi:10.1006/abio.2001.5313 0003-2697/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

Florian Raible, Michael Brand
Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development.
Mech Dev, 107(1-2) 105-117 (2001)
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Several molecules of the Fibroblast growth factor family have been implicated in the development of the vertebrate brain, but the effectors of these molecules remain largely unknown. Here we study Erm and Pea3, two ETS domain transcription factors, and show that their expression correlates closely with the domains of fgf8 and fgf3 expression. In situ hybridization analysis in wild-type and acerebellar (ace) mutant embryos defective for fgf8 demonstrates a requirement of Fgf8 for normal expression levels of erm and pea3 transcripts in and close to various domains of Fgf8 action, including the prospective midbrain-hindbrain region, the somites, the neural crest, the forebrain, and developing eyes. Morpholino-oligomer-assisted gene knock-down experiments targeted against fgf8 and fgf3 suggest that Fgf3 and Fgf8 are co-regulators of these genes in the early forebrain anlage. Furthermore, inhibition of Fgf signaling by overexpression of sprouty4 or application of the Fgf inhibitor SU5402 leads to a loss of all erm and pea3 expression domains. Conversely, ectopically provided fgf3 mRNA or implanted beads coated with Fgf8 elicit ectopic transcription of erm and pea3. Both activation and loss of transcripts can be observed within short time frames. We conclude that both the transcriptional onset and maintenance of these factors are tightly coupled to Fgf signaling and propose that erm and pea3 transcription is a direct readout of cells to Fgf levels. Given the knowledge that has accumulated on the posttranslational control of ETS domain factors and their combinatorial interactions with other transcription factors, we suggest that the close coupling of erm and pea3 transcription to Fgf signaling might serve to integrate Fgf signaling with other signals to establish refined patterns in embryonic development.

B Berkhout, Nadine Vastenhouw, B I Klasens, H Huthoff
Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription.
RNA, 7(8) 1097-1114 (2001)

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.

Karen Echeverri, Jon D W Clarke, Elly M. Tanaka
In vivo imaging indicates muscle fiber dedifferentiation is a major contributor to the regenerating tail blastema.
Dev Biol, 236(1) 151-164 (2001)
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During tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema.

Fabian Feiguin, Michael Hannus, Marek Mlodzik, Suzanne Eaton
The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization.
Dev Cell, 1(1) 93-101 (2001)
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During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. This work identifies a tissue polarity gene, diego, that comprises six ankyrin repeats and colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. Our data suggest that Frizzled acts through Diego to promote local clustering of Flamingo, and that clustering of Diego and Flamingo in one cell nonautonomously propagates to others.

Isato Araki, Michael Brand
Morpholino-induced knockdown of fgf8 efficiently phenocopies the acerebellar (ace) phenotype.
Genesis, 30(3) 157-159 (2001)
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Morpholino-Induced Knockdown of fgf8 Efficiently Phenocopies the Acerebellar (Ace) Phenotype Isato Araki and Michael Brand* Max-Planck-Institute for Molecular Cell Biology and Genetics, Dresden, Germany Received 1 June 2001; Accepted 18 June 2001 Homozygous acerebellar (ace) embryos lack their cerebellum and the midbrain hindbrain boundary (MHB) organizer, and in addition have defects in forebrain and heart development (Brand et al., 1996; Picker et al., 1999; Reifers et al., 1998, Shanmugalingam et al. (2000); Raible and Brand, 2001; Araki and Brand, unpublished data), suggesting that this may be because of functional redundancy between Fgfs. Because morpholinos might help to resolve such issues, we sought to phenocopy the known defects caused by absence of Ace/Fgf8 through injection of a morpholino against fgf8 (MO-fgf8). In spite of the complexity of the ace phenotype, we find that MO-fgf8 efficiently and uniformly phenocopies the ace mutant in MHB, forebrain, and heart development. We designed an antisense morpholino against fgf8, covering the translational start codon (Fig. 1J). After injections into one- to eight-cell-stage wild-type embryos, we find that MO-fgf8 effectively phenocopies the ace phenotype. We used between 0.5 and 4 g/ l of MOfgf8 for the injection (Table 1), which delivers between 1.6 and 12.6 ng of MO-fgf8 per embryo. Morphologically, embryos at the 24-h stage injected with 1 4 g/ l lacked the cerebellum and the MHB organizer, as do ace mutants at the same stage (Fig. 1A I). A morpholino with four mismatched base pairs against fgf8 (control MO in Fig. 1J) had no effect, nor did it show any nonspecific effects at the same concentration (Table 1). To examine how closely the ace phenotype is mimicked, we stained the injected embryos with a probe for pax2.1 which reveals several of the tissues where Fgf8 functions. pax2.1 expression at the MHB is initially normal in ace mutants, but is not properly maintained (Reifers et al., 1998; Lun and Brand, 1998). At 24 h, MO-fgf8 injected embryos either lacked pax2.1 expression at the MHB completely, or it was reduced to a small dorsal patch, as seen in ace mutants at the same stage (Fig. 1D I and not shown; Reifers et al., 1998). Because ace mutants show defects also in forebrain and heart development, we analyzed with molecular markers whether MO-fgf8 injection can phenocopy the ace mutant also in these tissues. Ten hours after fertilization, injected embryos have reduced and perturbed expression of emx1, an early telencephalic marker, as in ace mutants (Fig. 1K M; Shanmugalingam et al., 2000). Similarly, at 24 h expression of pax2.1 in the optic stalk is reduced, and in the optic chiasm is reduced or missing (not shown), as found previously for ace mutants (Shanmugalingam et al., 2000). Injected embryos stained with nkx2.5, an early marker for heart primordium, showed downregulation of nkx2.5, as is observed in ace mutants (Fig. 1N P; Reifers et al., 2000a). We did not detect any nonspecific effect of MO-fgf8. We conclude that MO-fgf8 injection efficiently phenocopies the known loss-of-function phenotype of acerebellar in MHB, telencephalon, optic stalk, and heart development, thus validating the usefulness of this method. Because morpholino injection is thought to prevent translation (which we assume to be true, but have not tested, for fgf8), these findings also support (Reifers et al., 1998) that ace is a null allele. To further test this notion, we examined whether the phenotype of homozygous acerebellar mutants can be enhanced by Mo-fgf8 injection. Morphologically and after examining pax2.1 expression in injected embryos at the tailbud, two-somite, five-somite, and 24-h stage, we did not observe a difference between ace homozygotes and their wild-type siblings in the same clutch (Table 1 and not shown), showing that the ace phenotype cannot be further enhanced and therefore most likely represents the null phenotype. Morpholinos against other fgf mR-NAs alone or in combination with Mo-fgf8 will help to resolve the problem of redundancy that is often found with signaling by Fgfs. ACKNOWLEDGMENTS We thank Florian Raible and Steffen Scholpp for helpful comments, and Muriel Rhinn for technical advice. I.A. is supported by the Alexander von Humboldt Stiftung. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the EU Biotech Program, and the Max-Planck Society to M.B. LITERATURE CITED Brand M, Heisenberg C-P, Jiang Y-J, Beuchle D, Lun K, van Eeden FJM, Furutani-Seiki M, Granato M, Haffter P, Hammerschmidt M, Kane Contract grant sponsors: Alexander von Humboldt Stiftung, Deutsche Forschungsgemeinschaft, EU Biotech Program, and Max-Planck Society. * Correspondence to: Michael Brand, Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr 108, D-01307 Dresden, Germany. E-mail: brand@mpi-cbg.de 2001 Wiley-Liss, Inc. genesis 30:157 159 (2001) FIG. 1. Injection of a morpholino against fgf8 phenocopies the ace phenotype. (A K) MO-fgf8 phenocopied ace phenotype in the midbrain hindbrain boundary (MHB). Injected embryos or ace embryos lack the structure of MHB (arrowheads in A), the conspicuous constriction at MHB (compare arrowheads in G and H/I), and pax2.1 expression at MHB. The embryos were injected with MO-fgf8 at 2 g/ l in this figure. The embryos in A, D, and G are wild type; the embryos in B, E, and H are ace mutants. The embryos in D I were stained with probe for pax2.1 mRNA. The pictures in A F are lateral view, whereas the ones in G I are dorsal view. In A I, embryos were fixed at 24 h after fertilization. (J) The nucleotide/amino acid sequence around the initiation codon (underlined) of fgf8 cDNA and the sequences of the morpholino against fgf8 (MO-fgf8) and its negative control (control MO) are shown. The control-MO contains four single nucleotide exchanges (underlined) that are predicted to lead to a strongly reduced target binding (Gene Tools, LLC, Corvallis, OR). (K P) MO-fgf8 phenocopied ace phenotype also in other tissues. The embryos in K and N are wild type; the embryos in L and O are ace mutant; the embryos in M and P are MO-fgf8-injected. The embryos were stained with an emx1 probe and a nkx2.5 probe in K M and N P, respectively. The pictures in K P are dorsal views. Embryos in K M were fixed at 10 h after fertilization; at 12 h in N P. All embryos in this figure are oriented with rostral to the left. OS, optic stalk; OV, otic vesicle; TC, tectum. Methods: Crystallized morpholino oligos (Gene Tools, LLC) were dissolved in water at 40 g/ l, and this stock solution was stored at 20 C. The working solution consisted of 5 mM HEPES (pH 7.5), 0.1% phenol red and the appropriate concentration of morpholinos. Injection was performed between one-cell to eight-cell stage. The injected/control embryos were raised at 28 C. Table 1 Dose Response to a Morpholino Against Fgf8 Morpholino Concentration ( g/ l) Dose (ng) n pax2.1 staining at MHB Absent Reduced Normal MO-Fgf8 0.5 1.6 30 20 (67%) 9 (30%) 1 3.1 103 50 (49%) 53 (51%) 0 2 6.3 101 60 (59%) 41 (41%) 0 4 12.6 24 10 (42%) 14 (58%) 0 4 12.6 7 7 (100%) 0 0 Injection into ace/ace homozygotes 2 6.3 63 58 (93%) 5 (8%) 0 Control MO 2 6.3 22 0 0 22 (100%) 4 12.6 19 0 0 19 (100%) Mock 0 0 23 0 0 23 (100%) The readout was in situ hybridizations with pax2.1 probe at 24 h after fertilization. 158 ARAKI AND BRAND DA, Kelsh RN, Mullins MC, Odenthal J, Nu sslein-Volhard C. 1996. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123:179 190. Lun K, Brand M. 1998. A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125: 3049 3062. Picker A, Brennan C, Reifers F, Clarke JD, Holder N, Brand M. 1999. Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection. Development 126:2967 2978. Raible F, Brand M. 2001. Tight transcriptional control of the ETS domain factors Erm and Pea3 by FGF signaling during early zebrafish nervous system development. Mech Dev (in press). Reifers F, Bo hli H, Walsh EC, Crossley PH, Stainier DYR, Brand M. 1998. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125:2381 2395. Reifers F, Walsh EC, Le ger S, Stainier DYR, Brand M. 2000a. Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar). Development 127:225 235. Reifers F, Adams J, Mason IJ, Schulte-Merker S, Brand M. 2000b. Overlapping and distinct functions provided by Fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs. Mech Dev 99:39 49. Shanmugalingam S, Houart C, Picker A, Reifers F, Macdonald R, Barth A, Griffin K, Brand M, Wilson SW. 2000. Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon. Development 127:2549 2561. 159 KNOCKDOWN OF FGF8 PHENOCOPIES ACE PHENOTYPE

Sandrine Uttenweiler-Joseph, Gitte Neubauer, Savvas Christoforidis, Marino Zerial, Matthias Wilm
Automated de novo sequencing of proteins using the differential scanning technique.
Proteomics, 1(5) 668-682 (2001)
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Despite the progress in genomic DNA sequencing de novo sequencing of peptides is still required in a biological research environment since many experiments are done in organisms whose genomes are not sequenced. A way to unambiguously retrieve a peptide sequence from a tandem mass spectrum is to assign the correct ion type to the fragments. Here we describe a method which improves the specificity in y-ion assignment throughout the spectrum. The differential scanning technique requires that the peptides are partially 18O labelled at their C-terminus and that two fragment spectra are acquired for each peptide, one selecting the 16O/18O isotopic cluster and a second fragmenting only the 18O labelled ions. When the spectra are acquired with a quadrupole time of flight mass spectrometer y-ions can be very specifically filtered from the spectrum using a computer algorithm. Partial or complete peptide sequences can be assigned automatically simply by finding the most abundant series of fragments spaced by amino acid residue masses. This method was used extensively in a project investigating vesicular transport in bovine brain cells. Human or mouse homologues to the bovine proteins were found in EST databases facilitating rapid cloning of the human homologues.

Sari Lusa^*, Titta S. Blom^*, Eeva-Liisa Eskelinen, Esa Kuismanen, Jan-Eric Månsson, Kai Simons, Elina Ikonen
Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.
J Cell Sci, 114(Pt 10) 1893-1900 (2001)
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In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Andrej Shevchenko, Shamil Sunyaev, Alexander Loboda, Anna Shevchenko, Peer Bork, Werner Ens, Kenneth G. Standing
Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching.
Anal Chem, 73(9) 1917-1926 (2001)
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MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown. The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra. A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions. This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.

Frank Lafont, Kai Simons
Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling.
Proc Natl Acad Sci U.S.A., 98(6) 3180-3184 (2001)
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The high affinity receptor for IgE, FcepsilonRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcepsilonRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcepsilonRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcepsilonRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcepsilonRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcepsilonRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcepsilonRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

J C Venter, M D Adams, E W Myers, P W Li, Richard J Mural, Granger G Sutton, H O Smith, Mark D Yandell, Cheryl A Evans, Robert A Holt, Jeannine D Gocayne, Peter G Amanatides, R M Ballew, Daniel H. Huson, Jennifer R Wortman, Qinyu Zhang, Chinnappa Kodira, X H Zheng, L Chen, M Skupski, G Subramanian, P D Thomas, J Zhang, George L Gabor Miklos, C Nelson, Samuel Broder, Andrew Clark, Joe Nadeau, V A McKusick, N Zinder, A J Levine, R J Roberts, M Simon, C Slayman, Michael W Hunkapiller, R A Bolanos, A L Delcher, I M Dew, D P Fasulo, M J Flanigan, Liliana Florea, Aaron L. Halpern, Sridhar Hannenhalli, S A Kravitz, S Levy, C M Mobarry, Knut Reinert, K A Remington, J Abu-Threideh, E M Beasley, Kendra Biddick, V Bonazzi, R C Brandon, M Cargill, I Chandramouliswaran, Rosane Charlab, Kabir Chaturvedi, Zuoming Deng, V Di Francesco, P Dunn, K Eilbeck, Carlos C Evangelista, Andrei E Gabrielian, Weiniu Gan, W Ge, F Gong, Z Gu, Ping Guan, T J Heiman, M E Higgins, Rui-Ru Ji, Z Ke, K A Ketchum, Z Lai, Y Lei, Z Li, J Li, Y Liang, X Lin, F Lu, Gennady V Merkulov, Natalia V Milshina, H M Moore, Ashwinikumar K Naik, V A Narayan, B Neelam, D R Nusskern, Douglas B Rusch, Steven L Salzberg, W Shao, Bixiong Chris Shue, J Sun, Z Wang, A Wang, X Wang, J Wang, M Wei, Ron Wides, C Xiao, C Yan, Alison Yao, Jane Ye, M Zhan, W Zhang, H Zhang, Q Zhao, L Zheng, F Zhong, W Zhong, S Zhu, Senming Zhao, D Gilbert, S Baumhueter, G Spier, Crystal N. Carter, Anibal Cravchik, T Woodage, F Ali, Hui-Jin An, A Awe, Danita Baldwin, Holly Baden, Mary Barnstead, I Barrow, Karen Y Beeson, D Busam, Amy Carver, Angela Center, M L Cheng, L Curry, Steven Danaher, Lionel B Davenport, Raymond Desilets, Susanne M Dietz, Kristina L Dodson, Lisa Doup, Steven Ferriera, N Garg, Andres Gluecksmann, Brit Hart, J Haynes, C Haynes, C Heiner, Susanne L Hladun, D Hostin, Jarrett T Houck, Tim Howland, Chinyere Ibegwam, J Johnson, Francis Kalush, Leslie Kline, S Koduru, A Love, F Mann, David May, S McCawley, T McIntosh, I McMullen, M Moy, L Moy, B Murphy, K Nelson, Cynthia Pfannkoch, Eric C Pratts, V Puri, Hina Qureshi, Matthew S Reardon, R Rodriguez, Y H Rogers, Deanna L Romblad, B Ruhfel, R Scott, Cynthia D Sitter, Michella Smallwood, E Stewart, Renee Strong, E Suh, R Thomas, Ni Ni Tint, S Tse, C Vech, G Wang, J Wetter, S Williams, M Williams, Sandra M Windsor, E Winn-Deen, Keriellen Wolfe, Jayshree Zaveri, K Zaveri, Josep F Abril, R Guigó, M J Campbell, K V Sjolander, B Karlak, A Kejariwal, H Mi, B Lazareva, T Hatton, A Narechania, K Diemer, A Muruganujan, N Guo, S Sato, V Bafna, Sorin Istrail, Ross Lippert, R Schwartz, Brian Walenz, Shibu Yooseph, D Allen, A Basu, J Baxendale, L Blick, M Caminha, J Carnes-Stine, Parris M Caulk, Y H Chiang, My D Coyne, Carl E Dahlke, A Mays, M Dombroski, Michael T Donnelly, D Ely, S Esparham, Carl R Fosler, Harold C Gire, S Glanowski, K Glasser, Anna Glodek, M Gorokhov, K Graham, Barry Gropman, M Harris, J Heil, S Henderson, Jeffrey Hoover, D Jennings, C M Jordan, J Jordan, J Kasha, L Kagan, C Kraft, Alex Levitsky, M Lewis, X Liu, J Lopez, D Ma, William H Majoros, J McDaniel, S Murphy, Matthew H Newman, T Nguyen, N Nguyen, Marc Nodell, S Pan, J Peck, M Peterson, W Rowe, Robert Sanders, J Scott, M Simpson, T Smith, Andrew Sprague, T Stockwell, R Turner, E Venter, M Wang, M Wen, D Wu, M Wu, Ashley C Xia, A Zandieh, X Zhu
The sequence of the human genome.
Science, 291(5507) 1304-1351 (2001)
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A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Muriel Rhinn, Michael Brand
The midbrain--hindbrain boundary organizer.
Curr Opin Neurobiol, 11(1) 34-42 (2001)
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Cell fate in the cephalic neural primordium is controlled by an organizer located at the midbrain-hindbrain boundary. Studies in chick, mouse and zebrafish converge to show that mutually repressive interactions between homeodomain transcription factors of the Otx and Gbx class position this organizer in the neural primordium. Once positioned, independent signaling pathways converge in their activity to drive organizer function. Fibroblast growth factors secreted from the organizer are necessary for, and sufficient to mimic, organizer activity in patterning the midbrain and anterior hindbrain, and are tightly controlled by feedback inhibition.

Carsten Janke, Jennifer Ortiz, Johannes Lechner, Anna Shevchenko, Andrej Shevchenko, Maria M. Magiera, C Schramm, E Schiebel
The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control.
EMBO J, 20(4) 777-791 (2001)
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Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

Stephan W. Grill, Pierre Gönczy, Ernst H K Stelzer, Anthony A. Hyman
Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo.
Nature, 409(6820) 630-633 (2001)
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Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.

Sibylle D. Müller, Ivo F. Sbalzarini, Jens H. Walther, Petros Koumoutsakos
Evolution Strategies for the Optimization of Microdevices
In: Proceedings of the Congress on Evolutionary Computation (CEC 2001) (2001), Piscataway, N.J., IEEE (2001), 302-309
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Jonathon Howard
Mechanics of Motor Proteins and the Cytoskeleton
Sunderland, USA, Sinauer Associates (2001), 384 S.
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Torsten Wittmann, Anthony A. Hyman, Arshad Desai
The spindle: a dynamic assembly of microtubules and motors.
Nat Cell Biol, 3(1) 28-34 (2001)
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In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.

Daniel J. Müller, Norbert A. Dencher, Thomas Meier, Peter Dimroth, Kitaru Suda, Henning Stahlberg, Andreas Engel, Holger Seelert, Ulrich Matthey
ATP synthase: constrained stoichiometry of the transmembrane rotor
FEBS Lett, 504(3) 219-222 (2001)
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Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.

Daniel H. Huson, Knut Reinert, S A Kravitz, K A Remington, A L Delcher, I M Dew, M J Flanigan, Aaron L. Halpern, Z Lai, C M Mobarry, Granger G Sutton, E W Myers
Design of a compartmentalized shotgun assembler for the human genome.
Bioinformatics, 17 Suppl 1 132-139 (2001)
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Two different strategies for determining the human genome are currently being pursued: one is the "clone-by-clone" approach, employed by the publicly funded project, and the other is the "whole genome shotgun assembler" approach, favored by researchers at Celera Genomics. An interim strategy employed at Celera, called compartmentalized shotgun assembly, makes use of preliminary data produced by both approaches. In this paper we describe the design, implementation and operation of the "compartmentalized shotgun assembler".

Harald Stenmark, Marino Zerial
Molecular mechanisms of membrane fusion in the endocytic pathway
In: Endocytosis. (Eds.) Mark Marsh Frontiers in molecular biology ; 36., Oxford, UK, Oxford University Press (2001), 94-110 Ch. 5
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Pierre Gönczy, Stephan W. Grill, Ernst H K Stelzer, Matthew Kirkham, Anthony A. Hyman
Spindle positioning during the asymmetric first cell division of Caenorhabditis elegans embryos.
Novartis Found Symp, 237 164-175 (2001)
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Cell division during development in many cases generates daughter cells that differ not only in fate, but also in size. We investigate the mechanisms that ensure proper spindle positioning during such asymmetric divisions using the one-cell stage Caenorhabditis elegans embryo as a model system. We utilized a UV laser microbeam as an in vivo microtubule-severing device to probe the forces driving spindle positioning. Our results indicate that extra-spindle pulling forces acting on the spindle poles dictate spindle position along the anterior-posterior embryonic axis. Importantly, forces acting on the posterior spindle pole appear more extensive than those acting on the anterior one, thus explaining the overall posterior spindle displacement that leads to the asymmetric division of the wild-type one-cell stage embryo. In separate work, we analysed a locus called zyg-8, which plays a key role in ensuring proper spindle positioning. Our data show that zyg-8 is required to promote microtubule growth and/or stability during anaphase. We identified the molecular nature of the zyg-8 locus in the course of a large-scale RNAi-based functional genomics screen. ZYG-8 harbours two notable protein domains: a Ca2+/calmodulin-dependent kinase domain, and a domain related to doublecortin, a human microtubule-associated protein involved in neuronal migration.

K Ladjali-Mohammedi, Anne Grapin-Botton, M A Bonnin, N M Le Douarin
Distribution of HOX genes in the chicken genome reveals a new segment of conservation between human and chicken.
Cytogenet. Cell Genet., 92(1-2) 157-161 (2001)
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Homeobox genes play an important role in the regulation of early embryonic development. They represent a family of evolutionarily highly conserved transcription factors. In this work, several genes that belong to the four HOX gene clusters are assigned by in situ hybridization to four distinct chicken chromosomes. The four gene clusters are mapped to 2p2.1 (HOXA), 3q3.1 (HOXB), 1q3.1 (HOXC) and 7q1.3--> q1.4 (HOXD). We confirm partial homologies already detected by genetic mapping between chicken chromosomes 1, 2 and 7 and human chromosomes 12, 7 and 2 and we describe a new conserved segment between chicken chromosome 3 and human chromosome 17. These results represent the first data that confirm the physical linkage between chicken HOX genes and may improve our understanding of phylogenetic relationships and genome evolution.

Jürg Zumbrunn, Kazuhisa Kinoshita, Anthony A. Hyman, Inke S. Näthke
Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation.
Curr Biol, 11(1) 44-49 (2001)
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Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

Eugeni V. Entchev, Anja Schwabedissen, Marcos González-Gaitán
Gradient formation of the TGF-beta homolog Dpp.
Cell, 103(6) 981-991 (2000)
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Secreted morphogens such as the Drosophila TGF-beta homolog Decapentaplegic (Dpp) are thought to spread through target tissues and form long-range concentration gradients providing positional information. Using a GFP-Dpp fusion, we monitored a TGF-beta family member trafficking in situ throughout the target tissue and forming a long-range concentration gradient. Evidence is presented that long-range Dpp movement involves Dpp receptor and Dynamin functions. We also show that the rates of endocytic trafficking and degradation determine Dpp signaling range. We propose a model where the gradient is formed via intracellular trafficking initiated by receptor-mediated endocytosis of the ligand in receiving cells with the gradient slope controlled by endocytic sorting of Dpp toward recycling versus degradation.

Yanzhuang Wang, Christoph Thiele, Wieland B. Huttner
Cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-Golgi network.
Traffic, 1(12) 952-962 (2000)
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We studied the role of cholesterol in regulated protein secretion in neuroendocrine cells by manipulating the cholesterol content of AtT-20 cells. Depletion of cellular cholesterol levels caused a reversible block of immature secretory granule biogenesis at the level of the trans-Golgi-network, whereas increased cholesterol levels promoted immature secretory granule formation. Cholesterol depletion also blocked the formation of constitutive secretory vesicles, but did not inhibit the transport between the endoplasmic reticulum and the Golgi complex. Our results indicate that the assembly of cholesterol-based lipid microdomains is required for the biogenesis of both regulated and constitutive secretory vesicles from the trans-Golgi-network in neuroendocrine cells.

Frank Reifers, Jan Adams, Ivor J. Mason, Stefan Schulte-Merker, Michael Brand
Overlapping and distinct functions provided by fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs.
Mech Dev, 99(1-2) 39-49 (2000)
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Members of the fibroblast growth factor (Fgf) family are important signaling molecules in several inductive and patterning processes, and act as brain organizer-derived signals during formation of the early vertebrate nervous system. We isolated a new member of the Fgf8/17/18 subgroup of Fgfs from the zebrafish, and studied its expression and function during somitogenesis, optic stalk and midbrain-hindbrain boundary (MHB) development. In spite of a slightly higher aminoacid similarity to Fgf8, expression analysis and mapping to a chromosome stretch that is syntenic with mammalian chromosomes shows that this gene is orthologous to mammalian Fgf17. These data provide a further example of conserved chromosomal organization between zebrafish and mammalian genomes. Using an mRNA injection assay, we show that fgf17 can act similar to fgf8 during gastrulation, when fgf17 is not normally expressed. Direct comparison of the expression patterns of fgf17 and fgf8 suggest however a possible cooperation of these Fgfs at later stages in several tissues requiring Fgf signaling. Analysis of zebrafish MHB mutants demonstrates a gene-dosage dependent requirement of fgf17 expression for the no isthmus// pax2.1 gene, showing that no isthmus/pax2.1 functions upstream of fgf17 at the MHB in a haplo-insufficient manner, similar to what has been reported for mammalian pax2 mutants. In contrast, only maintenance of fgf17 expression is disturbed at the MHB of acerebellar/fgf8 mutants. Consistent with a requirement for fgf8 function, implantation of FGF8-soaked beads induces fgf17 expression, and expression is upregulated in aussicht mutants, which display upregulation of the Fgf8 signaling pathway. Taken together, our results argue that Fgf8 and Fgf17 act as hierarchically organized signaling molecules during development of the MHB organizer and possibly other organizers in the developing nervous system.

Kai Simons, Jean Gruenberg
Jamming the endosomal system: lipid rafts and lysosomal storage diseases.
Trends Cell Biol, 10(11) 459-462 (2000)
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Some lysosomal storage diseases result from the accumulation of lipids in degradative compartments of the endocytic pathway. Particularly striking is the example of the Niemann-Pick (NP) syndrome. NP syndromes types A and B are characterized by the accumulation of sphingomyelin, whereas cholesterol typically accumulates in NP type C. These two different lipids, sphingomyelin and cholesterol, are normal constituents of specific lipid microdomains called rafts. Because accumulation of raft lipids is observed not only in NP diseases but also in many other lipidoses, we forward the hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes.

Letizia Lanzetti, Vladimir Rybin, Maria Grazia Malabarba, Savvas Christoforidis, Giorgio Scita, Marino Zerial, Pier Paolo Di Fiore
The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5.
Nature, 408(6810) 374-377 (2000)
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How epidermal growth factor receptor (EGFR) signalling is linked to EGFR trafficking is largely unknown. Signalling and trafficking involve small GTPases of the Rho and Rab families, respectively. But it remains unknown whether the signalling relying on these two classes of GTPases is integrated, and, if it is, what molecular machinery is involved. Here we report that the protein Eps8 connects these signalling pathways. Eps8 is a substrate of the EGFR, which is held in a complex with Sos1 by the adaptor protein E3bl (ref. 2), thereby mediating activation of Rac. Through its src homology-3 domain, Eps8 interacts with RN-tre. We show that RN-tre is a Rab5 GTPase-activating protein, whose activity is regulated by the EGFR. By entering in a complex with Eps8, RN-tre acts on Rab5 and inhibits internalization of the EGFR. Furthermore, RN-tre diverts Eps8 from its Rac-activating function, resulting in the attenuation of Rac signalling. Thus, depending on its state of association with E3b1 or RN-tre, Eps8 participates in both EGFR signalling through Rac, and trafficking through Rab5.

Kai Simons
A voice for European life scientists. An interview with Kai Simons, President of the European Life Science Organisation and Director of the Max Planck Institute for Molecular Cell Biology and Genetics in Dresden.
EMBO Rep, 1(5) 381-383 (2000)
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Sanna Lehtonen, Ari Ora, Vesa M Olkkonen, Lin Geng, Marino Zerial, Stefan Somlo, Eero Lehtonen
In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2.
J Biol Chem, 275(42) 32888-32893 (2000)
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We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.

Martin Klemke, H. Amalia Pasolli, Ralph H. Kehlenbach, Stefan Offermanns, Günter Schultz, Wieland B. Huttner
Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties.
J Biol Chem, 275(43) 33633-33640 (2000)
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In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.

Marcos González-Gaitán, Herbert Jäckle
Tip cell-derived RTK signaling initiates cell movements in the Drosophila stomatogastric nervous system anlage.
EMBO Rep, 1(4) 366-371 (2000)
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The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

Maja Adamska, Sophie Léger, Michael Brand, Thorsten Hadrys, Thomas Braun, Eva Bober
Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace.
Mech Dev, 97(1-2) 161-165 (2000)
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An orthologue of the mouse homeobox gene Nkx5-1 was cloned and characterized in the zebrafish. As in the mouse and chick, the zebrafish Nkx5-1 gene is expressed in the ear placode and vesicle and in cells forming the vestibulo-acoustic ganglion. In addition, a novel expression domain, the lateral line, appears in the zebrafish, supporting a common precursor hypothesis for these two organs. In the FGF8 zebrafish mutant ace, expression of Nkx5-1 in the otic structures is diminished. The most significant reduction of zfNkx5-1 expression was observed in cells of the vestibulo-acoustic ganglion.

Andreas Engel, Daniel J. Müller
Observing single biomolecules at work with the atomic force microscope.
Nat Struct Biol, 7(9) 715-718 (2000)
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Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.

Katja Röper, Denis Corbeil, Wieland B. Huttner
Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane.
Nat Cell Biol, 2(9) 582-592 (2000)
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Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

L M Veenhoff, Eric R Geertsma, J Knol, B Poolman
Close approximation of putative alpha -helices II, IV, VII, X, and XI in the translocation pathway of the lactose transport protein of Streptococcus thermophilus.
J Biol Chem, 275(31) 23834-23840 (2000)
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The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and IV have been implicated in cation binding and the coupled transport of the substrate and the cation. Here, the analysis of site-directed mutants shows that a positive and negative charge at positions 64 and 71 in helix II are essential for transport, but not for lactose binding. The conservation of charge/side-chain properties is less critical for Glu-67 and Ile-70 in helix II, and Asp-133 and Lys-139 in helix IV, but these residues are important for the coupled transport of lactose together with a proton. The analysis of second-site suppressor mutants indicates an ion pair exists between helices II and IV, and thus a close approximation of these helices can be made. The second-site suppressor analysis also suggests ion pairing between helix II and the intracellular loops 6-7 and 10-11. Because the C-terminal region of the transmembrane domain, especially helix XI and loop 10-11, is important for substrate binding in this family of proteins, we propose that sugar and proton binding and translocation are performed by the joint action of these regions in the protein. Indeed, substrate protection of maleimide labeling of single cysteine mutants confirms that alpha-helices II and IV are directly interacting or at least conformationally involved in sugar binding and/or translocation. On the basis of new and published data, we reason that the helices II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation pathway in the transporters of the galactosides-pentosides-hexuronides family.

Mercedes Blázquez, Christoph Thiele, Wieland B. Huttner, Kevin Docherty, Kathleen I. J. Shennan
Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway.
Biochem J, 349 Pt 3 843-852 (2000)
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Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.

Clemens Möller, Georg Büldt, Norbert A. Dencher, Andreas Engel, Daniel J. Müller
Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine.
J Mol Biol, 301(4) 869-879 (2000)
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Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Jürgen B. Heymann, Matthias Pfeiffer, Volker Hildebrandt, H Ronald Kaback, Dimitrios Fotiadis, Bert de Groot, Andreas Engel, Dieter Oesterhelt, Daniel J. Müller
Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin.
Structure, 8(6) 643-653 (2000)
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BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.

Shantha Shanmugalingam, Corinne Houart, Alexander Picker, Frank Reifers, Rachel Macdonald, A Barth, Kevin Griffin, Michael Brand, Stephen W. Wilson
Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon.
Development, 127(12) 2549-2561 (2000)
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Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.

F. J. Ahmad, Jessica Hughey, Torsten Wittmann, Anthony A. Hyman, Marion Greaser, Peter W. Baas
Motor proteins regulate force interactions between microtubules and microfilaments in the axon.
Nat Cell Biol, 2(5) 276-280 (2000)
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It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.

Birte Sönnichsen, Stefano de Renzis, Erik Nielsen, Jens Rietdorf, Marino Zerial
Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.
J Cell Biol, 149(4) 901-914 (2000)
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Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

Arindam Majumdar, Klaus Lun, Michael Brand, Iain A. Drummond
Zebrafish no isthmus reveals a role for pax2.1 in tubule differentiation and patterning events in the pronephric primordia.
Development, 127(10) 2089-2098 (2000)
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Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, we have used the zebrafish pax2.1 mutant no isthmus to investigate the role for pax2.1 in development of the pronephros. We demonstrate a requirement for pax2.1 in multiple aspects of pronephric development including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi(- )larvae specifically lack pronephric tubules while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na(+)/K(+) ATPase (&agr;)1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron.

Silke Pichler, Pierre Gönczy, Heinke Schnabel, Andrei I. Pozniakovsky, Anthony J. Ashford, Ralf Schnabel, Anthony A. Hyman
OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos.
Development, 127(10) 2063-2073 (2000)
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Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.

Derek K Toomre, Jürgen A. Steyer, Patrick Keller, Wolfhard Almers, Kai Simons
Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy.
J Cell Biol, 149(1) 33-40 (2000)
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Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

M D Adams, Susan E Celniker, Robert A Holt, Cheryl A Evans, Jeannine D Gocayne, Peter G Amanatides, Steven E Scherer, P W Li, Roger A Hoskins, R F Galle, Reed A George, S E Lewis, Stephen Richards, Michael Ashburner, S Henderson, Granger G Sutton, Jennifer R Wortman, Mark D Yandell, Qinyu Zhang, L X Chen, R C Brandon, Y H Rogers, R G Blazej, Mark Champe, B D Pfeiffer, Kenneth Wan, C Doyle, E G Baxter, G Helt, C R Nelson, G L Gabor, Josep F Abril, A Agbayani, Hui-Jin An, C Andrews-Pfannkoch, Danita Baldwin, R M Ballew, A Basu, J Baxendale, L Bayraktaroglu, E M Beasley, Karen Y Beeson, P V Benos, Benjamin P Berman, D Bhandari, S Bolshakov, D Borkova, M R Botchan, J Bouck, P Brokstein, P Brottier, K C Burtis, D Busam, H Butler, E Cadieu, Angela Center, I Chandra, J M Cherry, S Cawley, Carl E Dahlke, Lionel B Davenport, P Davies, B de Pablos, A L Delcher, Zuoming Deng, A Mays, I M Dew, Susanne M Dietz, Kristina L Dodson, Lisa Doup, M Downes, S Dugan-Rocha, B C Dunkov, P Dunn, K J Durbin, Carlos C Evangelista, C Ferraz, Steven Ferriera, W Fleischmann, Carl R Fosler, Andrei E Gabrielian, N Garg, W M Gelbart, K Glasser, Anna Glodek, F Gong, J H Gorrell, Z Gu, Ping Guan, M Harris, N L Harris, D Harvey, T J Heiman, J R Hernandez, Jarrett T Houck, D Hostin, K A Houston, Tim Howland, M H Wei, Chinyere Ibegwam, Mena Jalali, Francis Kalush, Gary H Karpen, Z Ke, J A Kennison, K A Ketchum, B E Kimmel, Chinnappa Kodira, C Kraft, S A Kravitz, D Kulp, Z Lai, P Lasko, Y Lei, Alex Levitsky, J Li, Z Li, Y Liang, X Lin, X Liu, B Mattei, T C McIntosh, M P McLeod, D McPherson, Gennady V Merkulov, Natalia V Milshina, C M Mobarry, J Morris, A Moshrefi, S M Mount, M Moy, B Murphy, L Murphy, Donna M Muzny, D L Nelson, D R Nelson, K A Nelson, K Nixon, D R Nusskern, Joanne M Pacleb, M Palazzolo, G S Pittman, S Pan, J Pollard, V Puri, M G Reese, Knut Reinert, K A Remington, R D Saunders, F Scheeler, H Shen, Bixiong Chris Shue, I Sidén-Kiamos, M Simpson, M Skupski, T Smith, E Spier, A C Spradling, Mark Stapleton, Renee Strong, E Sun, Robert R Svirskas, C Tector, R Turner, E Venter, A H Wang, X Wang, Z Y Wang, D A Wassarman, George Weinstock, Jean Weissenbach, S M Williams, S M WoodageT, K C Worley, D Wu, S Yang, Q A Yao, Jane Ye, R F Yeh, J S Zaveri, M Zhan, G Zhang, Q Zhao, L Zheng, X H Zheng, F N Zhong, W Zhong, X Zhou, S Zhu, X Zhu, H O Smith, Richard A Gibbs, E W Myers, G M Rubin, J C Venter
The genome sequence of Drosophila melanogaster.
Science, 287(5461) 2185-2195 (2000)
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The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Michel Bagnat, Sirkka Keränen, Anna Shevchenko, Andrej Shevchenko, Kai Simons
Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast.
Proc Natl Acad Sci U.S.A., 97(7) 3254-3259 (2000)
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Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

A Pralle, Patrick Keller, E L Florin, Kai Simons, J K Hörber
Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells.
J Cell Biol, 148(5) 997-1008 (2000)
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To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.

Paul Verkade, Thomas Harder, Frank Lafont, Kai Simons
Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells.
J Cell Biol, 148(4) 727-739 (2000)
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In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits.Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

Denis Corbeil^*, Katja Röper^*, Andrea Hellwig, Manuela Tavian, Sheri Miraglia, Suzanne M. Watt, Paul J. Simmons, Bruno Peault, David W. Buck, Wieland B. Huttner
The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions.
J Biol Chem, 275(8) 5512-5520 (2000)
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The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.

Frank Reifers, Emily C. Walsh, Sophie Léger, Didier Y.R. Stainier, Michael Brand
Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar).
Development, 127(2) 225-235 (2000)
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Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. We describe here that the secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4, but not gata6, expression in cardiac precursors. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 RNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signalling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independently of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors.

Katja Röper^*, Denis Corbeil^*, Wieland B. Huttner
Distinct Lipid Microdomains within the Apical Plasma Membrane of Polarized Epithelial Cells
In: Protein, Lipid and Membrane Traffic: Pathways and Targeting. (Eds.) Jos A. F. Op den Kamp NATO Science Series: Life Sciences ; 322., Amsterdam, The Netherlands, IOS Press (2000), 73-84 Ch. 10
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William O. Hancock, Jonathon Howard
Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.
Proc Natl Acad Sci U.S.A., 96(23) 13147-13152 (1999)
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Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.

Pierre Gönczy, Silke Pichler, Matthew Kirkham, Anthony A. Hyman
Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo.
J Cell Biol, 147(1) 135-150 (1999)
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We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150(Glued) were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.

Joachim Füllekrug, Peter Scheiffele, Kai Simons
VIP36 localisation to the early secretory pathway.
J Cell Sci, 112 ( Pt 17) 2813-2821 (1999)
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VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localised to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al., 1994). Here we provide evidence that endogenous VIP36 is localised to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalise with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalisation with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.

N Paricio, Fabian Feiguin, Michael Boutros, Suzanne Eaton, Marek Mlodzik
The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.
EMBO J, 18(17) 4669-4678 (1999)
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The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.

Marcos González-Gaitán, Herbert Jäckle
The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.
Mech Dev, 87(1-2) 143-151 (1999)
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Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.

David L. Coy, William O. Hancock, Michael Wagenbach, Jonathon Howard
Kinesin's tail domain is an inhibitory regulator of the motor domain.
Nat Cell Biol, 1(5) 288-292 (1999)
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When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.

Alexander Picker, Caroline Brennan, Frank Reifers, Jon D W Clarke, Nigel Holder, Michael Brand
Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.
Development, 126(13) 2967-2978 (1999)
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The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.

Jürgen Benting, Anton Rietveld, Kai Simons
N-Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.
J Cell Biol, 146(2) 313-320 (1999)
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Glycosyl-phosphatidylinositol (GPI)- anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells. It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts. We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells. The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein. However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretory proteins. In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly. Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.

Anne Grapin-Botton, F Cambronero, H L Weiner, M A Bonnin, L Puelles, N M Le Douarin
Patterning signals acting in the spinal cord override the organizing activity of the isthmus.
Mech Dev, 84(1-2) 41-53 (1999)
DOI

The regionalization of the neural tube along the anteroposterior axis is established through the action of patterning signals from the endomesoderm including the organizer. These signals set up a pre-pattern which is subsequently refined through local patterning events. The midbrain-hindbrain junction, or isthmus, is endowed with such an organizing activity. It is able to induce graded expression of the Engrailed protein in the adjacent mesencephalon and rhombencephalon, and subsequently elicits the development of tectal and cerebellar structures. Ectopically grafted isthmus was also shown to induce Engrailed expression in diencephalon and otic and pre-otic rhombencephalon. Fgf8 is a signalling protein which is produced by the isthmus and which is able to mimic most isthmic properties. We show here that the isthmus, when transposed to the level of either rhombomere 8 or the spinal cord, loses its ability to induce Engrailed and cerebellar development in adjacent tissues. This is accompanied by the down-regulation of fgf8 expression in the grafted isthmus and by the up-regulation of a marker of the recipient site, Hoxb-4. Moreover, these changes in gene activity in the transplant are followed by a transformation of the fate of the grafted cells which adjust to their novel environment. These results show that the fate of the isthmus is not determined at 10-somite stage and that the molecular loop of isthmic maintenance can be disrupted by exogenous signals.

Denis Corbeil^*, Katja Röper^*, Matthew Hannah, Andrea Hellwig, Wieland B. Huttner
Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions.
J Cell Sci, 112 ( Pt 7) 1023-1033 (1999)
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Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

Pierre Gönczy, Heinke Schnabel, Titus Kaletta, Ana Duran Amores, Anthony A. Hyman, Ralf Schnabel
Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis.
J Cell Biol, 144(5) 927-946 (1999)
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To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.

Savvas Christoforidis, Heidi M. McBride, Robert D. Burgoyne, Marino Zerial
The Rab5 effector EEA1 is a core component of endosome docking.
Nature, 397(6720) 621-625 (1999)
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Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5. Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

Daniel J. Müller, Hans-Jürgen Sass, Shirley A Müller, Georg Büldt, Andreas Engel
Surface structures of native bacteriorhodopsin depend on the molecular packing arrangement in the membrane
J Mol Biol, 285(5) 1903-1909 (1999)
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Bacteriorhodopsin is the one of the best-studied models of an ion pump. Five atomic models are now available, yet their comparison reveals differences of some loops connecting the seven transmembrane alpha-helices. In an attempt to resolve this enigma, topographs were recorded in aqueous solution with the atomic force microscope (AFM) to reveal the most native surface structure of bacteriorhodopsin molecules in the purple membrane. Individual peptide loops were observed with a lateral resolution of between 4.5 A and 5.8 A, and a vertical resolution of about 1 A. The AFM images demonstrate for the first time, that the shape, the position, and the flexibility of individual polypeptide loops depend on the packing arrangement of bacteriorhodopsin molecules in the lipid bilayer.

Simon Scheuring, Daniel J. Müller, Philippe Ringler, Jürgen B. Heymann, Andreas Engel
Imaging streptavidin 2D crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope.
J Microsc, 193(1) 28-35 (1999)
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Streptavidin crystals were grown on biotinylated lipid monolayers at an air/water interface and transferred onto highly oriented pyrolytic graphite (HOPG). These arrays could be imaged to a resolution below 1 nm using the atomic force microscope. The surface topographs obtained were compared with negative-stain electron microscopy images and the atomic model as determined by X-ray crystallography. The streptavidin tetramer (60 kDa) exposes two free biotin-binding sites to the buffer solution, while two are occupied by linkage to the lipid monolayer. Therefore, the streptavidin 2D crystals can be used as nanoscale matrices for binding biotinylated compounds. Furthermore, this HOPG-based preparation method provides a general novel approach to study the structure of protein arrays assembled on lipid monolayers with the AFM.

Simon Scheuring, Philippe Ringler, Mario Borgnia, Henning Stahlberg, Daniel J. Müller, Peter Agre, Andreas Engel
High resolution AFM topographs of the Escherichia coli water channel aquaporin Z
EMBO Rep, 18(18) 4981-4987 (1999)
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Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Peter Scheiffele, Anton Rietveld, Thomas Wilk, Kai Simons
Influenza viruses select ordered lipid domains during budding from the plasma membrane.
J Biol Chem, 274(4) 2038-2044 (1999)
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During the budding of enveloped viruses from the plasma membrane, the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the host membrane. Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus family, vesicular stomatitis virus (VSV), and Semliki Forest virus (SFV). Analysis of detergent extractability of proteins, cholesterol, phosphoglycerolipids, and sphingomyelin in virions showed that FPV contains high amounts of detergent-insoluble complexes, whereas such complexes are largely absent from VSV or SFV. Cholesterol depletion from the viral envelope by methyl-beta-cyclodextrin results in increased solubility of sphingomyelin and of the glycoproteins in the FPV envelope. This biochemical behavior suggests that so-called raft-lipid domains are selectively incorporated into the influenza virus envelope. The "fluidity" of the FPV envelope, as measured by the fluorescence polarization of diphenylhexatriene, was significantly lower than compared with VSV or SFV. Furthermore, influenza virus hemagglutinin incorporated into the envelope of recombinant VSV was largely detergent-soluble, indicating the depletion of raft-lipid assemblies from this membrane. The results provide a model for lipid selectivity during virus budding and support the view of lipid rafts as cholesterol-dependent, ordered domains in biological membranes.

Torsten Wittmann, Anthony A. Hyman
Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes
In: Mitosis and meiosis. (Eds.) Conly L. Rieder Methods in cell biology ; 61., Amsterdam, Netherlands, Academic Press (1999), 137-143
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Anton Rietveld, Kai Simons
The differential miscibility of lipids as the basis for the formation of functional membrane rafts.
Biochim Biophys Acta, 1376(3) 467-479 (1998)
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The formation of sphingolipid-cholesterol microdomains in cellular membranes has been proposed to function in sorting and transport of lipids and proteins as well as in signal transduction. An increasing number of cell biological and biochemical studies now supports this concept. Here we discuss the structural properties of lipids in a cell biological context. The sphingolipid-cholesterol microdomains or rafts are described as dispersed liquid ordered phase domains. These domains are dynamic assemblies to which specific proteins are selectively sequestered while others are excluded. The proteins associated to rafts can act as organizers and can modulate raft size and function.

Elina Ikonen, Kai Simons
Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells.
Semin Cell Dev Biol, 9(5) 503-509 (1998)
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The targeting of proteins and lipids to the cell surface domains of polarized cells is not a simple bulk flow process but requires sorting into distinct apical and basolateral pathways from the trans-Golgi network. Here, we describe the sorting determinants in the cargo molecules, the cellular sorting machineries responsible for the hierarchical read-out of the signals, and the mechanisms of cargo delivery. Furthermore, we discuss the implications of these findings for protein-lipid interactions in other cellular machineries.

Christoph Thiele, Wieland B. Huttner
Protein and lipid sorting from the trans-Golgi network to secretory granules-recent developments.
Semin Cell Dev Biol, 9(5) 511-516 (1998)
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Secretory granules are the cellular organelles mediating storage and regulated secretion of proteins. Their biogenesis involves sorting of secretory protein cargo and membrane constituents, which takes place at two distinct levels, the trans-Golgi network and the immature secretory granule. At both levels, sorting is accomplished by cargo aggregation and cargo-membrane recognition. Given not only the aggregative properties of the regulated secretory proteins but also the ability of lipids to form distinct membrane microdomains, self-organization of both lumenal and membrane constituents is proposed to play a crucial role in secretory granule biogenesis.

G Couly, Anne Grapin-Botton, P Coltey, B Ruhin, N M Le Douarin
Determination of the identity of the derivatives of the cephalic neural crest: incompatibility between Hox gene expression and lower jaw development.
Development, 125(17) 3445-3459 (1998)

In addition to pigment cells, and neural and endocrine derivatives, the neural crest is characterized by its ability to yield mesenchymal cells. In amniotes, this property is restricted to the cephalic region from the mid-diencephalon to the end of rhombomere 8 (level of somites 4/5). The cephalic neural crest is divided into two domains: an anterior region corresponding to the diencephalon, mesencephalon and metencephalon (r1, r2) in which expression of Hox genes is never observed, and a posterior domain in which neural crest cells exhibit (with a few exceptions) the same Hox code as the rhombomeres from which they originate. By altering the normal distribution of neural crest cells in the branchial arches through appropriate embryonic manipulations, we have investigated the relationships between Hox gene expression and the level of plasticity that neural crest cells display when they are led to migrate to an ectopic environment. We made the following observations. (i) Hox gene expression is not altered in neural crest cells by their transposition to ectopic sites. (ii) Expression of Hox genes by the BA ectoderm does not depend upon an induction by the neural crest. This second finding further supports the concept of segmentation of the cephalic ectoderm into ectomeres (Couly and Le Douarin, 1990). According to this concept, metameres can be defined in large bands of ectoderm including not only the CNS and the neural crest but also the corresponding superficial ectoderm fated to cover craniofacial primordia. (iii) The construction of a lower jaw requires the environment provided by the ectomesodermal components of BA1 or BA2 associated with the Hox gene non-expressing neural crest cells. Hox gene-expressing neural crest cells are unable to yield the lower jaw apparatus including the entoglossum and basihyal even in the BA1 environment. In contrast, the posterior part of the hyoid bone can be constructed by any region of the neural crest cells whether or not they are under the regulatory control of Hox genes. Such is also the case for the neural and connective tissues (including those comprising the cardiovascular system) of neural crest origin, upon which no segmental restriction is imposed. The latter finding confirms the plasticity observed 24 years ago (Le Douarin and Teillet, 1974) for the precursors of the PNS.

Peter L. Pfeffer, Thomas Gerster, Klaus Lun, Michael Brand, Meinrad Busslinger
Characterization of three novel members of the zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function.
Development, 125(16) 3063-3074 (1998)
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The mammalian Pax2, Pax5 and Pax8 genes code for highly related transcription factors, which play important roles in embryonic development and organogenesis. Here we report the characterization of all members of the zebrafish Pax2/5/8 family. These genes have arisen by duplications before or at the onset of vertebrate evolution. Due to an additional genome amplification in the fish lineage, the zebrafish contains two Pax2 genes, the previously known Pax[b] gene (here renamed as Pax2.1) and a novel Pax2.2 gene. The zebrafish Pax2.1 gene most closely resembles the mammalian Pax2 gene in its expression pattern, as it is transcribed first in the midbrain-hindbrain boundary region, then in the optic stalk, otic system, pronephros and nephric ducts, and lastly in specific interneurons of the hindbrain and spinal cord. Pax2.2 differs from Pax2.1 by the absence of expression in the nephric system and by a delayed onset of transcription in other Pax2.1 expession domains. Pax8 is also expressed in the same domains as Pax2.1, but its transcription is already initiated during gastrulation in the primordia of the otic placode and pronephric anlage, thus identifying Pax8 as the earliest developmental marker of these structures. The zebrafish Pax5 gene, in contrast to its mouse orthologue, is transcribed in the otic system in addition to its prominent expression at the midbrain-hindbrain boundary. The no isthmus (noi) mutation is known to inactivate the Pax2.1 gene, thereby affecting the development of the midbrain-hindbrain boundary region, pronephric system, optic stalk and otic region. Although the different members of the Pax2/5/8 family may potentially compensate for the loss of Pax2.1 function, we demonstrate here that only the expression of the Pax2.2 gene remains unaffected in noi mutant embryos. The expression of Pax5 and Pax8 is either not initiated at the midbrain-hindbrain boundary or is later not maintained in other expression domains. Consequently, the noi mutation of zebrafish is equivalent to combined inactivation of the mouse Pax2 and Pax5 genes with regard to the loss of midbrain-hindbrain boundary development.

Klaus Lun, Michael Brand
A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary.
Development, 125(16) 3049-3062 (1998)
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Generation of cell diversity in the vertebrate central nervous system starts during gastrulation stages in the ectodermal germ layer and involves specialized cell groups, such as the organizer located at the midbrain-hindbrain boundary (MHB). Mutations in the zebrafish no isthmus (noi) gene alter development of the MHB, and affect the pax2.1 gene (formerly pax(zf-b)). Analysis of the structure of pax2.1 reveals at least 12 normal splice variants. The noi alleles can be arranged, by molecular and phenotypic criteria, into a series of five alleles of differing strength, ranging from a null allele to weak alleles. In keeping with a role in development of the MHB organizer, gene expression is already affected in the MHB primordium of the gastrula neural ectoderm in noi mutants. eng3 activation is completely and eng2 activation is strongly dependent on noi function. In contrast, onset of wnt1, fgf8 and her5 expression occurs normally in the null mutants, but is eliminated later on. Our observations suggest that three signaling pathways, involving pax2.1, wnt1 and fgf8, are activated independently in early anterior-posterior patterning of this area. In addition, analysis of the allelic series unexpectedly suggests that noi activity is also required during dorsal-ventral patterning of the MHB in somitogenesis stages, and possibly in a later eng expression phase. We propose that noi/pax2.1 participates in sequential signaling processes as a key integrator of midbrain-hindbrain boundary development.

V Nataf, Anne Grapin-Botton, D Champeval, A Amemiya, M Yanagisawa, N M Le Douarin
The expression patterns of endothelin-A receptor and endothelin 1 in the avian embryo.
Mech Dev, 75(1-2) 145-149 (1998)
DOI

We investigated the expression pattern of the endothelin-A receptor and endothelin 1 genes, the mutations of which affect the development of the mesectodermal derivatives of the neural crest. We show here that endothelin 1 is expressed by the environment of the cephalic neural crest cells invading branchial arches. Later on, while the neural crest-derived tissues of the head continue to express endothelin-A receptor, endothelin 1 is no longer expressed in their environment.

A C Sharman, Michael Brand
Evolution and homology of the nervous system: cross-phylum rescues of otd/Otx genes.
Trends Genet, 14(6) 211-214 (1998)
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C O M M E N T Evolution and homology of the nervous system: cross-phylum rescues of otd/Otx genes ANNA C . SHARMAN AND MICHAEL BRAND anna.sharman@urz.uni-heidelberg.de brand@sun0.urz.uni-heidelberg.de I NSTITUTE OF N EUROBIOLOGY , U NIVERSITY OF H EIDELBERG , I N N EUENHEIMER F ELD 345, 69120 H EIDELBERG , G ERMANY . The homeotic or Hox genes were the first gene family to be shown to act in similar, probably homologous, ways in insect and mammalian development. They are thought to form a combinatorial code specifying the identity of different segments. They are clustered in the genome, and expressed in a nested pattern along the anterior posterior axis in an order that is colinear with their chromosomal order 1 . Since then, perhaps a surprisingly high number of developmental gene families have also been shown to have conserved expression patterns over several phyla. One such family is the Otx genes, which like the Hox genes encode homeodomain-containing DNA-binding proteins 2,3 . Flies and amphioxus have a single gene in the family, called orthodenticle (otd ) in Drosophila, while vertebrates typically have two Otx genes. Mouse Otx1 and Otx2, together with another family of homeobox genes, Emx1 and Emx2, are expressed in nested patterns in the fore- and mid-brain 3 , while their fly homologues otd and ems are expressed in the most anterior segments 4 6 . It has been suggested 3,7 that the Otx and Emx genes specify segmental identity in insects and vertebrates, fulfilling a role for the anterior brain similar to that of the Hox genes in the hindbrain and spinal cord. This hypothesis is supported by knockout phenotypes and mutations 8 12 : in Otx2 +/ Otx1 / mice the midbrain and posterior diencephalon are completely missing (interpretation of single Otx mutant phenotypes is complicated in mice by variability, an early role for Otx2 in gastrulation and by redundancy between Otx1 and Otx2). It also appears that in mammals, Otx function influences development of the midbrain hindbrain boundary, known to be an organizer of cell fate in the midbrain and anterior hindbrain 8 13 . In Emx knockouts discrete parts of the forebrain are missing 14,15 . So expression and mutation of Otx/otd and Hox genes show that the anterior posterior (AP) patterning mechanisms in insects and vertebrates share many features. Does this mean that these mechanisms are homologous, that is, that the common ancestor had these mechanisms too? The fact that amphioxus, a primitive chordate, also has anterior Otx expression 2 indicates that the mechanism is quite widespread, although otd expression in echinoderms 16 is quite different and variable. It is first of all necessary to test whether the genes are acting in the same way in both groups of animals. This can be done experimentally by expressing vertebrate genes in flies mutant for their homologues, or vice versa, and asking whether the foreign gene can rescue the mutant phenotype. This has already been done for some Hox genes 17 19 , showing that they can function in an equivalent manner, but it has not until now been tried for the Otx/otd family. A second factor also needs to be considered. The nervous system of vertebrates has been compared to the early ectoderm of insects, at a stage long before the central nervous system (CNS) is formed. A question mark has hung over interpretations of AP patterning similarities: could these two systems really be homologous, when they referred to such different stages of development? Two responses have been given towards the latter question. One (called the auricularia hypothesis) was to propose that the nervous system of chordates is homologous to the entire outer ectoderm of insects while the insect nervous system and the chordate outer ectoderm have arisen independently 20 . This was supported by studies of the anatomy of larval echinoderms (particularly auricularia larvae) and urochordates, which are proposed to be intermediates in the transition between the arthropodand chordate-type body plans, and by the fact that insect Hox genes are expressed in the surface ectoderm, whereas they are not in vertebrates. The nervous systems of insects and vertebrates have long been assumed to have evolved independently, because they are organized differently and are on opposite sides of the embryo 21 . The other response grew out of recent work showing that the dorsoventral axis is in fact patterned by the same set of genes in similar relationships in insects and in vertebrates, but with the dorsoventral axis inverted 21,22 . Many developmental genes, such as the achaete/scute homologues, Nkx2, Msx and Hox homeobox genes and netrins are expressed in similar patterns in insect and vertebrate nervous systems 21,23 . It is a reasonable hypothesis that the dorsoventral axis inverted at some point in evolution, and thus that the nervous systems of insects and vertebrates are homologous (Fig. 1). Which of these two theories is correct? Four recent papers 24 27 address the question of Otx functional equivalence, and supply further evidence for homology of the insect and vertebrate nervous systems. Three of them 24 26 show that the Otx and otd genes have a conserved function, as well as conserved expression patterns. Previously, the expression of otd and ems in the fly nervous system was determined (Ref. 6 and Fig. 2), and the fourth paper 27 finds that Hox gene expression there is more similar to expression of vertebrate Hox genes than was previously suspected. Functional equivalence of Otx and otd genes Two papers describe the overexpression of human OTX1 and OTX2 genes in flies defective in otd function. Nagao et al. 26 found that the human OTX genes could rescue the ocelliless phenotype (oc is a regulatory mutation causing defective otd expression in the fly pupa) just as well as otd could rescue. Not only the final phenotype, but also gene expression TIG J UNE 1998 V OL . 14 N O . 6 211 Copyright 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01488-7 C O M M E N T in the eye-antennal imaginal discs, is rescued. Leuzinger et al. 25 used a similar approach to drive transient ubiquitous expression of otd, OTX1 or OTX2, to look at the better-studied embryonic function of otd. According to several criteria, otd expression can rescue the otd mutant phenotype, though in a variable proportion of embryos. OTX2 also rescues, though at a lower frequency than otd, and OTX1 rescues less efficiently still. The third paper 24 goes in the other direction: the mouse Otx1 coding region was replaced by that of Drosophila otd. Many of the defects of Otx1 / mice were rescued by otd, for example several brain regions previously missing are restored. Interestingly, the rescue was less efficient nearer to the MHB, for example the mesencephalon was never completely normal, and cerebellar foliation remained abnormal. In the sense organs, most defects were rescued, but the lateral semicircular canal in the inner ear was not. Hox genes in the fly nervous system Hirth et al. 27 examine the expression and function of the Drosophila homeotic (Hox) genes in the CNS. In the mouse, the group 2 Hox genes have the most anterior expression boundaries, while group 1 Hox genes are expressed in restricted domains, one rhombomere posterior to the group 2 anterior boundary (Ref. 1 and Fig. 2). This lapse in the rule of colinearity has so far only been noted in vertebrates 28 ; in the Drosophila ectoderm, strict colinearity is adhered to. Previous descriptions of homeotic gene expression in the fly nervous system put the anterior boundary of labial (lab ; a group 1 Hox gene) and proboscipedia (pb ; group 2) at the same level, within the deutocerebrum 29 . However, Hirth et al. have re-examined this expression using neural segmental markers, and now show that lab is actually expressed only in the posterior of brain segment b3 (the tritocerebrum), posterior to the anterior boundary of pb expression in segment b2 (the deutocerebrum) (Ref. 27; Fig. 2). The same exception to colinearity therefore occurs in the fly CNS as well as in vertebrates. Homology of nervous systems It is important to realize that the ability of homologous genes to replace each other functionally does not necessarily show that the regions in which they are expressed are homologous. The Otx and otd gene products, for example, are transcription factors. It is quite possible that the ability of OTX proteins to bind a particular DNA sequence has been conserved, while the downstream genes regulated by this binding have altered. This explanation would still allow the proteins to replace each other, but they would be regulating different downstream genes in each animal. The formal possibility has not been ruled out that expression of otd and Otx in the anterior brain could have arisen independently in insects and chordates. However, it is now clear that the similar expression of Otx genes in the two nervous systems has functional relevance; the similarity cannot be explained by the gain of a few enhancer elements, and thus is less likely to have arisen by convergent evolution. These papers also show that, even though Hox and Otx genes are expressed in the early blastoderm in flies, this does not (as the auricularia hypothesis 20 suggests) mean that the ectoderm wall is homologous to the vertebrate neural tube, because the same genes are also expressed in the fly CNS, and in a very similar manner to vertebrates. Together with other evidence 17,22,30,31 , these results might be the final nail in the coffin for theories like the auricularia hypothesis that support an independent origin for the insect and vertebrate nervous systems. Significance of otd/Otx functional equivalence The fly otd gene can replace mouse Otx1 astoundingly well. We would have perhaps expected that Otx1 would have evolved new functions to do with vertebrate-specific developmental programs, that otd could not perform. It appears that only the homeobox and the regulatory elements, plus perhaps some less well conserved regions like the acidic activation domains, are sufficient for most of the functions of Otx1. New expression domains acquired since the arthropod/vertebrate split might account for much of the differences between otd and Otx1; these differences can only be detected experimentally by examining and possibly replacing regulatory elements rather than just coding regions. Replacement of regions of the gene outside the homeobox would test whether these regions, or only the homeobox, is important. Although otd can replace most functions of Otx1, some cannot be replaced. This could simply be a quantitative effect: Acampora et al. 9 have shown that in brain development Otx `dosage' is more important than which individual Otx gene is present, although Otx2 does appear to be more `potent' than Otx1, since the Otx2 mutant has a stronger phenotype. In the rescue experiments of Leuzinger et al. 25 , Otx1 is less effective at rescuing the otd phenotype than Otx2. otd could simply be less TIG J UNE 1998 V OL . 14 N O . 6 212 dpp dpp sog Ventral Dorsal Drosophila Ventral Dorsal Mouse msh vnd vnd ms h ms x Nkx2 Nkx2 msx chordin Bmp4 Bmp4 Midline, netrin + AS-C/ash F IGURE 1. Transverse sections through the fly and vertebrate central nervous system primordia, showing similar dorsoventral regulation of pattern by the sog (short gastrulation)/chordin, dpp (decapentaplegic)/BMP4, Msx/msh, Nkx2/vnd, AS-C (achaete-scute complex)/ash (AS-C homologues) and netrin gene families. (Redrawn from Ref. 23, with extra information from Ref. 22.) C O M M E N T `potent' in the mouse than either Otx1 or Otx2, as is supported by the fact that mice with two copies of Otx2 and no Otx1 show a weaker phenotype than mice 24 with one copy of each gene (Ref. 32). Some of the lack of rescue cannot be explained in this way, however, and could reflect truly novel functions of Otx1 that depend on its coding region. One example is the lateral semicircular canal phenotype of Otx1, which cannot be rescued by otd, suggesting that Otx1 has taken on a role in development of this canal in the inner ear after duplication. Significantly, this canal evolved around the time when the Otx genes are thought to have duplicated, at the origin of jawed vertebrates 33 (agnathan fish have only two semicircular canals, and lack the lateral one 24 ). The evolution of the midbrain hindbrain boundary A second way in which otd cannot replace Otx1 is shown in the graded level of rescue in the brain: the midbrain hindbrain boundary (MHB), midbrain and cerebellum, at the posterior of the Otx domain, are most sensitive to a low level of otd/Otx function and thus can be less easily rescued, while the telencephalon (at the anterior of the Otx domain) is least sensitive 24 . The vertebrate MHB is known to act as an organizer, producing signals that pattern the midbrain and anterior hindbrain 35,36 , but no such organizer has been described in the brain of Drosophila. Like the lateral semicircular canal, the organizer may have arisen early in vertebrate evolution, since lampreys and all jawed vertebrates, but not hagfish or cephalochordates, have an undeniable midbrain and cerebellum 34 . Thus the inability of Drosophila otd to rescue the midbrain hindbrain boundary phenotype of mouse Otx1 may be because the MHB is a new structure that evolved, or was at least greatly elaborated, early in the vertebrate lineage. The Otx genes themselves may have helped in the evolution of the MHB organizer; changing Otx expression patterns could have produced a gap between Otx and Hox expression domains (otd and Hox are adjacent in the fly; Fig. 2), which was then filled by MHB-specific transcription factors and signalling molecules such as EN, PAX2, PAX5, PAX8, WNT1 and FGF8 (Ref. 36). A thorough analysis of the development of this region in a range of vertebrates is clearly needed. Perspectives The papers reviewed here answer some questions while raising others. The list of known similarities between the vertebrate and arthropod nervous systems has been expanded significantly, suggesting that they are probably homologous structures. If this is so, has the auricularia hypothesis been disproved? Enteropneusts, a group of invertebrate chordates, have both a dorsal neural tube and a ventral nerve plexus, and it has been argued 37 that this shows that the vertebrate dorsal neural tube cannot be homologous to the invertebrate ventral nervous system. However, it is possible to argue that they are, if one of the enteropneust nervous systems is an independently-derived novelty, or if the two enteropneust nervous systems arose from a duplication of the single ancestral one. Investigation of expression patterns of developmental genes in enteropneusts may resolve the discrepancy. Additionally, it is still not resolved whether the functional equivalence of genes, as shown by their ability to replace each other, really demonstrates that the genes are doing the same thing in the two animals concerned. It would be interesting, for example, to test whether mammalian Hoxb2 can rescue the phenotype of Drosophila lab mutants (lab being a group 1 Hox gene, Hoxb2 a group 2). So far, genes in the same subfamily have mostly been tested for rescuing ability [otd and Otx1 (Refs 24 26), hh and shh (Ref. 30) or Hoxb1 and labial (Ref. 17)], but this ability may simply show that the important coding-region functions of a whole gene family have remained constant, rather than that two genes are the closest homologues within the family. The ability to rescue should be tested for a range of related genes, not just the ones suspected to be most closely related to each other. Certainly, these studies emphasize the need to compare regulatory elements as much as coding regions between species, since it appears that evolution has relied predominantly on regulatory changes. References 1 McGinnis, W. and Krumlauf, R. (1992) Cell 68, 283 302 2 Williams, N.A. and Holland, P.W.H. (1996) Nature 383, 490 3 Simeone, A. et al. (1992) Nature 358, 687 690 4 Younossi-Hartenstein, A. et al. (1997) Dev. Biol. 182, 270 283 TIG J UNE 1998 V OL . 14 N O . 6 213 1 2 3 4 5 7 6 M P2 P3 T Rhombomeres Mouse 8 P1 B1 B2 B3 S1 S2 S3 ems lab (Hox1) pb (Hox2 ) otd Drosophila Dfd (Hox4 ) Scr (Hox5 ) Antp (Hox6 ) Ubx (Hox7 ) Hoxb1 Hoxb2 Otx2 Otx1 Emx2 Emx1 Hoxb3 Hoxb4 Hoxb5 Hoxb6 Hoxb7 F IGURE 2. Anteroposterior gene expression in the fly and mouse central nervous systems showing Hox, Otx/otd and Emx/ems expression patterns. (Redrawn from Ref. 27, with extra information from Refs 6, 29, 38 and H. Reichert, pers. commun.) Arrow denotes the midbrain hindbrain boundary; B1 B3, brain segments (proto-, deuto- and trito-cerebrum, respectively); S1 S3, mandibular, maxillary and labial segments, respectively; P1 P3, prosomeres in the diencephalon; T, telencephalon; M, mesencephalon. C O M M E N T TIG J UNE 1998 V OL . 14 N O . 6 214 Copyright 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01500-5 5 Finkelstein, R. and Boncinelli, E. (1994) Trends Genet. 10, 310 315 6 Hirth, F. et al. (1995) Neuron 15, 769 778 7 Holland, P., Ingham, P. and Krauss, S. (1992) Nature 358, 627 628 8 Matsuo, I. et al. (1995) Genes Dev. 9, 2646 2658 9 Acampora, D. et al. (1997) Development 124, 3639 3650 10 Ang, S.L. et al. (1996) Development 122, 243 252 11 Acampora, D. et al. (1995) Development 121, 3279 3290 12 Acampora, D. et al. (1996) Nat. Genet. 14, 218 222 13 Marin, F. and Puelles, L. (1994) Dev. Biol. 163, 19 37 14 Pellegrini, M. et al. (1996) Development 122, 3893 3898 15 Qiu, M. et al. (1996) Dev. Biol. 178, 174 178 16 Lowe, C.J. and Wray, G.A. (1997) Nature 389, 718 721 17 Lutz, B. et al. (1996) Genes Dev. 10, 176 184 18 Malicki, J., Schughart, K. and McGinnis, W. (1990) Cell 63, 961 967 19 Zhao, J.J., Lazzarini, R.A. and Pick, L. (1993) Genes Dev. 7, 343 354 20 Lacalli, T.C. (1994) Am. Zool. 34, 533 541 21 Arendt, D. and N bler-Jung, K. (1996) BioEssays 18, 255 259 22 DeRobertis, E.M. and Sasai, Y. (1996) Nature 380, 37 40 23 D'Alessio, M. and Frasch, M. (1996) Mech. Dev. 58, 217 231 24 Acampora, D. et al. Development (in press) 25 Leuzinger, S. et al. Development (in press) 26 Nagao, T. et al. Proc. Natl. Acad. Sci. U. S. A. (in press) 27 Hirth, F., Hartmann, B. and Reichert, H. Development (in press) 28 Prince, V.E. et al. (1998) Development 125, 393 406 29 Diederich, R.J. et al. (1989) Genes Dev. 3, 399 414 30 Krauss, S., Concordet, J.P. and Ingham, P.W. (1993) Cell 75, 1431 1444 31 Halder, G., Callaerts, P. and Gehring, W.J. (1995) Science 267, 1788 1792 32 Suda, Y., Matsuo, I., Kuratani, S. and Aizawa, S. (1996) Genes Cells 1, 1031 1044 33 Williams, N.A. and Holland, P.W.H. Mol. Biol. Evol. (in press) 34 Butler, A.B. and Hodos, W. (1996) Comparative Vertebrate Neuroanatomy, Wiley-Liss 35 Marin, F. and Puelles, L. (1995) Eur. J. Neurosci. 7, 1714 1738 36 Joyner, A.L. (1996) Trends Genet. 12, 15 20 37 Peterson, K.J. (1995) Nature 373, 111 112 38 Puelles, L. and Rubenstein, J.L. (1993) Trends Neurosci. 16, 472 479 Last month's issue of Trends in Genetics featured a review of the disease-related potential of the transcriptional cofactors CREB-binding protein (CBP) and the adenovirus E1A-associated protein, p300 (Ref. 1). Shortly after the publication of this review, the combined efforts of David Livingston's laboratory (Harvard Medical School, Boston, MA, USA) and Richard Eckner's laboratory (Univ. of Zu rich, Switzerland) appeared in Cell, describing the deleterious effects of inactivating one or both murine CBP and/or p300 alleles 2 . The results-at-a-glance are presented in Table 1. When a single p300 allele is inactivated, the resultant embryos suffer a significantly reduced viability (up to 55% died in utero, depending on genetic background), although heterozygotes that do survive do not suffer from further p300-insufficiency after birth. Mice homozygous for p300 mutations always die in utero, between days 9 and 11.5 of gestation. These nullizygous embryos are much smaller than their littermates and exhibit severe open neural tube and heart defects. Interestingly, cells removed from the p300 homozygous mutants displayed poor proliferation properties, implying that p300 is required for growth stimulation, an idea that is contrary to the general opinion that CBP and p300 are tumor suppressor proteins. Unlike p300, CBP heterozygous mutant mice, described earlier 3 , manifest skeletal abnormalities consistent with the human congenital Rubinstein Taybi syndrome, in which one CBP allele is inactivated 4 . CBP homozygous mutant mice, however, strongly resemble the p300 mutants, and also die in utero, between days 9 and 11.5 of gestation. Crossing the p300 and CBP heterozygous mutants produced double heterozygous CBP/p300 mutant embryos, which died in utero but otherwise shared phenotypic similarities to both CBP and p300 homozygous mutants. This remarkable result suggests that the two proteins exert certain common embryonic survival functions and that the combined dose of CBP and p300 is critical for mouse embryonic development. Although CBP and p300 are not completely redundant physiologically, these results suggest that a 25% drop in combined CBP/p300 levels (through the loss of one CBP or p300 allele) is enough to interfere seriously with embryonic development, while a 50% drop results invariably in embryonic death. References 1 Giles, R.H., Peters, D.J.M. and Breuning, M.H. (1998) Trends Genet. 14, 178 183 2 Yao, T.P. et al. (1998) Cell 93, 361 372 3 Tanaka, Y. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10215 10220 4 Petrij, F. et al. (1995) Nature 376, 348 351 Rachel H. Giles rachel@ruly46.medfac.leidenuniv.nl Department of Human Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Update CBP/p300 transgenic mice T ABLE 1. Mouse models for CBP and p300 mutations CBP p300 Phenotype Refs ++ ++ Normal + ++ Skeletal abnormalities 3 ++ Embryonic lethal 2, 3 ++ + Reduced viability 2 ++ Embryonic lethal 2 + + Embryonic lethal 2 Abbreviations: +, normal allele; , inactive allele.

A M Mager, Anne Grapin-Botton, K Ladjali, D Meyer, C M Wolff, P Stiegler, M A Bonnin, P Remy
The avian fli gene is specifically expressed during embryogenesis in a subset of neural crest cells giving rise to mesenchyme.
Int. J. Dev. Biol., 42(4) 561-572 (1998)

The ets-family of transcription factors is involved in the development of endothelial and hematopoietic cells. Among these genes, fliwas shown to be responsible for erythroblastomas and Ewing's sarcomas. Its involvement in Ewing's sarcoma, a putative neurectodermal tumor, as well as the in situ hybridization studies performed in mice and Xenopus suggested a role in neural crest development. We cloned quail fli cDNA in order to analyze in more detail its expression in neural crest cells, which have been extensively studied in avian species. Fli gene maps on chicken chromosome 1 to band q31->q33. Two RNAs are transcribed, most likely arising from two different promoters. The analysis of its expression in neural crest cells reveals that it is expressed rather late, when the neural crest cells reach their target. Among the various lineages derived from the crest, it is restricted to the mesenchymal one. It is maintained at later stages in the cartilage of neural crest but also of mesodermal origin. In addition, fli is expressed in several mesoderm-derived cells: endothelial cells as well as intermediate and splanchnopleural mesoderm.

Gaetano Vitale, Vladimir Rybin, Savvas Christoforidis, Per-Ove Thornqvist, Mary McCaffrey, Harald Stenmark, Marino Zerial
Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.
EMBO J, 17(7) 1941-1951 (1998)
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Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

Helene Gournier, Harald Stenmark, Vladimir Rybin, Roger Lippé, Marino Zerial
Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.
EMBO J, 17(7) 1930-1940 (1998)
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Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Anne Grapin-Botton, M A Bonnin, M Sieweke, N M Le Douarin
Defined concentrations of a posteriorizing signal are critical for MafB/Kreisler segmental expression in the hindbrain.
Development, 125(7) 1173-1181 (1998)

It has been shown by using the quail/chick chimera system that Hox gene expression in the hindbrain is influenced by positional signals arising from the environment. In order to decipher the pathway that leads to Hox gene induction, we have investigated whether a Hox gene regulator, the leucine zipper transcription factor MafB/Kr, is itself transcriptionally regulated by the environmental signals. This gene is normally expressed in rhombomeres (r) 5 and 6 and their associated neural crest. MafB/Kr expression is maintained in r5/6 when grafted into the environment of r3/4. On the contrary, the environment of rhombomeres 7/8 represses MafB/Kr expression. Thus, as previously shown for the expression of Hox genes, MafB/Kr expression is regulated by a posterior-dominant signal, which in this case induces the loss of expression of this gene. We also show that the posterior signal can be transferred to the r5/6 neuroepithelium by posterior somites (somites 7 to 10) grafted laterally to r5/6. At the r4 level, the same somites induce MafB/Kr in r4, leading it to behave like r5/6. The posterior environment regulates MafB/Kr expression in the neural crest as it does in the corresponding hindbrain level, showing that some positional regulatory mechanisms are shared by neural tube and neural crest cells. Retinoic acid beads mimic the effect produced by the somites in repressing MafB/Kr in r5/6 and progressively inducing it more rostrally as its concentration increases. We therefore propose that the MafB/Kr expression domain is defined by a molecule unevenly distributed in the paraxial mesoderm. This molecule would allow the expression of the MafB/Kr gene in a narrow window of concentration by activating its expression at a definite threshold and repressing it at higher levels, accounting for its limited domain of expression in only two rhombomeres. It thus appears that the regulation of MafB/Kr expression in the rhombomeres could be controlled by the same posteriorizing factor(s) as Hox genes.

William O. Hancock, Jonathon Howard
Processivity of the motor protein kinesin requires two heads.
J Cell Biol, 140(6) 1395-1405 (1998)
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A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.

S Levy, L Compagnoni, E W Myers, G D Stormo
Xlandscape: the graphical display of word frequencies in sequences.
Bioinformatics, 14(1) 74-80 (1998)
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To provide a graphical interface for the generation, display and manipulation of a sequence landscape that will run on all X-windows-based Unix workstations.

Harald Stenmark, Marino Zerial
Transient Expression Using the T7 RNA Polymerase Recombinant Vaccinia Virus System
In: Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 4. (Eds.) Julio E. Celis, Amsterdam, Netherlands, Academic Press (1998), 201-203 Ch. 14
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Christoph Thiele, Wieland B. Huttner
The disulfide-bonded loop of chromogranins, which is essential for sorting to secretory granules, mediates homodimerization.
J Biol Chem, 273(2) 1223-1231 (1998)
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Chromogranins A and B, two widespread neuroendocrine secretory proteins, contain a homologous N-terminal disulfide-bonded loop that is required for sorting to secretory granules. Here we have investigated the role of this loop in the oligomerization of chromogranin A. Reduction of the disulfide bond or the addition of an excess of an N-terminal chromogranin A fragment containing the loop (CgA1-60) resulted in the dissociation into monomers of the chromogranin A dimer found at pH 7.4 and 6.4 and of the chromogranin tetramer found at pH 5.4. The addition of an excess of a synthetic peptide corresponding to the conserved C-terminal domain of chromogranin A (CgA406-431) had no effect on the chromogranin dimers at pH 7.4 and 6.4 and resulted in the dissociation of the chromogranin A tetramers at pH 5.4 into dimers. Fluorescence energy transfer experiments using fluorescently labeled CgA1-60 showed that the N-terminal disulfide-bonded loop has a high affinity for homodimerization (KD = 20 nM at pH 6.4), which was sufficient to mediate dimerization of full-length chromogranin A. Association and dissociation of loop-mediated chromogranin A dimerization approached completion within a few seconds. Our results imply that chromogranin A homodimerizes shortly after synthesis in the endoplasmic reticulum and that the loop-mediated homodimeric state is an essential prerequisite for its sorting, in the trans-Golgi-network, to secretory granules.

A Eichmann, Anne Grapin-Botton, L Kelly, T Graf, N M Le Douarin, M Sieweke
The expression pattern of the mafB/kr gene in birds and mice reveals that the kreisler phenotype does not represent a null mutant.
Mech Dev, 65(1-2) 111-122 (1997)
DOI

The recessive mouse mutation kreisler affects hindbrain segmentation and inner ear development in homozygous mice. The mouse gene affected by the mutation was found to encode a basic domain leucine-zipper (bZIP)-type transcription factor of the Maf-family named kr (Cordes, S.P. and Barsh, G.S. (1994) Cell 79, 1025-1034). The avian bZIP transcription factor mafB, which shows high homology to kr, has been identified as an interaction partner of c-Ets 1 (Sieweke, M.H., Tekotte, M.H., Frampton, J. and Graf, T. (1996) Cell 85, 49-60). Here we demonstrate by Southern blot analysis that mafB is the avian homologue of kr, and present a detailed pattern of its expression during avian and murine embryonic development. Consistent with the kreisler phenotype, mafB is expressed in avians in the tissues which are affected by the mouse mutation: rhombomeres 5 and 6 (r5 and r6) and the neural crest derived from these rhombomeres. However, our analysis reveals a variety of additional expression sites: mafB/kr expression persists in vestibular and acoustic nuclei and is also observed in differentiating neurons of the spinal cord and brain stem. Restricted expression sites are found in the mesonephros, the perichondrium, and in the hemopoietic system. Since these expression sites are conserved between mouse and chicken we reexamined homozygous kreisler mice for unrevealed phenotypes in the hemopoietic system. However, peritoneal macrophages from homozygous kreisler mice were found to be functionally normal and still expressed mafB/kr. Other adult tissues examined from homozygous kreisler mice had also not lost mafB/kr expression. Our results thus indicate that the kreisler mutation involves a tissue specific gene inactivation and suggest additional roles for mafB/kr in later developmental and differentiation processes that are not revealed by the mutation.

Rachel Macdonald, John Scholes, Uwe Strähle, Caroline Brennan, Nigel Holder, Michael Brand, Stephen W. Wilson
The Pax protein Noi is required for commissural axon pathway formation in the rostral forebrain.
Development, 124(12) 2397-2408 (1997)
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No-isthmus (Noi) is a member of the zebrafish Pax family of transcriptional regulators that is expressed in restricted domains of the developing CNS. In the developing eye and optic nerve, the Noi+ cells are primitive glial cells that line the choroid fissure and optic stalk/nerve to its junction with the optic tract. This pattern of Noi expression is retained in the adult, defining the optic nerve astroglia, which wrap the left and right nerves separately at the midline, thus forming the bodily crossed optic chiasm found in fish. In embryos carrying mutations in the noi gene, the choroid fissure fails to close, glial cells of the optic nerve fail to differentiate and optic axons exhibit abnormal trajectories exiting the eye and at the midline of the diencephalon. Optic axons select inappropriate pathways into the contralateral optic nerve, rostrally towards the anterior commissure and along the ipsilateral optic tract. Noi+ cells also border the pathway of axons in the postoptic commissure, which is located adjacent to the optic chiasm. These postoptic commissural axons are defasciculated and also exhibit pathfinding defects in noi- embryos. These results indicate that Noi is required in cells that line the pathways taken by optic and non-optic commissural axons for guidance across the midline of the diencephalon. We find that expression of two members of the Netrin family of axon guidance molecules and the signalling protein Sonic hedgehog is disturbed in noi- embryos, whereas several members of the Eph family of receptors and ligands show no obvious alterations in expression at the diencephalic midline.

Anne Grapin-Botton, M A Bonnin, N M Le Douarin
Hox gene induction in the neural tube depends on three parameters: competence, signal supply and paralogue group.
Development, 124(4) 849-859 (1997)

It has been previously shown that Hox gene expression in the rhombencephalon is controlled by environmental cues. Thus posterior transposition of anterior rhombomeres to the r7/8 level results in the activation of Hox genes of the four first paralog groups and in homeotic transformations of the neuroepithelial fate according to its position along the anteroposterior axis. We demonstrate here that although the anteroposterior levels of r2 to r6 express Hox genes they do not have inducing activity on more anterior territories. If transposed at the posterior rhombencephalon and trunk level, however, the same anterior regions are able to express Hox gene such as Hoxa-2, a-3 or b-4. We also provide evidence that these signals are transferred by two paths: one vertical, arising from the paraxial mesoderm, and one planar, travelling in the neural epithelium. The competence to express Hox genes extends up to the forebrain and midbrain but expression of Hox genes does not preclude Otx2 expression in these territories and results only in slight changes in their phenotypes. Similarly, rhombomeres transplanted to posterior truncal levels turned out to be able to express posterior genes of the first eight paralog groups to the exclusion of others located downstream in the Hox genes genomic clusters. This suggests that the neural tube is divided into large territories characterized by different Hox gene regulatory features.

N M Le Douarin, A Grapin-Botton
Contrôle génétique du développement du rhombencéphale par les gènes Hox étudié chez l'embryon d'oiseau par la méthode des chimères caille-poulet [Genetic control of rhombencephalon development by Hox genes studied in bird embryo by the quail-chick chimera method].
C R Seances Soc Biol Fil, 191(1) 29-42 (1997)

The rhombencephalic neural tube is transiently segmented along the anteroposterior axis into 8 rhombomeres. Each rhombomere, as well as its derived neural crest cells, is characterized by the expression of a specific set of Hox genes which constitute its Hox code. This code is supposed to define the morphogenetic program of these cells according to their position. We took advantage of the quail/chick chimera system to study the regulation of Hox gene expression in neural tube and neural crest cells. We have therefore ectopically transplanted the presumptive territories of the future rhombomeres and studied the evolution of their Hox code. We evidence in the posterior rhombencephalon and the spinal cord a posteriorising signal able to induce Hox gene expression, to repress anterior molecular markers and to control the subsequent development of the neural tube. This signal is conveyed horizontally in the plane of the neuroepithelium and vertically from the mesoderm to the ectoderm. The anteroposterior identity of the neural crest cells seem independent from this inducer after formation of the neural fold.

Suzanne Eaton, Roger Wepf, K Simons
Roles for Rac1 and Cdc42 in planar polarization and hair outgrowth in the wing of Drosophila.
J Cell Biol, 135(5) 1277-1289 (1996)
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The wing of Drosophila melanogaster is covered by an array of distally pointing hairs. A hair begins as a single membrane outgrowth from each wing epithelial cell, and its distal orientation is determined by the restriction of outgrowth to a single distal site on the cell circumference (Wong, L., and P. Adler. 1993. J. Cell Biol. 123:209-211.). We have examined the roles of Cdc42 and Rac1 in the formation of wing hairs. We find that Cdc42 is required for localized actin polymerization in the extending hair. Interfering with Cdc42 activity by expression of a dominant negative protein abolishes both localized actin polymerization and hair outgrowth. In contrast, Rac1 is important for restricting the site at which hairs grow out. Cells expressing the dominant negative Rac1N17 fail to restrict outgrowth to a single site and give rise to multiple wing hairs. This polarity defect is associated with disturbances in the organization of junctional actin and also with disruption of an intricate microtubule network that is intimately associated with the junctional region. We also find that apical junctions and microtubules are involved in structural aspects of hair outgrowth. During hair formation, the apical microtubules that point distally elongate and fill the emerging wing hair. As the hair elongates, junctional proteins are reorganized on the proximal and distal edges of each cell.

G Couly, Anne Grapin-Botton, P Coltey, N M Le Douarin
The regeneration of the cephalic neural crest, a problem revisited: the regenerating cells originate from the contralateral or from the anterior and posterior neural fold.
Development, 122(11) 3393-3407 (1996)

The mesencephalic and rhombencephalic levels of origin of the hypobranchial skeleton (lower jaw and hyoid bone) within the neural fold have been determined at the 5-somite stage with a resolution corresponding to each single rhombomere, by means of the quail-chick chimera technique. Expression of certain Hox genes (Hoxa-2, Hoxa-3 and Hoxb-4) was recorded in the branchial arches of chick and quail embryos at embryonic days 3 (E3) and E4. This was a prerequisite for studying the regeneration capacities of the neural crest, after the dorsal neural tube was resected at the mesencephalic and rhombencephalic level. We found first that excisions at the 5-somite stage extending from the midmesencephalon down to r8 are followed by the regeneration of neural crest cells able to compensate for the deficiencies so produced. This confirmed the results of previous authors who made similar excisions at comparable (or older) developmental stages. When a bilateral excision was followed by the unilateral homotopic graft of the dorsal neural tube from a quail embryo, thus mimicking the situation created by a unilateral excision, we found that the migration of the grafted unilateral neural crest (quail-labelled) is bilateral and compensates massively for the missing crest derivatives. The capacity of the intermediate and ventral neural tube to yield neural crest cells was tested by removing the chick rhombencephalic neural tube and replacing it either uni- or bilaterally with a ventral tube coming from a stage-matched quail. No neural crest cells exited from the ventral neural tube but no deficiency in neural crest derivatives was recorded. Crest cells were found to regenerate from the ends of the operated region. This was demonstrated by grafting fragments of quail neural fold at the extremities of the excised territory. Quail neural crest cells were seen migrating longitudinally from both the rostral and caudal ends of the operated region and filling the branchial arches located inbetween. Comparison of the behaviour of neural crest cells in this experimental situation with that showed by their normal fate map revealed that crest cells increase their proliferation rate and change their migratory behaviour without modifying their Hox code.

Kurt I. Anderson, Yu-Li Wang, J Victor Small
Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body.
J Cell Biol, 134(5) 1209-1218 (1996)
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We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.

Jonathon Howard, J A Spudich
Is the lever arm of myosin a molecular elastic element?
Proc Natl Acad Sci U.S.A., 93(9) 4462-4464 (1996)
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37), while the strongly bound state time, t s , determines the velocity at which movement occurs (16 18). Thus, the elevated ATPase is not a reflection of a change in t s and thus is not relevant to velocity considerations. Importantly, we can conclude that none of the mutated myosins have been hampered in their ability to hydrolyze ATP, which we take as an indication that the mutations did not have generally deleterious effects on the myosin. The mutant and wild-type myosins were then subjected to an in vitro sliding filament motility assay (32, 33, 41). The sliding velocities increased with increasing number of light chain binding sites for wild-type and mutant myosins (Fig. 4 Left), consistent with the swinging neck-lever model. Most significantly, the 2xELCBS mutant form moved faster than the wild-type myosin (the range was 21 33% faster in four experiments), and the most straightforward interpretation of making the enzyme move faster is that the neck behaves like a lever arm. If one makes the further (undoubtedly oversimplified) assumptions that, first, all of the stroke derives from the movement of a relatively rigid lever arm that rotates about some fulcrum point, and, second, that the 2xELCBS mutant has a lever arm that is elongated by the linear insertion of one extra ELC binding domain, then one can extrapolate the points in Fig. 4 Left back to zero lever arm length. This ``fulcrum point'' in the structure is shown by the red dot in Fig. 1, and the sliding velocities are now proportional to the length of lever arm when the same set of data is replotted against the length measured from this putative fulcrum point (Fig. 4 Right). Milligan and colleagues (13, 14) provided complementary evidence for a fulcrum point in this region by comparing helically reconstituted actomyosin structures between the ADP-bound and rigor (no nucleotide) states. Interestingly, this putative fulcrum point is very near to what has been called the reactive thiol region in skeletal muscle myosin, which undergoes dramatic changes in structure during the ATPase cycle (38, 39). There are other, albeit more complicated, explanations for the velocity results shown in Fig. 4. For example, it is possible that there is another minor but independent mechanism to generate movement, such as a change in binding angle between actin and the myosin head at the actin myosin binding face, as has been long postulated (40). Thus, the fulcrum point of the swinging motion of the lever arm may be to the right of the red dot in Fig. 1 Upper, closer to the ELC binding domain. Another possibility is that t s is linearly related to the number of light chain binding sites, and this contributes to the changes in velocity since v d t s . This possibility can be tested in the future. For example, the feedback-enhanced laser trap assay (6) can be used to determine t s directly as a measure of the duration time of the myosin displacement. In summary, the linear relationship between sliding velocity and the neck length strongly supports the swinging neck-lever model. It is particularly noteworthy that we were able to create a mutant motor that moves faster than the wild type in a way the model predicts. An interesting point to consider (see Appendix) is that this lever arm of the S1 will have a certain bending stiffness and may be the structural equivalent of the elastic element that has long been known to be part of the actin myosin system, as elucidated by tension-transient experiments using muscle fibers (40). APPENDIX: Is the lever arm of myosin a molecular elastic element? J ONATHON H OWARD * AND J AMES A. S PUDICH *Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290; and Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 The classic experiments of Huxley and Simmons (40) defined an elastic element in muscle that has been attributed to the myosin molecule. They measured the tension drop when a stimulated muscle held at a fixed length is rapidly shortened through a small distance and found that a component of the system behaves like a linear spring. Such an elastic element is fundamental to force generation because it allows strain to develop within the motor prior to movement of the cargo; relief of this strain then drives the relative displacement of the motor and the track along which it moves. While diagrammatic representations often show this elastic spring as being part of the myosin rod beyond the light-chain binding domain of the molecule, we consider here that the elastic element is the light-chain binding domain itself and may account quantitatively for the cross-bridge stiffness observed in muscle experiments. The head domain of myosin, commonly called subfragment 1 or S1, is the only part of the myosin molecule required for movement in vitro (33) and for production of force similar to that seen in intact muscle (9, 42). An unusual structural feature of S1 is the 8-nm-long light-chain binding domain that is at F IG . 4. Sliding velocities of mutant and wild-type myosins. Bars indicate standard deviation. (Left) Sliding velocity as a function of the number of light chain binding sites. These data are representative of four independent experiments with different preparations of proteins over a period of a year. (Right) The same set of data is replotted against the length of the putative lever arm. The lever arm lengths for wild type and each mutant were measured from the fulcrum point shown as a red dot in Fig. 1 to the 90 bend at the C terminus of the long heavy chain -helix (shown in violet in Fig. 1) that makes up the neck domain--these lengths are 3-D computer-graphic measurements based on the crystal structure (2). 4462 Biophysics: Uyeda et al. Proc. Natl. Acad. Sci. USA 93 (1996) the C terminus of the S1 moiety (2, 3). It has been suggested that this region of the myosin head could serve as a lever arm to amplify smaller conformational changes elsewhere in the motor domain (5, 13 15, 19 21, 43, and this paper). Indeed, fluorescence polarization experiments have shown that the light-chain binding region changes orientation by a minimum of 3 relative to the filament axis in muscle in response to quick length changes and during the transitions between states of the cross-bridge cycle associated with active force production (15). While this angle change would appear to be too small to account for a unitary displacement of several nanometers (6), it is a minimum value for technical reasons, and two other complementary studies strongly support the lever arm hypothesis. First, electron microscopy of decorated actin filaments showed that a rotation of the light-chain binding domain through 23 accounts well for the two different conformations that S1 adopts depending on whether ADP is bound at the active site; the difference could account for as much as 3.5 nm of movement of the far C terminus of S1 (13, 14). Second, this paper used molecular genetic approaches to shorten, and importantly, to elongate the lever arm and demonstrate a linear relationship between the lever arm length and the velocity with which the myosin moves in vitro. We argue here that the lever arm could also be the elastic element referred to above, since the elasticity of the light-chain binding domain is expected to be comparable to that measured in the rapid shortening experiments. Furthermore, the nature of the light chains and their interaction with the 8-nm-long -helical stretch of the heavy chain at the C terminus of S1 may determine the spring constant of the light-chain binding domain and therefore affect the force that the molecular motor can produce. Consider a very simple model of the lever arm as a clamped beam of length L and flexural rigidity (the resistance to bending forces) equal to EI. If a transverse force F is applied at the free end, then this end will move through a distance x such that: F 3EI L 3 x (44). In other words, the beam has a stiffness 3EI L 3 3kTL p L 3 , where L p EI kT is the persistence length (45), k is the Boltzmann constant, and T is temperature. The light-chain binding domain has a length of 8 nm. It seems reasonable to consider that the lever arm, which has two light chains wrapped around the long -helix, has a rigidity similar to that of a coiled coil, which has two -helices wrapped around each other. The persistence length of a coiled coil is 100 nm (J.H., unpublished measurements derived from the coiled-coil myosin rod domain). For comparison, the L p of DNA, which has a dimension similar to these two protein structures, is 50 nm (46). Substituting L 8 nm, L p 100 nm, and kT 4 pN nm, we obtain 2 pN nm. On the other hand, the rapid shortening experiments indicate a muscle stiffness equal to 0.27 pN nm when normalized to the total number of myosin heads per half sarcomere [a shortening of 6 nm per half sarcomere drops the force from 1.6 pN per head to zero (47)]. Since only about half the compliance in muscle resides in the myosin heads and the other half resides in the actin filaments (e.g., see ref. 48), this value for the stiffness needs to be doubled to 0.5 pN nm per myosin head. If only a quarter of the myosin heads were attached during isometric contraction (duty ratio of 0.25; refs. 6 and 16), then the stiffness per attached head would be 2 pN nm, equal to that derived above! Clearly, this equality could be fortuitous given the large uncertainties in both the experimental and theoretical stiffnesses. The assumptions made, however, are not unreasonable, and the calculations do show that it is quite plausible that the elasticity of myosin resides within the light-chain binding domain, which corresponds to the lever arm. Indeed, one expects the light-chain binding domain to contribute some compliance to the myosin molecule. There are three interesting predictions that follow from the hypothesis that the lever arm is the elastic element. (i) The motor force should be inversely proportional to the square of the length of the lever arm. To see this, let the force-generating conformational change be a rotation, through an angle , of the insertion point of the lever into the motor domain. Thus, in the absence of a restoring force, the tip of the lever arm (the C terminus of S1) would move through a distance x L , On the other hand if there were a restoring force (F max ) that prevented the C terminus of the lever arm from moving, then F max 3kTL p L 3 x 3kTL p L 3 L 3kTL p L 2 . Since the angular change is independent of the length of the lever arm, it follows that the maximum force is proportional to L 2 . On the other hand, if the lever arm acted as a rigid rod and the elasticity were due to a pivotal spring (49) located at the point of insertion into the motor domain, then the maximum force would depend on L 1 . (ii) The maximum work should be inversely proportional to the lever length (L 1 ). To see this, note that if the restoring force (F o ) is less than the maximum force, then the tip will move through a distance x F o (the working stroke), and the amount of work done will equal W F o x F o F o x F o2 . The maximum work occurs when F o F max 2, and W max F max x 4 3 4 kTL p 2 L. That is, the maximum work is inversely proportional to the lever length. This leads to a paradox at the shortest lever arm lengths where the work might get so large as to exceed the theoretical maximum force. Presumably a motor with a very short lever arm will fail at high forces (the rotation through would not take place). (iii) The maximum force will depend on the stiffness of the lever arm. For example, if the link between the ELC and the catalytic domain of S1 and or the link between the ELC and RLC domains were flexible, we would expect a smaller stiffness and thus a smaller force. Thus, the properties of the light chains may affect the flexural rigidity of the lever arm, thereby regulating the force produced by a particular myosin isoform. The establishment of laser trap technologies to measure directly the force and work produced by a single myosin molecule (6, 50) and systems that allow genetic engineering of the molecular motor myosin to produce myosins with different lever arm lengths (this paper) should allow critical testing of whether force production is inversely proportional to the lever arm length squared, as predicted by the elastic lever arm model. The same approaches should allow testing of the concept that the nature of the light chains modulates the spring constant of the elastic lever arm and therefore the amount of force that can be produced by different isoforms of myosin, Biophysics: Uyeda et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4463 which have different light chains. Indeed, even skeletal myosin binds two alternate forms of RLC, for reasons that have been unclear. Moreover, myosin light chains are altered by posttranslational modifications, such as phosphorylation in the case of smooth muscle myosin and Dictyostelium myosin (for a review, see ref. 51) and binding of Ca 2 in the case of scallop myosin (52). One goal then is to use molecular genetics and laser trap technology to gain detailed molecular information about the physiological relevance of altered myosin types. We thank members of the Spudich laboratory for stimulating discussions and advice, and K. Zaita for technical assistance. This work is supported by a Human Frontier Science Program short-term fellowship to T.Q.P.U. and National Institutes of Health Grant GM33289 to J.A.S. 1. Itakura, S., Yamakawa, H., Toyoshima, Y. Y., Ishijima, A., Kojima, T., Harada, Y., Yanagida, T., Wakabayashi, T. & Sutoh, K. (1993) Biochem. Biophys. Res. Commun. 196, 1504 1510. 2. Rayment, I., Rypniewski, W., Schmidt-Base, K., Smith, R., Tomchick, D., Benning, M., Winkelmann, D., Wesenberg, G. & Holden, H. (1993) Science 261, 50 58. 3. Xie, X., Harrison, D. H., Schlichting, I., Sweet, R. M., Kalabokis, V. N., Szent-Gyorgyi, A. G. & Cohen, C. (1994) Nature (London) 368, 306 312. 4. Cooke, R., Crowder, M. S., Wendt, C. H., Bamett, V. A. & Thomas, D. D. (1984) Adv. Exp. Med. Biol. 170, 413 427. 5. Vibert, P. & Cohen, C. (1988) J. Muscle Res. Cell Motil. 9, 296 305. 6. Finer, J. T., Simmons, R. M. & Spudich, J. A. (1994) Nature (London) 368, 113 119. 7. Ishijima, A., Harada, Y., Kojima, H., Funatsu, T., Higuchi, H. & Yanagida, T. 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Gene Myers, Mudita Jain
Going Against the Grain
In: Proceedings of the Third South American Workshop on String Processing : Recife, Brazil (1996), Recife, Brazil, South American Workshop on String Processing (1996), 203-213
PDF

Nicole M. Le Douarin, Anne Grapin-Botton, Martin Catala
Patterning of the neural primordium in the avian embryo.
Semin Cell Dev Biol, 7(2) 157-167 (1996)

Suzanne Eaton, P Auvinen, L Luo, Y N Jan, K Simons
CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium.
J Cell Biol, 131(1) 151-164 (1995)
DOI

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.

Jonathon Howard, Edgar Meyhöfer
The force generated by a single kinesin molecule against an elastic load.
Proc Natl Acad Sci U.S.A., 92(2) 574-578 (1995)
PDF

To probe the mechanism by which the motor protein kinesin moves along microtubules, we have developed a highly sensitive technique for measuring the force exerted by a single motor molecule. In this technique, one end of a microtubule is attached to the tip of a flexible glass fiber of calibrated stiffness. The other end of the microtubule makes contact with a surface sparsely coated with kinesin. By imaging the tip of the glass fiber on a photodiode detector, displacement of the microtubule by kinesin through as little as 1 nm can be detected and forces as small as 1 pN resolved. Using this force-fiber apparatus we have characterized the mechanical output of this molecular motor. The speed at which a molecule of kinesin moved along the surface of a microtubule decreased linearly as the elastic force was increased. The force required to stop a single kinesin molecule was 5.4 +/- 1.0 pN (mean +/- SD; n = 16), independent of the stiffness of the fiber, the damping from the fluid, and whether the ATP concentration was high or low.

Carolyn J Lawrence, S Honda, N W Parrott, T C Flood, L Gu, L Zhang, Mudita Jain, S Larson, E W Myers
The genome reconstruction manager: a software environment for supporting high-throughput DNA sequencing.
Genomics, 23(1) 192-201 (1994)
PDF DOI

A new software system designed for use in high-throughput DNA sequencing laboratories is described. The Genome Reconstruction Manager (GRM) was developed from requirements derived from ongoing large-scale DNA sequencing projects. Object-oriented principles were followed in designing the system, and tools supporting object-oriented system development were employed for its implementation. GRM provides several advances in software support for high-throughput DNA sequencing: support for random, directed, and mixed sequencing strategies; a novel system for fragment assembly; a commercial object data-base management system for data storage; a client/server architecture for using network computational servers; and an underlying data model that can evolve to support fully automatic sequence reconstruction. GRM is currently being deployed for production use in high-throughput DNA sequencing projects.

T Tabata, Suzanne Eaton, T B Kornberg
The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation.
Genes Dev, 6(12B) 2635-2645 (1992)
DOI

cDNAs were isolated that represent transcripts of the Drosophila segment polarity gene, hedgehog (hh). Sequence analysis reveals a motif characteristic of a transmembrane domain, suggesting that the hh protein is membrane-associated. hh expression in epidermal cells is confined to the posterior compartments and coincides precisely with that of engrailed (en). Despite the similar patterns of expression in the cellular blastoderm, hh expression is independent of en, but hh expression becomes sensitive to and dependent on en during the extended germ band stage. The ectopic expression of hh that is normally induced in patched (ptc) mutant embryos does not appear in ptc en double mutants. We discuss these findings in terms of the relationship between en and hh, and the role of the hh function.

C Bucci, Robert G. Parton, I H Mather, Hendrik G Stunnenberg, K Simons, Bernard Hoflack, Marino Zerial
The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.
Cell, 70(5) 715-728 (1992)
DOI

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.

E W Myers, D W Mount
Computer program for the IBM personal computer which searches for approximate matches to short oligonucleotide sequences in long target DNA sequences.
Nucleic Acids Res, 14(1) 501-508 (1986)
Open Access PDF

We describe a program which may be used to find approximate matches to a short predefined DNA sequence in a larger target DNA sequence. The program predicts the usefulness of specific DNA probes and sequencing primers and finds nearly identical sequences that might represent the same regulatory signal. The program is written in the C programming language and will run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The program has been integrated into an existing software package for the IBM personal computer (see article by Mount and Conrad, this volume). Some examples of its use are given.

Eugene Myers
AN O(ElogE+i) EXPECTED TIME ALGORITHM FOR THE PLANAR SEGMENT INTERSECTION PROBLEM
SIAM J Sci Comput, 14(3) 625-637 (1985)
PDF

ItisanopenquestionincomputationalgeometryastowhetherthereexistsanO(ElogE+I) algorithm to determine the I intersections of a collection of E line segments in the plane. An approach utilizing a work list bubble sort and a distribution-based search is presented. The resulting algorithm has O(E log E + I) expected time complexity. In the worst case the algorithm has the same complexity as the algorithm of Bentley and Ottmann [IEEE Trans. Comput., 28 (1979), pp. 643-647]: O(E log E + I log E). The algorithm requires only O(E) space and in contrast to prior work, no restrictions are placed upon the nature of the intersections.

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