* joint first author # joint corresponding author

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.

Mrityunjoy Kar, Laura T Vogel, Gaurav Chauhan, Suren Felekyan, Hannes Ausserwöger, Timothy J Welsh, Furqan Dar, Anjana R Kamath, Tuomas P J Knowles, Anthony Hyman#, Claus A M Seidel, Rohit V Pappu#
Solutes unmask differences in clustering versus phase separation of FET proteins.
Nat Commun, 15(1) Art. No. 4408 (2024)
Open Access DOI
Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Aaron D Wolfe, John N Koberstein, Chadwick B Smith, Melissa L Stewart, Ian J Gonzalez, Marc Hammarlund, Anthony Hyman, Philip J S Stork, Richard H Goodman#, Daniel A Colón-Ramos#
Local and dynamic regulation of neuronal glycolysis in vivo.
Proc Natl Acad Sci U.S.A., 121(3) Art. No. e2314699121 (2024)
Open Access DOI
Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.

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.

Shaon Basu, Paula Martínez-Cristóbal, Marta Frigolé-Vivas, Mireia Pesarrodona, Michael Lewis, Elzbieta Szulc, C Adriana Bañuelos, Carolina Sánchez-Zarzalejo, Stasė Bielskutė, Jiaqi Zhu, Karina Pombo-García, Carla Garcia-Cabau, Levente Zodi, Hannes Dockx, Jordann Smak, Harpreet Kaur, Cristina Batlle, Borja Mateos, Mateusz Biesaga, Albert Escobedo, Lídia Bardia, Xavier Verdaguer, Alessandro Ruffoni, Nasrin R Mawji, Jun Wang, Jon K Obst, Teresa Tam, Isabelle Brun-Heath, Salvador Ventura, David Meierhofer, Jesús García, Paul Robustelli, Travis H Stracker, Marianne D Sadar, Antoni Riera#, Denes Hnisz#, Xavier Salvatella#
Rational optimization of a transcription factor activation domain inhibitor.
Nat Struct Mol Biol, 30(12) 1958-1969 (2023)
Open Access DOI
Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.

Xuezhao Feng✳︎, Daxiao Sun✳︎#, Yanchang Li✳︎, Jinpei Zhang✳︎, Shiyu Liu, Dachuan Zhang, Jingxiang Zheng, Qing Xi, Haisha Liang, Wenkang Zhao, Ying Li, Mengbo Xu, Jiayu He, Tong Liu, Ayshamgul Hasim, Meisheng Ma, Ping Xu#, Na Mi#
Local membrane source gathering by p62 body drives autophagosome formation.
Nat Commun, 14(1) Art. No. 7338 (2023)
Open Access DOI
Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

Hao Yin, Yin-Lei Han, Xiao Yan#, Yi-Xin Guan#
Hematoxylin modulates tau-RD protein fibrillization and ameliorates Alzheimer's disease-like symptoms in a yeast model.
Int J Biol Macromol, 250 Art. No. 126140 (2023)
Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases with no effective treatment options available. The formation of insoluble amyloid fibrils of the hyperphosphorylated tau protein is intimately associated with AD, hence the tau protein has been a key target for AD drug development. In this work, hematoxylin was discovered as a dual functional compound, that is, acting in the inhibition of repeat domain of tau (tau-RD) protein fibrillogenesis and remodeling of pre-formed tau-RD fibrils in vitro. Meanwhile, hematoxylin was able to reduce the accumulation of tau-RD aggregates in Saccharomyces cerevisiae. Experimental and computational studies indicated that hematoxylin directly interacts with tau-RD protein through hydrophobic forces, hydrogen bonds, π-cation interactions, and π-π stackings. In addition, cellular viability assays showed that hematoxylin greatly reduced cytotoxicity induced by tau-RD aggregates. In summary, hematoxylin might be a promising candidate for further development as a potential therapeutic drug for AD patients.

Rohit V Pappu, Samuel R Cohen, Furqan Dar, Mina Farag, Mrityunjoy Kar
Phase Transitions of Associative Biomacromolecules.
Chem. Rev., 123(14) 8945-8987 (2023)
Multivalent proteins and nucleic acids, collectively referred to as multivalent associative biomacromolecules, provide the driving forces for the formation and compositional regulation of biomolecular condensates. Here, we review the key concepts of phase transitions of aqueous solutions of associative biomacromolecules, specifically proteins that include folded domains and intrinsically disordered regions. The phase transitions of these systems come under the rubric of coupled associative and segregative transitions. The concepts underlying these processes are presented, and their relevance to biomolecular condensates is discussed.

Esteban Meca#, Anatol W Fritsch, Juan M Iglesias-Artola, Simone Reber, Barbara Wagner#
Predicting disordered regions driving phase separation of proteins under variable salt concentration.
Front Phys, 11 Art. No. 1213304 (2023)
Open Access DOI
We investigate intrinsically disordered regions (IDRs) of phase separating proteins regarding their impact on liquid-liquid phase separation (LLPS) of the full protein. Our theoretical approach uses a mean-field theory that accounts for sequence-dependent electrostatic interactions via a Random Phase Approximation (RPA) and in addition allows for variable salt concentration for the condensed and dilute protein phases. The numerical solution of the complete phase diagrams together with the tie lines that we derive for this model system leaves two parameters to be determined by fitting experimental data on concentrations of all species involved in the system. For our comparisons, we focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that its long IDR near the C-terminus promotes LLPS, which we validate through direct comparison with in vitro experimental results under the same physiological conditions. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS has been intensively studied. Apart from the LC domain we here investigate theoretically two IDRs, one near the N-terminus and another near the C-terminus. Our theoretical analysis of these domains predict that the IDR at the N-terminus (aa 1-285) is the main driver of LLPS of FUS by comparison to in vitro experiments of the full length protein under the same physiological temperature and salt conditions.

Nadia Rostam, Soumyadeep Ghosh, Chi Fung Willis Chow, Anna Hadarovich, Cedric Landerer, Rajat Ghosh, HongKee Moon, Lena Hersemann, Diana M Mitrea, Isaac A Klein, Anthony Hyman, Agnes Toth-Petroczy
CD-CODE: crowdsourcing condensate database and encyclopedia.
Nat Methods, 20(5) 673-676 (2023)
Open Access DOI
The discovery of biomolecular condensates transformed our understanding of intracellular compartmentalization of molecules. To integrate interdisciplinary scientific knowledge about the function and composition of biomolecular condensates, we developed the crowdsourcing condensate database and encyclopedia ( cd-code.org ). CD-CODE is a community-editable platform, which includes a database of biomolecular condensates based on the literature, an encyclopedia of relevant scientific terms and a crowdsourcing web application. Our platform will accelerate the discovery and validation of biomolecular condensates, and facilitate efforts to understand their role in disease and as therapeutic targets.

Xiaojie Zhang✳︎, Sindhuja Sridharan✳︎, Ievgeniia Zagoriy, Christina Eugster Oegema, Cyan Ching, Tim Pflaesterer, Herman K H Fung, Isabelle Becher, Ina Poser, Christoph W Müller, Anthony Hyman, Mikhail M Savitski#, Julia Mahamid#
Molecular mechanisms of stress-induced reactivation in mumps virus condensates.
Cell, 186(9) 1877-1894 (2023)
Open Access DOI
Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

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.

William E Arter✳︎, Runzhang Qi✳︎, Nadia A Erkamp✳︎, Georg Krainer✳︎, Kieran Didi, Timothy J Welsh, Julia Acker, Jonathan Nixon-Abell, Seema Qamar, Jordina Guillén-Boixet, Titus Franzmann, David Kuster, Anthony Hyman, Alexander Borodavka, Peter St George-Hyslop, Simon Alberti, Tuomas P J Knowles
Biomolecular condensate phase diagrams with a combinatorial microdroplet platform.
Nat Commun, 13(1) Art. No. 7845 (2022)
Open Access DOI
The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.

Jonathan Bauermann✳︎, Sudarshana Laha✳︎, Patrick M McCall, Frank Jülicher#, Christoph A. Weber#
Chemical Kinetics and Mass Action in Coexisting Phases.
J Am Chem Soc, 144(42) 19294-19304 (2022)
The kinetics of chemical reactions are determined by the law of mass action, which has been successfully applied to homogeneous, dilute mixtures. At nondilute conditions, interactions among the components can give rise to coexisting phases, which can significantly alter the kinetics of chemical reactions. Here, we derive a theory for chemical reactions in coexisting phases at phase equilibrium. We show that phase equilibrium couples the rates of chemical reactions of components with their diffusive exchanges between the phases. Strikingly, the chemical relaxation kinetics can be represented as a flow along the phase equilibrium line in the phase diagram. A key finding of our theory is that differences in reaction rates between coexisting phases stem solely from phase-dependent reaction rate coefficients. Our theory is key to interpreting how concentration levels of reactive components in condensed phases control chemical reaction rates in synthetic and biological systems.

Shovamayee Maharana✳︎#, Stefanie Kretschmer✳︎#, Susan Hunger, Xiao Yan, David Kuster, Sofia Traikov, Thomas Zillinger, Marc Gentzel, Shobha Elangovan, Padmanava Dasgupta, Nagaraja Chappidi, Nadja Lucas, Katharina Isabell Maser, Henrike Maatz, Alexander Rapp, Virginie Marchand, Young-Tae Chang, Yuri Motorin, Norbert Hubner, Gunther Hartmann, Anthony Hyman, Simon Alberti, Min Ae Lee-Kirsch#
SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.
Mol Cell, 82(19) 3712-3728 (2022)
Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

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.

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.

Mrityunjoy Kar, Furqan Dar, Timothy J Welsh, Laura T Vogel, Ralf Kühnemuth, Arindam Majumdar, Georg Krainer, Titus Franzmann, Simon Alberti, Claus A M Seidel, Tuomas P J Knowles, Anthony Hyman#, Rohit V Pappu#
Phase-separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions.
Proc Natl Acad Sci U.S.A., 119(28) Art. No. e2202222119 (2022)
Open Access DOI
Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.

Damian Wollny#, Benjamin Vernot, Jie Wang, Maria Hondele, Aram Safrastyan, Franziska Aron, Julia Micheel, Zhisong He, Anthony Hyman, Karsten Weis, J Gray Camp, T-Y Dora Tang#, Barbara Treutlein#
Characterization of RNA content in individual phase-separated coacervate microdroplets.
Nat Commun, 13(1) Art. No. 2626 (2022)
Open Access DOI
Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Juan M Iglesias-Artola, Björn Drobot, Mrityunjoy Kar, Anatol Fritsch, Hannes Mutschler, T-Y Dora Tang, Moritz Kreysing
Charge-density reduction promotes ribozyme activity in RNA-peptide coacervates via RNA fluidization and magnesium partitioning.
Nat Chem, 14(4) 407-416 (2022)
Open Access PDF DOI
It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA-peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes.

Roman Renger, Jose A. Morin, Regis P. Lemaitre, Martine Ruer-Gruss, Frank Jülicher, Andreas Hermann, Stephan W. Grill
Co-condensation of proteins with single- and double-stranded DNA.
Proc Natl Acad Sci U.S.A., 119(10) Art. No. e2107871119 (2022)
Open Access DOI
SignificanceBiomolecular condensates are intracellular organelles that are not bounded by membranes and often show liquid-like, dynamic material properties. They typically contain various types of proteins and nucleic acids. How the interaction of proteins and nucleic acids finally results in dynamic condensates is not fully understood. Here we use optical tweezers and fluorescence microscopy to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) condenses with individual molecules of single- and double-stranded DNA. We find that FUS adsorbs on DNA in a monolayer and hence generates an effectively sticky FUS-DNA polymer that collapses and finally forms a dynamic, reversible FUS-DNA co-condensate. We speculate that protein monolayer-based protein-nucleic acid co-condensation is a general mechanism for forming intracellular membraneless organelles.

Jose A. Morin✳︎, Sina Wittmann✳︎, Sandeep Choubey✳︎, Adam Klosin, Stefan Golfier, Anthony A. Hyman#, Frank Jülicher#, Stephan W. Grill#
Sequence-dependent surface condensation of a pioneer transcription factor on DNA.
Nat Phys, 18(3) 271-276 (2022)
Open Access DOI
Biomolecular condensates are dense assemblies of proteins that form distinct biochemical compartments without being surrounded by a membrane. Some, such as P granules and stress granules, behave as droplets and contain many millions of molecules. Others, such as transcriptional condensates that form on the surface of DNA, are small and contain thousands of molecules. The physics behind the formation of small condensates on DNA surfaces is still under discussion. Here we investigate the nature of transcription factor condensates using the pioneer transcription factor Kruppel-like factor 4 (Klf4). We show that Klf4 can phase separate on its own at high concentrations, but at low concentrations, Klf4 only forms condensates on DNA. Using optical tweezers, we demonstrate that these Klf4 condensates form on DNA as a type of surface condensation. This surface condensation involves a switch-like transition from a thin adsorbed layer to a thick condensed layer, which shows hallmarks of a prewetting transition. The localization of condensates on DNA correlates with sequence, suggesting that the condensate formation of Klf4 on DNA is a sequence-dependent form of surface condensation. Prewetting together with sequence specificity can explain the size and position control of surface condensates. We speculate that a prewetting transition of pioneer transcription factors on DNA underlies the formation and positioning of transcriptional condensates and provides robustness to transcriptional regulation. A DNA-binding protein condenses on DNA via a switch-like transition. Surface condensation occurs at preferential DNA locations suggesting collective sequence readout and enabling sequence-specificity robustness with respect to protein concentration.

Frank Sauer, Anatol Fritsch, Steffen Grosser, Steve Pawlizak, Tobias Kießling, Martin Reiss-Zimmermann, Mehrgan Shahryari, Wolf C Müller, Karl-Titus Hoffmann, Josef A Käs, Ingolf Sack
Whole tissue and single cell mechanics are correlated in human brain tumors.
Soft Matter, 17(47) 10744-10752 (2021)
Biomechanical changes are critical for cancer progression. However, the relationship between the rheology of single cells measured ex-vivo and the living tumor is not yet understood. Here, we combined single-cell rheology of cells isolated from primary tumors with in vivo bulk tumor rheology in patients with brain tumors. Eight brain tumors (3 glioblastoma, 3 meningioma, 1 astrocytoma, 1 metastasis) were investigated in vivo by magnetic resonance elastography (MRE), and after surgery by the optical stretcher (OS). MRE was performed in a 3-Tesla clinical MRI scanner and magnitude modulus |G*|, loss angle φ, storage modulus G', and loss modulus G'' were derived. OS experiments measured cellular creep deformation in response to laser-induced step stresses. We used a Kelvin-Voigt model to deduce two parameters related to cellular stiffness (μKV) and cellular viscosity (ηKV) from OS measurements in a time regimen that overlaps with that of MRE. We found that single-cell μKV was correlated with |G*| (R = 0.962, p < 0.001) and G'' (R = 0.883, p = 0.004) but not G' of the bulk tissue. These results suggest that single-cell stiffness affects tissue viscosity in brain tumors. The observation that viscosity parameters of individual cells and bulk tissue were not correlated suggests that collective mechanical interactions (i.e. emergent effects or cellular unjamming) of many cancer cells, which depend on cellular stiffness, influence the mechanical dissipation behavior of the bulk tissue. Our results are important to understand the emergent rheology of active multiscale compound materials such as brain tumors and its role in disease progression.

Mrityunjoy Kar, Avery D Posey, Furqan Dar, Anthony Hyman#, Rohit V Pappu#
Glycine-Rich Peptides from FUS Have an Intrinsic Ability to Self-Assemble into Fibers and Networked Fibrils.
Biochemistry, 60(43) 3213-3222 (2021)
Glycine-rich regions feature prominently in intrinsically disordered regions (IDRs) of proteins that drive phase separation and the regulated formation of membraneless biomolecular condensates. Interestingly, the Gly-rich IDRs seldom feature poly-Gly tracts. The protein fused in sarcoma (FUS) is an exception. This protein includes two 10-residue poly-Gly tracts within the prion-like domain (PLD) and at the interface between the PLD and the RNA binding domain. Poly-Gly tracts are known to be highly insoluble, being potent drivers of self-assembly into solid-like fibrils. Given that the internal concentrations of FUS and FUS-like molecules cross the high micromolar and even millimolar range within condensates, we reasoned that the intrinsic insolubility of poly-Gly tracts might be germane to emergent fluid-to-solid transitions within condensates. To assess this possibility, we characterized the concentration-dependent self-assembly for three non-overlapping 25-residue Gly-rich peptides derived from FUS. Two of the three peptides feature 10-residue poly-Gly tracts. These peptides form either long fibrils based on twisted ribbon-like structures or self-supporting gels based on physical cross-links of fibrils. Conversely, the peptide with similar Gly contents but lacking a poly-Gly tract does not form fibrils or gels. Instead, it remains soluble across a wide range of concentrations. Our findings highlight the ability of poly-Gly tracts within IDRs that drive phase separation to undergo self-assembly. We propose that these tracts are likely to contribute to nucleation of fibrillar solids within dense condensates formed by FUS.

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.

Anatol Fritsch✳︎, Andrés F Diaz-Delgadillo✳︎, Omar Adame-Arana✳︎, Carsten Hoege, Matthäus Mittasch, Moritz Kreysing, Mark Leaver, Anthony Hyman, Frank Jülicher, Christoph A. Weber
Local thermodynamics govern formation and dissolution of Caenorhabditis elegans P granule condensates.
Proc Natl Acad Sci U.S.A., 118(37) Art. No. e2102772118 (2021)
Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm Caenorhabditis elegans that play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams that are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intracellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the nonequilibrium nature of the cytoplasm is manifested on larger scales.

Edgar Boczek✳︎, Julius Fürsch✳︎, Marie Laura Niedermeier, Louise Jawerth, Marcus Jahnel, Martine Ruer-Gruß, Kai-Michael Kammer, Paul J Heid, Laura Mediani, Jie Wang, Xiao Yan, Andrei Pozniakovski, Ina Poser, Daniel Mateju, Lars Hubatsch, Serena Carra, Dr Simon Alberti, Anthony Hyman, Florian Stengel
HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain.
Elife, 10 Art. No. e69377 (2021)
Open Access DOI
Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

Leilei Xu, Mahboob Ali, Wenxiu Duan, Xiao Yuan#, Fatima Garba, McKay Mullen, Binwen Sun, Ina Poser, Hequan Duan, Jianlin Lu, Ruijun Tian, Yushu Ge, Lingluo Chu, Weijun Pan, Dongmei Wang, Anthony Hyman, Hadiyah Green, Lin Li, Zhen Dou#, Dan Liu#, Xing Liu#, Xuebiao Yao#
Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation.
Cell Rep, 36(2) Art. No. 109343 (2021)
Open Access DOI
Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

Axel Freischmidt, Anand Goswami, Katharina Limm, Vitaly Zimyanin, Maria Demestre, Hannes Glaß, Karlheinz Holzmann, Anika M Helferich, Sarah J Brockmann, Priyanka Tripathi, Alfred Yamoah, Ina Poser, Peter J Oefner, Tobias M Böckers, Eleonora Aronica, Albert C Ludolph, Peter Refsing Andersen, Andreas Hermann, Joachim Weis, Jörg Reinders, Karin M Danzer, Jochen H Weishaupt
A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.
Brain, 144(4) 1214-1229 (2021)
Open Access DOI
Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

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.

Oliver D Schwich✳︎, Nicole Blümel✳︎, Mario Keller✳︎, Marius Wegener, Samarth Thonta Setty, Melinda Elaine Brunstein, Ina Poser, Igor Ruiz De Los Mozos, Beatrix Suess, Christian Münch, François McNicoll, Kathi Zarnack#, Michaela Müller-McNicoll#
SRSF3 and SRSF7 modulate 3'UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels.
Genome Biol, 22(1) Art. No. 82 (2021)
Open Access DOI
Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.

Georg Krainer, Timothy J Welsh, Jerelle A Joseph, Jorge R Espinosa, Sina Wittmann, Ella de Csilléry, Akshay Sridhar, Zenon Toprakcioglu, Giedre Gudiškytė, Magdalena A Czekalska, William E Arter, Jordina Guillén-Boixet, Titus Franzmann, Seema Qamar, Peter St George-Hyslop, Anthony Hyman, Rosana Collepardo-Guevara, Simon Alberti, Tuomas P J Knowles
Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions.
Nat Commun, 12(1) Art. No. 1085 (2021)
Open Access DOI
Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

Guido van Mierlo#, Jurriaan R G Jansen, Jie Wang, Ina Poser, Simon J van Heeringen#, Michiel Vermeulen#
Predicting protein condensate formation using machine learning.
Cell Rep, 34(5) Art. No. 108705 (2021)
Open Access DOI
Membraneless organelles are liquid condensates, which form through liquid-liquid phase separation. Recent advances show that phase separation is essential for cellular homeostasis by regulating basic cellular processes, including transcription and signal transduction. The reported number of proteins with the capacity to mediate protein phase separation (PPS) is continuously growing. While computational tools for predicting PPS have been developed, obtaining a proteome-wide overview of PPS probabilities has remained challenging. Here, we present a phase separation analysis and prediction (PSAP) machine-learning classifier that, based solely on the amino acid content of a training set of known PPS proteins, can determine the phase separation likelihood for each protein in a given proteome. Through comparison with PPS databases, existing predictors, and experimental evidence, we demonstrate the validity and advantages of the PSAP classifier. We anticipate that the PSAP predictor provides a useful tool for future research aimed at identifying phase separating proteins in health and disease.

Arnaud Rondelet✳︎, Andrei I. Pozniakovsky✳︎, Devika Namboodiri, Richard Cardoso da Silva, Divya Singh, Marit Leuschner, Ina Poser, Andrea Ssykor, Julian Berlitz, Nadine Schmidt, Lea Röhder, Gerben Vader, Anthony Hyman, Alexander W. Bird
ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.
Life Sci Alliance, 4(2) Art. No. e202000836 (2021)
Open Access DOI
Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

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)
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.

Anthony Hyman
ASCB Keith Porter Lecture.
Mol Biol Cell, 31(26) 2864-2867 (2020)
Although we attempt to plan the way we live, perhaps the best description of life is from the words of Yogi Berra: "It's tough to make predictions, especially about the future." A little over a year ago, I thought my future was predictable; after a fulfilling career, I would enjoy a last decade of research before a comfortable retirement. Then I lost my beloved wife and life partner, Suzanne Eaton to a senseless act of violence, and all assumptions were called into question.

Louise Jawerth, Elisabeth Fischer-Friedrich, Suropriya Saha, Jie Wang, Titus Franzmann, Xiaojie Zhang, Jenny Sachweh, Martine Ruer, Mahdiye Ijavi, Shambaditya Saha, Julia Mahamid, Anthony Hyman#, Frank Jülicher#
Protein condensates as aging Maxwell fluids.
Science, 370(6522) 1317-1323 (2020)
Protein condensates are complex fluids that can change their material properties with time. However, an appropriate rheological description of these fluids remains missing. We characterize the time-dependent material properties of in vitro protein condensates using laser tweezer-based active and microbead-based passive rheology. For different proteins, the condensates behave at all ages as viscoelastic Maxwell fluids. Their viscosity strongly increases with age while their elastic modulus varies weakly. No significant differences in structure were seen by electron microscopy at early and late ages. We conclude that protein condensates can be soft glassy materials that we call Maxwell glasses with age-dependent material properties. We discuss possible advantages of glassy behavior for modulation of cellular biochemistry.

Tina Wiegand, Anthony Hyman
Drops and fibers - how biomolecular condensates and cytoskeletal filaments influence each other.
Emerg Top Life Sci, 4(3) 247-261 (2020)
Open Access DOI
The cellular cytoskeleton self-organizes by specific monomer-monomer interactions resulting in the polymerization of filaments. While we have long thought about the role of polymerization in cytoskeleton formation, we have only begun to consider the role of condensation in cytoskeletal organization. In this review, we highlight how the interplay between polymerization and condensation leads to the formation of the cytoskeleton.

Jeong-Mo Choi, Anthony Hyman, Rohit V Pappu
Generalized models for bond percolation transitions of associative polymers.
Phys Rev E, 102(4-1) Art. No. 042403 (2020)
Polymers with stickers-and-spacers architectures can drive phase-separation-aided bond percolation transitions. Here, we present a generalized mean-field model to enable the calculation of bond percolation thresholds for polymers with multiple types of stickers. Further, using graph-based Monte Carlo simulations we demonstrate how cooperativity in bond formation can give rise to reentrant phase behavior. When combined with recent advances for modeling phase separation, our approaches for calculating percolation lines could be useful for modeling hardening transitions for multivalent proteins.

Racha Chouaib, Adham Safieddine, Xavier Pichon, Arthur Imbert, Oh Sung Kwon, Aubin Samacoits, Abdel-Meneem Traboulsi, Marie-Cécile Robert, Nikolay Tsanov, Emeline Coleno, Ina Poser, Christophe Zimmer, Anthony Hyman, Hervé Le Hir, Kazem Zibara, Matthias Peter, Florian Mueller, Thomas Walter, Edouard Bertrand
A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.
Dev Cell, 54(6) 773-791 (2020)
Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Kamran Hosseini, Leon Sbosny, Ina Poser, Elisabeth Fischer-Friedrich
Binding Dynamics of α-Actinin-4 in Dependence of Actin Cortex Tension.
Biophys J, 119(6) 1091-1107 (2020)
Mechanosensation of cells is an important prerequisite for cellular function, e.g., in the context of cell migration, tissue organization, and morphogenesis. An important mechanochemical transducer is the actin cytoskeleton. In fact, previous studies have shown that actin cross-linkers such as α-actinin-4 exhibit mechanosensitive properties in their binding dynamics to actin polymers. However, to date, a quantitative analysis of tension-dependent binding dynamics in live cells is lacking. Here, we present a, to our knowledge, new technique that allows us to quantitatively characterize the dependence of cross-linking lifetime of actin cross-linkers on mechanical tension in the actin cortex of live cells. We use an approach that combines parallel plate confinement of round cells, fluorescence recovery after photobleaching, and a mathematical mean-field model of cross-linker binding. We apply our approach to the actin cross-linker α-actinin-4 and show that the cross-linking time of α-actinin-4 homodimers increases approximately twofold within the cellular range of cortical mechanical tension, rendering α-actinin-4 a catch bond in physiological tension ranges.

SoRi Jang, Zhao Xuan, Ross C Lagoy, Louise Jawerth, Ian J Gonzalez, Milind Singh, Shavanie Prashad, Hee Soo Kim, Avinash Patel, Dirk R Albrecht, Anthony Hyman, Daniel A Colón-Ramos
Phosphofructokinase Relocalizes into Subcellular Compartments with Liquid-like Properties In Vivo.
Biophys J, 120(7) 1170-1186 (2020)
Open Access DOI
Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.

Isaac A Klein, Ann Boija, Lena K Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan E Henninger, Krishna Shrinivas, Benjamin R Sabari, Ido Sagi, Victoria E Clark, Jesse M Platt, Mrityunjoy Kar, Patrick M McCall, Alicia V Zamudio, John C Manteiga, Eliot L Coffey, Charles H Li, Nancy M Hannett, Yang Eric Guo, Tim-Michael Decker, Tong Ihn Lee, Tinghu Zhang, Jing-Ke Weng, Dylan J Taatjes, Arup Chakraborty, Phillip A Sharp, Young Tae Chang, Anthony Hyman, Nathanael S Gray, Richard A Young
Partitioning of cancer therapeutics in nuclear condensates.
Science, 368(6497) 1386-1392 (2020)
The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.

Christiane Iserman, Christine Desroches Altamirano, Ceciel Jegers, Ulrike Friedrich, Taraneh Zarin, Anatol Fritsch, Matthäus Mittasch, Antonio Domingues, Lena Hersemann, Marcus Jahnel, Doris Richter, Ulf-Peter Guenther, Matthias W. Hentze, Alan M Moses, Anthony Hyman, Günter Kramer, Moritz Kreysing, Titus Franzmann, Simon Alberti
Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.
Cell, 181(4) 818-831 (2020)
Open Access PDF DOI
Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

Jordina Guillén-Boixet✳︎, Andrii Kopach✳︎, Alex S Holehouse, Sina Wittmann, Marcus Jahnel, Raimund Schlüßler, Kyoohyun Kim, Irmela Trussina, Jie Wang, Daniel Mateju, Ina Poser, Shovamayee Maharana, Martine Ruer-Gruß, Doris Richter, Xiaojie Zhang, Young-Tae Chang, Jochen Guck, Alf Honigmann, Julia Mahamid, Anthony Hyman, Rohit V Pappu, Simon Alberti, Titus Franzmann
RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation.
Cell, 181(2) 346-361 (2020)
Open Access DOI
Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

Adam Klosin✳︎, Florian Oltsch✳︎, Tyler Harmon, Alf Honigmann, Frank Jülicher#, Anthony A. Hyman#, Christoph Zechner#
Phase separation provides a mechanism to reduce noise in cells.
Science, 367(6476) 464-468 (2020)
Expression of proteins inside cells is noisy, causing variability in protein concentration among identical cells. A central problem in cellular control is how cells cope with this inherent noise. Compartmentalization of proteins through phase separation has been suggested as a potential mechanism to reduce noise, but systematic studies to support this idea have been missing. In this study, we used a physical model that links noise in protein concentration to theory of phase separation to show that liquid droplets can effectively reduce noise. We provide experimental support for noise reduction by phase separation using engineered proteins that form liquid-like compartments in mammalian cells. Thus, phase separation can play an important role in biological signal processing and control.

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.

Johannes Baumgart✳︎, Marcel Kirchner✳︎, Stefanie Redemann, Alec Bond, Jeffrey Woodruff, Jean-Marc Verbavatz, Frank Jülicher, Thomas Müller-Reichert, Anthony Hyman, Jan Brugués
Soluble tubulin is significantly enriched at mitotic centrosomes.
J Cell Biol, 218(12) 3977-3985 (2019)
During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymeric tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in early C. elegans embryos. We show that a C. elegans one-cell stage centrosome at metaphase contains >10,000 microtubules with a total polymer concentration of 230 µM. Centrosomes concentrate soluble α/β tubulin by about 10-fold over the cytoplasm, reaching peak values of 470 µM, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 µM. These findings support in vitro data suggesting that microtubule nucleation in C. elegans centrosomes is driven in part by concentrating soluble tubulin.

Valerie Siahaan, Jochen Krattenmacher, Anthony Hyman, Stefan Diez, Amayra Hernández-Vega, Zdenek Lansky, Marcus Braun
Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes.
Nat Cell Biol, 21(9) 1086-1092 (2019)
Tau is an intrinsically disordered protein, which diffuses on microtubules1. In neurodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules are correlated with axonal degeneration2. Tau can protect microtubules from microtubule-degrading enzymes such as katanin3. However, how tau carries out this regulatory function is still unclear. Here, using in vitro reconstitution, we show that tau molecules on microtubules cooperatively form cohesive islands that are kinetically distinct from tau molecules that individually diffuse on microtubules. Dependent on the tau concentration in solution, the islands reversibly grow or shrink by addition or release of tau molecules at their boundaries. Shielding microtubules from kinesin-1 motors and katanin, the islands exhibit regulatory qualities distinct from a comparably dense layer of diffusible tau. Superprocessive kinesin-8 motors penetrate the islands and cause their disassembly. Our results reveal a microtubule-dependent phase of tau that constitutes an adaptable protective layer on the microtubule surface. We anticipate that other intrinsically disordered axonal proteins display a similar cooperative behaviour and potentially compete with tau in regulating access to the microtubule surface.

Edgar Boczek, Qi Luo, Marco Dehling, Michael Röpke, Sophie L Mader, Andreas Seidl, Ville R I Kaila, Johannes Buchner
Autophosphorylation activates c-Src kinase through global structural rearrangements.
J Biol Chem, 294(35) 13186-13197 (2019)
Open Access DOI
The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms.

Sadjad Arzash, Patrick M McCall, Jingchen Feng, Margaret L Gardel, Fred C MacKintosh
Stress relaxation in F-actin solutions by severing.
Soft Matter, 15(31) 6300-6307 (2019)
Networks of filamentous actin (F-actin) are important for the mechanics of most animal cells. These cytoskeletal networks are highly dynamic, with a variety of actin-associated proteins that control cross-linking, polymerization and force generation in the cytoskeleton. Inspired by recent rheological experiments on reconstituted solutions of dynamic actin filaments, we report a theoretical model that describes stress relaxation behavior of these solutions in the presence of severing proteins. We show that depending on the kinetic rates of assembly, disassembly, and severing, one can observe both length-dependent and length-independent relaxation behavior.

Laura Mediani, Jordina Guillén-Boixet, Jonathan Vinet, Titus Franzmann, Ilaria Bigi, Daniel Mateju, Arianna D Carrà, Federica F Morelli, Tatiana Tiago, Ina Poser, Simon Alberti, Serena Carra
Defective ribosomal products challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin.
EMBO J, 38(15) Art. No. e101341 (2019)
Open Access DOI
Nuclear protein aggregation has been linked to genome instability and disease. The main source of aggregation-prone proteins in cells is defective ribosomal products (DRiPs), which are generated by translating ribosomes in the cytoplasm. Here, we report that DRiPs rapidly diffuse into the nucleus and accumulate in nucleoli and PML bodies, two membraneless organelles formed by liquid-liquid phase separation. We show that nucleoli and PML bodies act as dynamic overflow compartments that recruit protein quality control factors and store DRiPs for later clearance. Whereas nucleoli serve as constitutive overflow compartments, PML bodies are stress-inducible overflow compartments for DRiPs. If DRiPs are not properly cleared by chaperones and proteasomes due to proteostasis impairment, nucleoli undergo amyloidogenesis and PML bodies solidify. Solid PML bodies immobilize 20S proteasomes and limit the recycling of free ubiquitin. Ubiquitin depletion, in turn, compromises the formation of DNA repair compartments at fragile chromosomal sites, ultimately threatening cell survival.

Peter Gross, K Vijay Kumar, Nathan Goehring, Justin Bois, Carsten Hoege, Frank Jülicher, Stephan W. Grill
Guiding self-organized pattern formation in cell polarity establishment.
Nat Phys, 15(3) 293-300 (2019)
Spontaneous pattern formation in Turing systems relies on feedback. Patterns in cells and tissues however often do not form spontaneously, but are under control of upstream pathways that provide molecular guiding cues. The relationship between guiding cues and feedback in controlled biological pattern formation remains unclear. We explored this relationship during cell polarity establishment in the one-cell-stage C. elegans embryo. We quantified the strength of two feedback systems that operate during polarity establishment, feedback between polarity proteins and the actomyosin cortex, and mutual antagonism amongst polarity proteins. We characterized how these feedback systems are modulated by guiding cues from the centrosome. By coupling a mass-conserved Turing-like reaction-diffusion system for polarity proteins to an active gel description of the actomyosin cortex, we reveal a transition point beyond which feedback ensures self-organized polarization even when cues are removed. Notably, the baton is passed from a guide-dominated to a feedback-dominated regime significantly beyond this transition point, which ensures robustness. Together, this reveals a general criterion for controlling biological pattern forming systems: feedback remains subcritical to avoid unstable behaviour, and molecular guiding cues drive the system beyond a transition point for pattern formation.

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)
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.

Yaser Atlasi, Wout Megchelenbrink, Tianran Peng, Ehsan Habibi, Onkar Joshi, Shuang-Yin Wang, Cheng Wang, Colin Logie, Ina Poser, Hendrik Marks, Hendrik G Stunnenberg
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.
Nat Cell Biol, 21(5) 568-578 (2019)
The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

Lara Marrone, Hannes C A Drexler, Jie Wang, Priyanka Tripathi, Tania Distler, Patrick Heisterkamp, Eric D Anderson, Sukhleen Kour, Anastasia Moraiti, Shovamayee Maharana, Rajat Bhatnagar, T Grant Belgard, Vadreenath Tripathy, Norman Kalmbach, Zohreh Hosseinzadeh, Valeria Crippa, Masin Abo-Rady, Florian Wegner, Angelo Poletti, Dirk Troost, Eleonora Aronica, Volker Busskamp, Joachim Weis, Udai Pandey, Anthony Hyman, Simon Alberti, Anand Goswami, Jared Sterneckert
FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.
Acta Neuropathol, 138(1) 67-84 (2019)
Open Access DOI
Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Ivan Ivanov#, Rafael B. Lira, T-Y Dora Tang, Titus Franzmann, Adam Klosin, Lucas Caire da Silva, Anthony Hyman, Katharina Landfester, Reinhard Lipowsky, Kai Sundmacher, Rumiana Dimova#
Directed Growth of Biomimetic Microcompartments.
Adv Biosys, 3(6) Art. No. 1800314 (2019)

Aruljothi Mariappan, Komal Soni, Kenji Schorpp, Fan Zhao, Amin Minakar, Xiangdong Zheng, Sunit Mandad, Iris Macheleidt, Anand Ramani, Tomáš Kubelka, Maciej Dawidowski, Kristina Golfmann, Arpit Wason, Chunhua Yang, Judith Simons, Hans-Günther Schmalz, Anthony Hyman, Ritu Aneja, Roland Ullrich, Henning Urlaub, Margarete Odenthal, Reinhardt Büttner, Haitao Li, Michael Sattler, Kamyar Hadian, Jay Gopalakrishnan
Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.
EMBO J, 38(2) Art. No. e99876 (2019)
Open Access DOI
Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.

Louise Jawerth, Mahdiye Ijavi, Martine Ruer, Shambaditya Saha, Marcus Jahnel, Anthony A. Hyman, Frank Jülicher#, Elisabeth Fischer-Friedrich#
Salt-Dependent Rheology and Surface Tension of Protein Condensates Using Optical Traps.
Phys Rev Lett, 121(25) Art. No. 258101 (2018)
An increasing number of proteins with intrinsically disordered domains have been shown to phase separate in buffer to form liquidlike phases. These protein condensates serve as simple models for the investigation of the more complex membraneless organelles in cells. To understand the function of such proteins in cells, the material properties of the condensates they form are important. However, these material properties are not well understood. Here, we develop a novel method based on optical traps to study the frequency-dependent rheology and the surface tension of P-granule protein PGL-3 condensates as a function of salt concentration. We find that PGL-3 droplets are predominantly viscous but also exhibit elastic properties. As the salt concentration is reduced, their elastic modulus, viscosity, and surface tension increase. Our findings show that salt concentration has a strong influence on the rheology and dynamics of protein condensates suggesting an important role of electrostatic interactions for their material properties.

Susana Montenegro Gouveia, Sihem Zitouni, Dong Kong, Paulo Duarte, Beatriz Ferreira Gomes, Ana Laura Sousa, Erin M Tranfield, Anthony Hyman, Jadranka Loncarek, Monica Bettencourt-Dias
PLK4 is a microtubule-associated protein that self-assembles promoting de novo MTOC formation.
J Cell Sci, 132(4) Art. No. jcs219501 (2018)
Open Access DOI
The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.

Simon Alberti, Shambaditya Saha, Jeffrey Woodruff, Titus Franzmann, Jie Wang, Anthony Hyman
A User's Guide for Phase Separation Assays with Purified Proteins.
J Mol Biol, 430(23) 4806-4820 (2018)
Open Access DOI
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

David Zwicker, Johannes Baumgart, Stefanie Redemann, Thomas Müller-Reichert, Anthony Hyman, Frank Jülicher
Positioning of Particles in Active Droplets.
Phys Rev Lett, 121(15) Art. No. 158102 (2018)
Chemically active droplets are nonequilibrium systems that combine phase separation with chemical reactions. We here investigate how the activity introduced by the chemical reactions influences solid particles inside such droplets. We find that passive particles are centered in active droplets governed by first-order reactions. In autocatalytic active droplets, only catalytically active particles can be centered. An example of such systems in biology are centrosomes. Our study can account for the observed positioning of centrioles and provides a general mechanism to control the position of particles within chemically active droplets.

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)
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.

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.

Edgar Boczek, Simon Alberti
Phase changes in neurotransmission.
Science, 361(6402) 548-549 (2018)

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)
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)
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.

Sonja Kroschwald, Matthias Munder, Shovamayee Maharana, Titus Franzmann, Doris Richter, Martine Ruer, Anthony Hyman, Simon Alberti
Different Material States of Pub1 Condensates Define Distinct Modes of Stress Adaptation and Recovery.
Cell Rep, 23(11) 3327-3339 (2018)
Open Access DOI
How cells adapt to varying environmental conditions is largely unknown. Here, we show that, in budding yeast, the RNA-binding and stress granule protein Pub1 has an intrinsic property to form condensates upon starvation or heat stress and that condensate formation is associated with cell-cycle arrest. Release from arrest coincides with condensate dissolution, which takes minutes (starvation) or hours (heat shock). In vitro reconstitution reveals that the different dissolution rates of starvation- and heat-induced condensates are due to their different material properties: starvation-induced Pub1 condensates form by liquid-liquid demixing and subsequently convert into reversible gel-like particles; heat-induced condensates are more solid-like and require chaperones for disaggregation. Our data suggest that different physiological stresses, as well as stress durations and intensities, induce condensates with distinct physical properties and thereby define different modes of stress adaptation and rates of recovery.

Richard Wheeler, Anthony Hyman
Controlling compartmentalization by non-membrane-bound organelles.
Philos Trans R Soc Lond B Biol Sci, 373(1747) Art. No. 20170193 (2018)
Open Access DOI
Compartmentalization is a characterizing feature of complexity in cells, used to organize their biochemistry. Membrane-bound organelles are most widely known, but non-membrane-bound liquid organelles also exist. These have recently been shown to form by phase separation of specific types of proteins known as scaffolds. This forms two phases: a condensate that is enriched in scaffold protein separated by a phase boundary from the cytoplasm or nucleoplasm with a low concentration of the scaffold protein. Phase separation is well known for synthetic polymers, but also appears important in cells. Here, we review the properties of proteins important for forming these non-membrane-bound organelles, focusing on the energetically favourable interactions that drive condensation. On this basis we make qualitative predictions about how cells may control compartmentalization by condensates; the partition of specific molecules to a condensate; the control of condensation and dissolution of condensates; and the regulation of condensate nucleation. There are emerging data supporting many of these predictions, although future results may prove incorrect. It appears that many molecules may have the ability to modulate condensate formation, making condensates a potential target for future therapeutics. The emerging properties of condensates are fundamentally unlike the properties of membrane-bound organelles. They have the capacity to rapidly integrate cellular events and act as a new class of sensors for internal and external environments.This article is part of the theme issue 'Self-organization in cell biology'.

Bruce Alberts, Tony Hyman, Christopher L. Pickett, Shirley Tilghman, Harold Varmus
Improving support for young biomedical scientists.
Science, 360(6390) 716-718 (2018)

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)
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.

Susanne Wegmann, Bahareh Eftekharzadeh, Katharina Tepper, Katarzyna M Zoltowska, Rachel E Bennett, Simon Dujardin, Pawel R Laskowski, Danny MacKenzie, Tarun Kamath, Caitlin Commins, Charles Vanderburg, Allyson D Roe, Zhanyun Fan, Amandine M Molliex, Amayra Hernandez-Vega, Daniel Muller, Anthony Hyman, Eckhard Mandelkow, J Paul Taylor, Bradley T Hyman
Tau protein liquid-liquid phase separation can initiate tau aggregation.
EMBO J, 37(7) Art. No. e98049 (2018)
Open Access DOI
The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid-liquid phase separation (LLPS) under cellular conditions and that phase-separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho-tau isolated from human Alzheimer brain. Droplet-like tau can also be observed in neurons and other cells. We found that tau droplets become gel-like in minutes, and over days start to spontaneously form thioflavin-S-positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.

Edgar Boczek, Simon Alberti
One domain fits all: Using disordered regions to sequester misfolded proteins.
J Cell Biol, 217(4) 1173-1175 (2018)
Small heat shock proteins (sHsps) are adenosine triphosphate-independent chaperones that protect cells from misfolded proteins. In this issue, Grousl et al. (2018.J. Cell Biol.https://doi.org/10.1083/jcb.201708116) show that the yeast sHsp Hsp42 uses a prion-like intrinsically disordered domain to bind and sequester misfolded proteins in protein deposition sites.

Radoslav Aleksandrov, Anton Dotchev, Ina Poser, Dragomir Krastev, Georgi Georgiev, Greta C Panova, Yordan Babukov, Georgi Danovski, Teodora Dyankova, Lars Hubatsch, Aneliya Ivanova, Aleksandar Atemin, Marina N Nedelcheva-Veleva, Susanne Hasse, Mihail Sarov, Frank Buchholz, Anthony Hyman, Stephan W. Grill, Stoyno Stoynov
Protein Dynamics in Complex DNA Lesions.
Mol Cell, 69(6) 1046-1061 (2018)
A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact.

FoSheng Hsu, Stephanie Spannl, Charles Ferguson, Anthony Hyman, Robert G. Parton, Marino Zerial
Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria.
Elife, 7 Art. No. e32282 (2018)
Open Access DOI
Mitochondrial stress response is essential for cell survival, and damaged mitochondria are a hallmark of neurodegenerative diseases. Thus, it is fundamental to understand how mitochondria relay information within the cell. Here, by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress. This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria. Interestingly, activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin, encoded by a gene mutated in amyotrophic lateral sclerosis (ALS). Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress. These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria. Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS.

Lara Marrone, Ina Poser, Ian Casci, Julia Japtok, Peter Reinhardt, Antje Janosch, Cordula Andree, Hyun-Ok Kate Lee, Claudia Moebius, Ellen Koerner, Lydia Reinhardt, Maria Elena Cicardi, Karl Hackmann, Barbara Klink, Angelo Poletti, Simon Alberti, Marc Bickle, Andreas Hermann, Udai Pandey, Anthony Hyman, Jared Sterneckert
Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy.
Stem Cell Rep, 10(2) 375-389 (2018)
Open Access DOI
Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments.

Jeffrey Woodruff, Anthony Hyman, Elvan Boke
Organization and Function of Non-dynamic Biomolecular Condensates.
Trends Biochem Sci, 43(2) 81-94 (2018)
Cells compartmentalize biochemical reactions using organelles. Organelles can be either membrane-bound compartments or supramolecular assemblies of protein and ribonucleic acid known as 'biomolecular condensates'. Biomolecular condensates, such as nucleoli and germ granules, have been described as liquid like, as they have the ability to fuse, flow, and undergo fission. Recent experiments have revealed that some liquid-like condensates can mature over time to form stable gels. In other cases, biomolecular condensates solidify into amyloid-like fibers. Here we discuss the assembly, organization, and physiological roles of these more stable condensates in cells, focusing on Balbiani bodies, centrosomes, nuclear pores, and amyloid bodies. We discuss how the material properties of these condensates can be explained by the principles of liquid-liquid phase separation and maturation.

Marcel Naumann, Arun Pal, Anand Goswami, Xenia Lojewski, Julia Japtok, Anne Vehlow, Maximilian Naujock, René Günther, Mengmeng Jin, Nancy Stanslowsky, Peter Reinhardt, Jared Sterneckert, Marie Frickenhaus, Francisco Pan-Montojo, Erik Storkebaum, Ina Poser, Axel Freischmidt, Jochen H Weishaupt, Karlheinz Holzmann, Dirk Troost, Albert C Ludolph, Tobias M Boeckers, Stefan Liebau, Susanne Petri, Nils Cordes, Anthony Hyman, Florian Wegner, Stephan W. Grill, Joachim Weis, Alexander Storch, Andreas Hermann
Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation.
Nat Commun, 9(1) Art. No. 335 (2018)
Open Access DOI
Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

Stephen Enos✳︎, Martin Dressler✳︎, Beatriz Ferreira Gomes, Anthony Hyman, Jeffrey Woodruff
Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit.
Biol Open, 7(1) Art. No. bio029777 (2018)
Open Access DOI
Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

Titus Franzmann, Marcus Jahnel, Andrei I. Pozniakovsky, Julia Mahamid, Alex S Holehouse, Elisabeth Nüske, Doris Richter, Wolfgang Baumeister, Stephan W. Grill, Rohit V Pappu, Anthony A. Hyman#, Simon Alberti#
Phase separation of a yeast prion protein promotes cellular fitness.
Science, 359(6371) Art. No. eaao5654 (2018)
Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.

Yusuke Toyoda✳︎, Cedric J Cattin✳︎, Martin P Stewart✳︎, Ina Poser, Mirko Theis, Teymuras V. Kurzchalia, Frank Buchholz, Anthony Hyman#, Daniel J. Müller#
Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding.
Nat Commun, 8(1) Art. No. 1266 (2017)
Open Access DOI
To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular pressure, which the contractile actomyosin cortex directs into shape. Here, we introduce a genome-scale microcantilever- and RNAi-based approach to phenotype the contribution of > 1000 genes to the rounding of single mitotic cells against confinement. Our screen analyzes the rounding force, pressure and volume of mitotic cells and localizes selected proteins. We identify 49 genes relevant for mitotic rounding, a large portion of which have not previously been linked to mitosis or cell mechanics. Among these, depleting the endoplasmic reticulum-localized protein FAM134A impairs mitotic progression by affecting metaphase plate alignment and pressure generation by delocalizing cortical myosin II. Furthermore, silencing the DJ-1 gene uncovers a link between mitochondria-associated Parkinson's disease and mitotic pressure. We conclude that mechanical phenotyping is a powerful approach to study the mechanisms governing cell shape.

Amayra Hernández-Vega, Marcus Braun, Lara Scharrel, Marcus Jahnel, Susanne Wegmann, Bradley T Hyman, Simon Alberti, Stefan Diez#, Anthony Hyman#
Local Nucleation of Microtubule Bundles through Tubulin Concentration into a Condensed Tau Phase.
Cell Rep, 20(10) 2304-2312 (2017)
Open Access DOI
Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that the concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that, under conditions of macro-molecular crowding, tau forms liquid-like drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles, which deform the drops while remaining enclosed by diffusible tau molecules exhibiting a liquid-like behavior. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.

Shambaditya Saha, Anthony Hyman
RNA gets in phase.
J Cell Biol, 216(8) 2235-2237 (2017)
Several neurological disorders are linked to tandem nucleotide repeat expansion in the mutated gene. Jain and Vale (2017. Nature. https://doi.org/10.1038/nature22386) show that, above a pathological threshold repeat number, base pairing interactions drive phase separation of RNA into membrane-less gels, suggesting that RNA can scaffold the assembly of phase-separated compartments that sequester proteins/RNAs causing toxicity.

Adam Klosin, Anthony Hyman
Molecular biology: A liquid reservoir for silent chromatin.
Nature, 547(7662) 168-170 (2017)

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)
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.

Beata E Mierzwa, Nicolas Chiaruttini, Lorena Redondo-Morata, Joachim Moser von Filseck, Julia König, Jorge Larios, Ina Poser, Thomas Müller-Reichert, Simon Scheuring, Aurélien Roux, Daniel W Gerlich
Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis.
Nat Cell Biol, 19(7) 787-798 (2017)
The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

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)
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.

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

Avinash Patel✳︎, Liliana Malinovska✳︎, Shambaditya Saha, Jie Wang, Simon Alberti, Yamuna Krishnan#, Anthony Hyman#
ATP as a biological hydrotrope.
Science, 356(6339) 753-756 (2017)
Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.

Salman F Banani✳︎, Hyun-Ok Kate Lee✳︎, Anthony Hyman, Michael K Rosen
Biomolecular condensates: organizers of cellular biochemistry.
Nat Rev Mol Cell Biol, 18(5) 285-298 (2017)
Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid-liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

Daniel Mateju, Titus Franzmann, Avinash Patel, Andrii Kopach, Edgar Boczek, Shovamayee Maharana, Hyun-Ok Kate Lee, Serena Carra, Anthony Hyman, Simon Alberti
An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function.
EMBO J, 36(12) 1669-1687 (2017)
Open Access PDF DOI
Stress granules (SG) are membrane-less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age-related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS-linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid-to-solid transition ofin vitroreconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.

David Zwicker✳︎, Rabea Seyboldt✳︎, Christoph A. Weber, Anthony A. Hyman, Frank Jülicher
Growth and division of active droplets provides a model for protocells
Nat Phys, 13(4) 408-413 (2017)
Nature Physics | Article Print Share/bookmark Growth and division of active droplets provides a model for protocells David Zwicker, Rabea Seyboldt, Christoph A. Weber, Anthony A. Hyman & Frank Jülicher Affiliations Contributions Corresponding author Nature Physics 13, 408–413 (2017) doi:10.1038/nphys3984 Received 29 April 2016 Accepted 10 November 2016 Published online 12 December 2016 Article tools PDF Citation Reprints Rights & permissions Article metrics Abstract Abstract• Introduction• Division of active droplets• Chemically active droplets as a model for protocells• Methods• References• Acknowledgements• Author information• Supplementary information It has been proposed that during the early steps in the origin of life, small droplets could have formed via the segregation of molecules from complex mixtures by phase separation. These droplets could have provided chemical reaction centres. However, whether these droplets could divide and propagate is unclear. Here we examine the behaviour of droplets in systems that are maintained away from thermodynamic equilibrium by an external supply of energy. In these systems, droplets grow by the addition of droplet material generated by chemical reactions. Surprisingly, we find that chemically driven droplet growth can lead to shape instabilities that trigger the division of droplets into two smaller daughters. Therefore, chemically active droplets can exhibit cycles of growth and division that resemble the proliferation of living cells. Dividing active droplets could serve as a model for prebiotic protocells, where chemical reactions in the droplet play the role of a prebiotic metabolism.

Qi Luo✳︎, Edgar Boczek✳︎, Qi Wang, Johannes Buchner, Ville R I Kaila
Hsp90 dependence of a kinase is determined by its conformational landscape.
Sci Rep, 7 Art. No. 43996 (2017)
Open Access DOI
Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken "control switch", resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence.

Marie Skogs, Charlotte Stadler, Rutger Schutten, Martin Hjelmare, Christian Gnann, Lars Björk, Ina Poser, Anthony Hyman, Mathias Uhlén, Emma Lundberg
Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.
J Proteome Res, 16(1) 147-155 (2017)
Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Nina Peel, Jyoti Iyer, Anar Naik, Michael P Dougherty, Markus Decker, Kevin F O'Connell
Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.
PLoS Genet, 13(1) Art. No. e1006543 (2017)
Open Access PDF DOI
In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

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)
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.

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)
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.

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.

Oliver Wueseke, David Zwicker, Anne Schwager, Yao Liang Wong, Karen Oegema, Frank Jülicher, Anthony Hyman, Jeffrey Woodruff
Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation.
Biol Open, 5(10) 1431-1440 (2016)
Open Access PDF DOI
Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM). In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1) phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-5(4A)) could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-5(4A) self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.

Cornelia G Spruijt, Martijn S Luijsterburg, Roberta Menafra, Rik G H Lindeboom, Pascal W T C Jansen, Raghu Ram Edupuganti, Marijke P Baltissen, Wouter W Wiegant, Moritz C Voelker-Albert, Filomena Matarese, Anneloes Mensinga, Ina Poser, Harmjan R Vos, Hendrik G Stunnenberg, Haico van Attikum, Michiel Vermeulen
ZMYND8 Co-localizes with NuRD on Target Genes and Regulates Poly(ADP-Ribose)-Dependent Recruitment of GATAD2A/NuRD to Sites of DNA Damage.
Cell Rep, 17(3) 783-798 (2016)
Open Access PDF DOI
NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles in transcription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide binding sites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, the MYND domain in ZMYND8 facilitates the rapid, poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes.

Simon Alberti, Anthony Hyman
Are aberrant phase transitions a driver of cellular aging?
Bioessays, 38(10) 959-968 (2016)
Open Access PDF DOI
Why do cells age? Recent advances show that the cytoplasm is organized into many membrane-less compartments via a process known as phase separation, which ensures spatiotemporal control over diffusion-limited biochemical reactions. Although phase separation is a powerful mechanism to organize biochemical reactions, it comes with the trade-off that it is extremely sensitive to changes in physical-chemical parameters, such as protein concentration, pH, or cellular energy levels. Here, we highlight recent findings showing that age-related neurodegenerative diseases are linked to aberrant phase transitions in neurons. We discuss how these aberrant phase transitions could be tied to a failure to maintain physiological physical-chemical conditions. We generalize this idea to suggest that the process of cellular aging involves a progressive loss of the organization of phase-separated compartments in the cytoplasm.

Shambaditya Saha, Christoph A. Weber, Marco Nousch, Omar Adame-Arana, Carsten Hoege, Marco Y Hein, Erin Osborne-Nishimura, Julia Mahamid, Marcus Jahnel, Louise Jawerth, Andrei Pozniakovski, Christian R. Eckmann, Frank Jülicher, Anthony Hyman
Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism.
Cell, 166(6) 1572-1584 (2016)
P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.

Jochen H Weishaupt, Tony Hyman, Ivan Dikic
Common Molecular Pathways in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.
Trends Mol Med, 22(9) 769-783 (2016)
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative diseases in which predominantly motor neurons and cerebral cortex neurons, respectively, are affected. Several novel ALS and FTD disease genes have been recently discovered, pointing toward a few overarching pathways in ALS/FTD pathogenesis. Nevertheless, a precise picture of how various cellular processes cause neuronal death, or how different routes leading to ALS and FTD are functionally connected is just emerging. Moreover, how the most recent milestone findings in the ALS/FTD field might lead to improved diagnosis and treatment is actively being explored. We highlight some of the most exciting recent topics in the field, which could potentially facilitate the identification of further links between the pathogenic ALS/FTD pathways related to autophagy, vesicle trafficking, and RNA metabolism.

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)
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.

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)
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.

Elvan Boke, Martine Ruer, Martin Wühr, Margaret Coughlin, Regis P. Lemaitre, Steven Gygi, Simon Alberti, David N. Drechsel, Anthony Hyman, Timothy J. Mitchison
Amyloid-like Self-Assembly of a Cellular Compartment.
Cell, 166(3) 637-650 (2016)
Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.

Sara Cuylen, Claudia Blaukopf, Antonio Z Politi, Thomas Müller-Reichert, Beate Neumann, Ina Poser, Jan Ellenberg, Anthony Hyman, Daniel W Gerlich
Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.
Nature, 535(7611) 308-312 (2016)
Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.

Diego Guerrera, Jimit Shah, Ekaterina Vasileva, Sophie Sluysmans, Isabelle Méan, Lionel Jond, Ina Poser, Matthias Mann, Anthony Hyman, Sandra Citi
PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.
J Biol Chem, 291(21) 11016-11029 (2016)
PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions.

Elke Gabriel, Arpit Wason, Anand Ramani, Li Ming Gooi, Patrick Keller, Andrei I. Pozniakovsky, Ina Poser, Florian Noack, Narasimha Swamy Telugu, Federico Calegari, Tomo Šarić, Juergen Hescheler, Anthony Hyman, Marco Gottardo, Giuliano Callaini, Fowzan Sami Alkuraya, Jay Gopalakrishnan
CPAP promotes timely cilium disassembly to maintain neural progenitor pool.
EMBO J, 35(8) 803-819 (2016)
Open Access DOI
A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.

Andrés Diaz Delgadillo
Temperature drives P granule formation in Caenorhabditis elegans
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2016)

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)
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.

Susanne Hasse, Anthony Hyman, Mihail Sarov
TransgeneOmics - A transgenic platform for protein localization based function exploration.
Methods, 96 69-74 (2016)
Open Access PDF DOI
The localization of a protein is intrinsically linked to its role in the structural and functional organization of the cell. Advances in transgenic technology have streamlined the use of protein localization as a function discovery tool. Here we review the use of large genomic DNA constructs such as bacterial artificial chromosomes as a transgenic platform for systematic tag-based protein function exploration.

Michaela Müller-McNicoll, Valentina Botti, Antonio Domingues, Holger Brandl, Oliver D Schwich, Michaela Steiner, Tomaz Curk, Ina Poser, Kathi Zarnack, Karla M. Neugebauer
SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.
Genes Dev, 30(5) 553-566 (2016)
Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.

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)
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.

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)
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.

Jan Arnold, Julia Mahamid, Vladan Lucic, Alex de Marco, Jose-Jesus Fernandez, Tim Laugks, Tobias Mayer, Anthony Hyman, Wolfgang Baumeister, Jürgen M Plitzko
Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.
Biophys J, 110(4) 860-869 (2016)
The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.

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.

Xiangdong Zheng, Anand Ramani, Komal Soni, Marco Gottardo, Shuangping Zheng, Li Ming Gooi, Wenjing Li, Shan Feng, Aruljothi Mariappan, Arpit Wason, Per Widlund, Andrei I. Pozniakovsky, Ina Poser, Haiteng Deng, Guangshuo Ou, Maria Riparbelli, Callaini Giuliano, Anthony Hyman, Michael Sattler, Jay Gopalakrishnan, Haitao Li
Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.
Nat Commun, 7 Art. No. 11874 (2016)
Open Access PDF DOI
Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.

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.

Barbara Sorce, Carlos Escobedo, Yusuke Toyoda, Martin P Stewart, Cedric J Cattin, Richard Newton, Indranil Banerjee, Alexander Stettler, Botond Roska, Suzanne Eaton, Anthony Hyman, Andreas Hierlemann, Daniel J. Müller
Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.
Nat Commun, 6 Art. No. 8872 (2015)
Open Access PDF DOI
Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Alexander Mietke, Oliver Otto, Salvatore Girardo, Philipp Rosendahl, Anna Taubenberger, Stefan Golfier, Elke Ulbricht, Sebastian Aland, Jochen Guck, Elisabeth Fischer-Friedrich
Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.
Biophys J, 109(10) 2023-2036 (2015)
Open Access PDF DOI
Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

Julia Mahamid, Ruud Schampers, Hans Persoon, Anthony Hyman, Wolfgang Baumeister, Jürgen M Plitzko
A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms.
J Struct Biol, 192(2) 262-269 (2015)
Cryo-electron tomography provides 3D views of cellular architecture with molecular resolution. A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. Recently it has been shown that cryo-focused ion beam milling of plunge-frozen eukaryotic cells can produce homogeneously thin, distortion free lamellas for cryo-electron tomography. Multicellular organisms and tissue cannot be properly vitrified and thinned using this technique because they are considerably thicker. High pressure freezing is therefore necessary to provide optimal preservation. Here, we describe a workflow for preparing lamellas from Caenorhabditis elegans worms using cryo-FIB applied to high pressure frozen samples. We employ cryo-planing followed by correlative cryo-fluorescence microscopy to navigate this large multicellular volume and to localize specific targets within. To produce vitreous lamellas amenable to cryo-TEM observations at these targeted locations, we have developed a dedicated lift-out procedure at cryogenic temperature.

Marco Y Hein, Nina C Hubner, Ina Poser, Jürgen Cox, Nagarjuna Nagaraj, Yusuke Toyoda, Igor A Gak, Ina Weisswange, Jorg Mansfeld, Frank Buchholz, Anthony Hyman, Matthias Mann
A Human Interactome in Three Quantitative Dimensions Organized by Stoichiometries and Abundances.
Cell, 163(3) 712-723 (2015)
The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

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)
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.

Li Ding, Maciej Paszkowski-Rogacz, Maria Winzi, Debojyoti Chakraborty, Mirko Theis, Sukhdeep Singh, Giovanni Ciotta, Ina Poser, Assen Roguev, Wai Kit Chu, Chunaram Choudhary, Matthias Mann, A. Francis Stewart, Nevan J Krogan, Frank Buchholz
Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells.
Cell Sys, 1(2) 141-151 (2015)

Simone Reber, Nathan Goehring
Intracellular Scaling Mechanisms.
Cold Spring Harb Perspect Biol, 7(12) Art. No. a019067 (2015)
Organelle function is often directly related to organelle size. However, it is not necessarily absolute size but the organelle-to-cell-size ratio that is critical. Larger cells generally have increased metabolic demands, must segregate DNA over larger distances, and require larger cytokinetic rings to divide. Thus, organelles often must scale to the size of the cell. The need for scaling is particularly acute during early development during which cell size can change rapidly. Here, we highlight scaling mechanisms for cellular structures as diverse as centrosomes, nuclei, and the mitotic spindle, and distinguish them from more general mechanisms of size control. In some cases, scaling is a consequence of the underlying mechanism of organelle size control. In others, size-control mechanisms are not obviously related to cell size, implying that scaling results indirectly from cell-size-dependent regulation of size-control mechanisms.

David Zwicker, Anthony Hyman, Frank Jülicher
Suppression of Ostwald ripening in active emulsions.
Phys Rev E, 92(1) Art. No. 012317 (2015)
Emulsions consisting of droplets immersed in a fluid are typically unstable since they coarsen over time. One important coarsening process is Ostwald ripening, which is driven by the surface tension of the droplets. Stability of emulsions is relevant not only in complex fluids but also in biological cells, which contain liquidlike compartments, e.g., germ granules, Cajal bodies, and centrosomes. Such cellular systems are driven away from equilibrium, e.g., by chemical reactions, and thus can be called active emulsions. In this paper, we study such active emulsions by developing a coarse-grained description of the droplet dynamics, which we analyze for two different chemical reaction schemes. We first consider the simple case of first-order reactions, which leads to stable, monodisperse emulsions in which Ostwald ripening is suppressed within a range of chemical reaction rates. We then consider autocatalytic droplets, which catalyze the production of their own droplet material. Spontaneous nucleation of autocatalytic droplets is strongly suppressed and their emulsions are typically unstable. We show that autocatalytic droplets can be nucleated reliably and their emulsions stabilized by the help of chemically active cores, which catalyze the production of droplet material. In summary, different reaction schemes and catalytic cores can be used to stabilize emulsions and to control their properties.

Jeffrey Woodruff, Oliver Wueseke, Valeria Viscardi, Julia Mahamid, Stacy D Ochoa, Jakob Bunkenborg, Per Widlund, Andrei I. Pozniakovsky, Esther Zanin, Shirin Bahmanyar, Andrea Zinke, Sun Hae Hong, Markus Decker, Wolfgang Baumeister, Jens S Andersen, Karen Oegema, Anthony Hyman
Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.
Science, 348(6236) 808-812 (2015)
The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

Ana Tomasovic, Nina Kurrle, Duran Sürün, Juliana Heidler, Koraljka Husnjak, Ina Poser, Frank Schnütgen, Susan Scheibe, Michael Seimetz, Peter Jaksch, Anthony Hyman, Norbert Weissmann, Harald von Melchner
Sestrin 2 Protein Regulates Platelet-derived Growth Factor Receptor β (Pdgfrβ) Expression by Modulating Proteasomal and Nrf2 Transcription Factor Functions.
J Biol Chem, 290(15) 9738-9752 (2015)
We recently identified the antioxidant protein Sestrin 2 (Sesn2) as a suppressor of platelet-derived growth factor receptor β (Pdgfrβ) signaling and Pdgfrβ signaling as an inducer of lung regeneration and injury repair. Here, we identified Sesn2 and the antioxidant gene inducer nuclear factor erythroid 2-related factor 2 (Nrf2) as positive regulators of proteasomal function. Inactivation of Sesn2 or Nrf2 induced reactive oxygen species-mediated proteasomal inhibition and Pdgfrβ accumulation. Using bacterial artificial chromosome (BAC) transgenic HeLa and mouse embryonic stem cells stably expressing enhanced green fluorescent protein-tagged Sesn2 at nearly endogenous levels, we also showed that Sesn2 physically interacts with 2-Cys peroxiredoxins and Nrf2 albeit under different reductive conditions. Overall, we characterized a novel, redox-sensitive Sesn2/Pdgfrβ suppressor pathway that negatively interferes with lung regeneration and is up-regulated in the emphysematous lungs of patients with chronic obstructive pulmonary disease (COPD).

Oliver Wüseke
Pericentriolar material assembly in Caenorhabditis elegans
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2015)

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.

Subramanian Ramanathan, Jonne Helenius, Martin P Stewart, Cedric J Cattin, Anthony Hyman, Daniel J. Müller
Cdk1-dependent mitotic enrichment of cortical myosin II promotes cell rounding against confinement.
Nat Cell Biol, 17(2) 148-159 (2015)
Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (<10 s) resistance to mechanical deformation, myosin sustains intracellular pressure for a longer duration (>60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.

Nurhan Özlü, Mohammad H Qureshi, Yusuke Toyoda, Bernhard Y Renard, Gürkan Mollaoglu, Nazlı E Özkan, Selda Bulbul, Ina Poser, Wiebke Timm, Anthony Hyman, Timothy J. Mitchison, Judith A Steen
Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.
EMBO J, 34(2) 251-265 (2015)
The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

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.

Mitsuhiro Yanagida, Anthony A. Hyman, Jonathon Pines
Mitosis : a subject collection from Cold Spring Harbor Perspectives in biology
Cold Spring Harbor, N.Y, Cold Spring Harbor Laboratory Press (2015), 203 S.

Jeffrey Woodruff, Anthony Hyman
Method: In vitro analysis of pericentriolar material assembly.
Methods Cell Biol, 129 369-382 (2015)
Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.

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

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)
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.

Amr El-Labban, A Zisserman, Yusuke Toyoda, Alexander W. Bird, Anthony A. Hyman
Temporal models for mitotic phase labelling.
Med Image Anal, 18(7) 977-988 (2014)
With the widespread use of time-lapse data to understand cellular function, there is a need for tools which facilitate high-throughput analysis of data. Fluorescence microscopy of genetically engineered cell lines in culture can be used to visualise the progression of these cells through the cell cycle, including distinctly identifiable sequential stages of cell division (mitotic phases). We present a system for automated segmentation and mitotic phase labelling using temporal models. This work takes the novel approach of using temporal features evaluated over the whole of the mitotic phases rather than over single frames, thereby capturing the distinctive behaviour over the phases. We compare and contrast three different temporal models: Dynamic Time Warping, Hidden Markov Models, and Semi Markov Models. A new loss function is proposed for the Semi Markov model to make it more robust to inconsistencies in data annotation near transition boundaries. The models are tested under two different experimental conditions to explore robustness to changes in biological conditions.

Jeffrey Woodruff, Oliver Wueseke, Anthony Hyman
Pericentriolar material structure and dynamics.
Philos Trans R Soc Lond B Biol Sci, 369(1650) Art. No. 20130459 (2014)
A centrosome consists of two barrel-shaped centrioles embedded in a matrix of proteins known as the pericentriolar material (PCM). The PCM serves as a platform for protein complexes that regulate organelle trafficking, protein degradation and spindle assembly. Perhaps most important for cell division, the PCM concentrates tubulin and serves as the primary organizing centre for microtubules in metazoan somatic cells. Thus, similar to other well-described organelles, such as the nucleus and mitochondria, the cell has compartmentalized a multitude of vital biochemical reactions in the PCM. However, unlike these other organelles, the PCM is not membrane bound, but rather a dynamic collection of protein complexes and nucleic acids that constitute the organelle's interior and determine its boundary. How is the complex biochemical machinery necessary for the myriad centrosome functions concentrated and maintained in the PCM? Recent advances in proteomics and RNAi screening have unveiled most of the key PCM components and hinted at their molecular interactions ( table 1). Now we must understand how the interactions between these molecules contribute to the mesoscale organization and the assembly of the centrosome. Among outstanding questions are the intrinsic mechanisms that determine PCM shape and size, and how it functions as a biochemical reaction hub.

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)
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.

David Zwicker, Markus Decker, Steffen Jaensch, Anthony Hyman, Frank Jülicher
Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.
Proc Natl Acad Sci U.S.A., 111(26) 2636-2645 (2014)
Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

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)

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)
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.

Anthony Hyman
Encouraging innovation.
Mol Biol Cell, 25(4) 427-428 (2014)
Innovation is central to the scientific endeavor, and yet the current system of funding in the United States discourages innovation, especially in the young. Subtle alterations to the funding system, guided in part by the success of the European Research Council, could have major effects on encouraging innovation.

Milan Esner, Felix Meyenhofer, Michael Kuhn, Melissa Thomas, Yannis Kalaidzidis, Marc Bickle
Development of a Kinetic Assay for Late Endosome Movement.
J Biomol Screen, 19(7) 1070-1078 (2014)
Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

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)
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.

Elisabeth Fischer-Friedrich, Anthony Hyman, Frank Jülicher, Daniel J. Müller, Jonne Helenius
Quantification of surface tension and internal pressure generated by single mitotic cells.
Sci Rep, 4 Art. No. 6213 (2014)
Open Access DOI
During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ≈ 40 Pa and 0.2?mNm(-1) during interphase to ≈ 400?Pa and 1.6?mNm(-1) during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

Anthony Hyman, Christian Weber, Frank Jülicher
Liquid-liquid phase separation in biology.
Annu Rev Cell Dev Biol, 30 39-58 (2014)
Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.

Xiangdong Zheng, Li Ming Gooi, Arpit Wason, Elke Gabriel, Narges Zare Mehrjardi, Qian Yang, Xingrun Zhang, Alain Debec, Marcus L Basiri, Tomer Avidor-Reiss, Andrei I. Pozniakovsky, Ina Poser, Tomo Saric, Anthony Hyman, Haitao Li, Jay Gopalakrishnan
Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis.
Proc Natl Acad Sci U.S.A., 111(3) 354-363 (2014)
Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th β-strands (β9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.

Britta Weber, Erin M Tranfield, Johanna L Höög, Daniel Baum, Claude Antony, Tony Hyman, Jean-Marc Verbavatz, Steffen Prohaska
Automated Stitching of Microtubule Centerlines across Serial Electron Tomograms.
PLoS ONE, 9(12) Art. No. e113222 (2014)
Open Access PDF DOI
Tracing microtubule centerlines in serial section electron tomography requires microtubules to be stitched across sections, that is lines from different sections need to be aligned, endpoints need to be matched at section boundaries to establish a correspondence between neighboring sections, and corresponding lines need to be connected across multiple sections. We present computational methods for these tasks: 1) An initial alignment is computed using a distance compatibility graph. 2) A fine alignment is then computed with a probabilistic variant of the iterative closest points algorithm, which we extended to handle the orientation of lines by introducing a periodic random variable to the probabilistic formulation. 3) Endpoint correspondence is established by formulating a matching problem in terms of a Markov random field and computing the best matching with belief propagation. Belief propagation is not generally guaranteed to converge to a minimum. We show how convergence can be achieved, nonetheless, with minimal manual input. In addition to stitching microtubule centerlines, the correspondence is also applied to transform and merge the electron tomograms. We applied the proposed methods to samples from the mitotic spindle in C. elegans, the meiotic spindle in X. laevis, and sub-pellicular microtubule arrays in T. brucei. The methods were able to stitch microtubules across section boundaries in good agreement with experts' opinions for the spindle samples. Results, however, were not satisfactory for the microtubule arrays. For certain experiments, such as an analysis of the spindle, the proposed methods can replace manual expert tracing and thus enable the analysis of microtubules over long distances with reasonable manual effort.

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)
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.

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)
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.

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)
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.

Esther Zanin, Arshad Desai, Ina Poser, Yusuke Toyoda, Cordula Andree, Claudia Moebius, Marc Bickle, Barbara Conradt, Alisa Piekny, Karen Oegema
A conserved RhoGAP limits M phase contractility and coordinates with microtubule asters to confine RhoA during cytokinesis.
Dev Cell, 26(5) 496-510 (2013)
During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis and cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M phase, leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions.

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)
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.

Ryota Uehara, Yuki Tsukada, Tomoko Kamasaki, Ina Poser, Kinya Yoda, Daniel W Gerlich, Gohta Goshima
Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase.
J Cell Biol, 202(4) 623-636 (2013)
The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B-dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.

Marija Zanic, Per Widlund, Anthony Hyman, Jonathon Howard
Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.
Nat Cell Biol, 15(6) 688-693 (2013)
In cells, a complex network of proteins regulates the dynamic growth of microtubules that is essential for division and migration. In vitro approaches with purified components have so far been unable to reconstitute fast microtubule growth observed in vivo . Here we show that two well-studied plus-end-binding proteins-end-tracking protein EB1 and microtubule polymerase XMAP215-act together to strongly promote microtubule growth to cellular rates. Unexpectedly, the combined effects of XMAP215 and EB1 are highly synergistic, with acceleration of growth well beyond the product of the individual effects of either protein. The synergistic growth promotion does not rely on any of the canonical EB1 interactions, suggesting an allosteric interaction through the microtubule end. This hypothesis is supported by the finding that taxol and XMAP215, which have non-overlapping binding sites on tubulin, also act synergistically on growth. The increase in growth rates is accompanied by a strong enhancement of microtubule catastrophe by EB1, thereby rendering the fast and dynamic microtubule behaviour typically observed in cells.

Carsten Hoege, Anthony Hyman
Principles of PAR polarity in Caenorhabditis elegans embryos.
Nat Rev Mol Cell Biol, 14(5) 315-322 (2013)
A hallmark of cell polarity in metazoans is the distribution of partitioning defective (PAR) proteins into two domains on the membrane. Domain boundaries are set by the collective integration of mechanical, biochemical and biophysical signals, and the resulting PAR domains define areas of cytosol specialization. However, the complexity of the signals acting on PAR proteins has been a barrier to uncovering the general principles of PAR polarity. We propose that physical studies, when combined with genetic data, provide new understanding of the mechanisms of polarity establishment in the Caenorhabditis elegans embryo and other organisms.

Jessie M Jeffery, Ilya Grigoriev, Ina Poser, Armando van der Horst, Nicholas Hamilton, Nigel Waterhouse, Jonathan Bleier, V Nathan Subramaniam, Ivan V Maly, Anna Akhmanova, Kum Kum Khanna
Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment.
Cell Cycle, 12(6) 899-906 (2013)
The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.

Zoltan Maliga, Magno Junqueira, Yusuke Toyoda, Andreas Ettinger, Felipe Mora-Bermúdez, Robin Klemm, Andrej Vasilj, E. Guhr, Itziar Ibarlucea-Benitez, Ina Poser, Enzio Bonifacio, Wieland B. Huttner, Andrej Shevchenko, Anthony Hyman
A genomic toolkit to investigate kinesin and myosin motor function in cells.
Nat Cell Biol, 15(3) 325-334 (2013)
Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

Michael C Bassik, Martin Kampmann, Robert Jan Lebbink, Shuyi Wang, Marco Y Hein, Ina Poser, Jimena Weibezahn, Max A Horlbeck, Siyuan Chen, Matthias Mann, Anthony A. Hyman, Emily M Leproust, Michael T McManus, Jonathan S. Weissman
A systematic Mammalian genetic interaction map reveals pathways underlying ricin susceptibility.
Cell, 152(4) 909-922 (2013)
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

Arne H Smits, Pascal W T C Jansen, Ina Poser, Anthony A. Hyman, Michiel Vermeulen
Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.
Nucleic Acids Res, 41(1) Art. No. e28 (2013)
Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

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

Anthony Hyman
Funding innovative science.
Science, 339(6116) 119-119 (2013)
Our objective was to explore self-management practices, health services use and information-seeking for type 2 diabetes care among adult men and women from four recent immigrant communities in Toronto.

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)
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.

Clifford Brangwynne, Anthony A. Hyman
The Origin of Life
Nature, 491(7425) 524-525 (2012)

Per Widlund, Marija Podolski, Simone Reber, Joshua Alper, Marko Storch, Anthony Hyman, Jonathon Howard, David N. Drechsel
One-step purification of assembly-competent tubulin from diverse eukaryotic sources.
Mol Biol Cell, 23(22) 4393-4401 (2012)
We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.

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)
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.

Daniël Splinter, David S Razafsky, Max A Schlager, Andrea Serra-Marques, Ilya Grigoriev, Jeroen Demmers, Nanda Keijzer, Kai Jiang, Ina Poser, Anthony Hyman, Casper C Hoogenraad, Stephen J King, Anna Akhmanova
BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures.
Mol Biol Cell, 23(21) 4226-4241 (2012)
Cytoplasmic dynein is the major microtubule minus-end-directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein-dynactin interaction are poorly understood. In this study, we focus on dynein-dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N-dynein-dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end-directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

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)
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.

Mihail Sarov, John I Murray, Kristin Schanze, Andrei Pozniakovski, Wei Niu, Karolin Angermann, Susanne Hasse, Michaela Rupprecht, Elisabeth Vinis, Matthew Tinney, Elicia A. Preston, Andrea Zinke, Susanne Enst, Tina Teichgraber, Judith Janette, Kadri Reis, Stephan Janosch, Siegfried Schloissnig, Radoslaw K Ejsmont, Cindie Slightam, Xiao Xu, Stuart K Kim, Valerie Reinke, A Francis Stewart, Michael Snyder, Robert H Waterston, Anthony A. Hyman
A Genome-Scale Resource for In Vivo Tag-Based Protein Function Exploration in C. elegans.
Cell, 150(4) 855-866 (2012)
Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.

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)
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.

Anthony A. Hyman, Kai Simons
Cell biology. Beyond oil and water--phase transitions in cells.
Science, 337(6098) 1047-1049 (2012)

Claudia Wurzenberger, Michael Held, Michael A Lampson, Ina Poser, Anthony A. Hyman, Daniel W Gerlich
Sds22 and Repo-Man stabilize chromosome segregation by counteracting Aurora B on anaphase kinetochores.
J Cell Biol, 198(2) 173-183 (2012)
During mitotic spindle assembly, Aurora B kinase is part of an error correction mechanism that detaches microtubules from kinetochores that are under low mechanical tension. During anaphase, however, kinetochore-microtubule attachments must be maintained despite a drop of tension after removal of sister chromatid cohesion. Consistent with this requirement, Aurora B relocates away from chromosomes to the central spindle at the metaphase-anaphase transition. By ribonucleic acid interference screening using a phosphorylation biosensor, we identified two PP1-targeting subunits, Sds22 and Repo-Man, which counteracted Aurora B-dependent phosphorylation of the outer kinetochore component Dsn1 during anaphase. Sds22 or Repo-Man depletion induced transient pauses during poleward chromosome movement and a high incidence of chromosome missegregation. Thus, our study identifies PP1-targeting subunits that regulate the microtubule-kinetochore interface during anaphase for faithful chromosome segregation.

Adam Frost, Marc G Elgort, Onn Brandman, Clinton Ives, Sean R. Collins, Lakshmi Miller-Vedam, Jimena Weibezahn, Marco Y Hein, Ina Poser, Matthias Mann, Anthony A. Hyman, Jonathan S. Weissman
Functional repurposing revealed by comparing S. pombe and S. cerevisiae genetic interactions.
Cell, 149(6) 1339-1352 (2012)
We present a genetic interaction map of pairwise measures including ∼40% of nonessential S. pombe genes. By comparing interaction maps for fission and budding yeast, we confirmed widespread conservation of genetic relationships within and between complexes and pathways. However, we identified an important subset of orthologous complexes that have undergone functional "repurposing": the evolution of divergent functions and partnerships. We validated three functional repurposing events in S. pombe and mammalian cells and discovered that (1) two lumenal sensors of misfolded ER proteins, the kinase/nuclease Ire1 and the glucosyltransferase Gpt1, act together to mount an ER stress response; (2) ESCRT factors regulate spindle-pole-body duplication; and (3) a membrane-protein phosphatase and kinase complex, the STRIPAK complex, bridges the cis-Golgi, the centrosome, and the outer nuclear membrane to direct mitotic progression. Each discovery opens new areas of inquiry and-together-have implications for model organism-based research and the evolution of genetic systems.

Katharina Ross✳︎, Anna Sedello✳︎, Gabriele Putz Todd, Maciej Paszkowski-Rogacz, Alexander W. Bird, Li Ding, Tatyana Grinenko, Kira Behrens, Nina Hubner, Matthias Mann, Claudia Waskow, Carol Stocking, Frank Buchholz
Polycomb group ring finger 1 cooperates with Runx1 in regulating differentiation and self-renewal of hematopoietic cells.
Blood, 119(18) 4152-4161 (2012)
The transcription factor runt-related transcription factor 1 (Runx1) is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cells. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. In the present study, we describe a genome-scale RNA interference screen to detect genes that cooperate with Runx1 in regulating hematopoietic stem and progenitor cells. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation and show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of lineage marker-negative cells in vitro. We found an up-regulation of HoxA cluster genes on Pcgf1 knock-down that possibly accounts for the increase in self-renewal. Moreover, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1, together with the epigenetic repressor Pcgf1, is necessary for terminal differentiation. The results of the present study uncover a link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation.

Nathan Goehring, Anthony A. Hyman
Organelle growth control through limiting pools of cytoplasmic components.
Curr Biol, 22(9) 330-339 (2012)
The critical importance of controlling the size and number of intracellular organelles has led to a variety of mechanisms for regulating the formation and growth of cellular structures. In this review, we explore a class of mechanisms for organelle growth control that rely primarily on the cytoplasm as a 'limiting pool' of available material. These mechanisms are based on the idea that, as organelles grow, they incorporate subunits from the cytoplasm. If this subunit pool is limited, organelle growth will lead to depletion of subunits from the cytoplasm. Free subunit concentration therefore provides a measure of the number of incorporated subunits and thus the current size of the organelle. Because organelle growth rates are typically a function of subunit concentration, cytoplasmic depletion links organelle size, free subunit concentration, and growth rates, ensuring that as the organelle grows, its rate of growth slows. Thus, a limiting cytoplasmic pool provides a powerful mechanism for size-dependent regulation of growth without recourse to active mechanisms to measure size or modulate growth rates. Variations of this general idea allow not only for size control, but also cell-size-dependent scaling of cellular structures, coordination of growth between similar structures within a cell, and the enforcement of singularity in structure formation, when only a single copy of a structure is desired. Here, we review several examples of such mechanisms in cellular processes as diverse as centriole duplication, centrosome and nuclear size control, cell polarity, and growth of flagella.

Britta Weber, Garrett Greenan, Steffen Prohaska, Daniel Baum, Hans-Christian Hege, Thomas Müller-Reichert, Anthony A. Hyman, Jean-Marc Verbavatz
Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos.
J Struct Biol, 178(2 SI) 129-138 (2012)
The ability to rapidly assess microtubule number in 3D image stacks from electron tomograms is essential for collecting statistically meaningful data sets. Here we implement microtubule tracing using 3D template matching. We evaluate our results by comparing the automatically traced centerlines to manual tracings in a large number of electron tomograms of the centrosome of the early Caenorhabditis elegans embryo. Furthermore, we give a qualitative description of the tracing results for three other types of samples. For dual-axis tomograms, the automatic tracing yields 4% false negatives and 8% false positives on average. For single-axis tomograms, the accuracy of tracing is lower (16% false negatives and 14% false positives) due to the missing wedge in electron tomography. We also implemented an editor specifically designed for correcting the automatic tracing. Besides, this editor can be used for annotating microtubules. The automatic tracing together with a manual correction significantly reduces the amount of manual labor for tracing microtubule centerlines so that large-scale analysis of microtubule network properties becomes feasible.

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)
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.

Cristina Aguirre-Portolés, Alexander W. Bird, Anthony A. Hyman, Marta Cañamero, Ignacio Pérez de Castro, Marcos Malumbres
Tpx2 controls spindle integrity, genome stability, and tumor development.
Cancer Res, 72(6) 1518-1528 (2012)
Tpx2 is a microtubule-associated protein that activates the cell-cycle kinase Aurora A and regulates the mitotic spindle. Overexpression of Tpx2 is associated with the development of different human tumors and strongly correlates with chromosomal instability. By analyzing a conditional null mutation in the mouse Tpx2 gene, we show here that Tpx2 expression is essential for spindle function and chromosome segregation in the mouse embryo. Conditional genetic ablation of Tpx2 in primary cultures resulted in deficient microtubule nucleation from DNA and aberrant spindles during prometaphase. These cells eventually exited from mitosis without chromosome segregation. In addition, Tpx2 haploinsufficiency led to the accumulation of aneuploidies in vivo and increased susceptibility to spontaneous lymphomas and lung tumors. Together, our findings indicate that Tpx2 is essential for maintaining genomic stability through its role in spindle regulation. Subtle changes in Tpx2 expression may favor tumor development in vivo.

Li Ding, Ina Poser, Maciej Paszkowski-Rogacz, Frank Buchholz
From RNAi screens to molecular function in embryonic stem cells
Stem cell rev, 8(1) 32-42 (2012)
The ability of embryonic stem (ES) cells to generate any of the around 220 cell types of the adult body has fascinated scientists ever since their discovery. The capacity to re-program fully differentiated cells into induced pluripotent stem (iPS) cells has further stimulated the interest in ES cell research. Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past. The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity. Here, we review the RNAi screens performed in ES cells to date and discuss the challenges associated with these large-scale experiments. Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

Heiko Dückert, Verena Pries, Vivek Khedkar, Sascha Menninger, Hanna Bruss, Alexander W. Bird, Zoltan Maliga, Andreas Brockmeyer, Petra Janning, Anthony A. Hyman, Stefan Grimme, Markus Schürmann, Hans Preut, Katja Hübel, Slava Ziegler, Kamal Kumar, Herbert Waldmann
Natural product-inspired cascade synthesis yields modulators of centrosome integrity.
Nat Chem Biol, 8(2) 179-184 (2012)
In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1.

Martin P Stewart, Yusuke Toyoda, Anthony A. Hyman, Daniel J. Müller
Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp.
Nat Protoc, 7(1) 143-154 (2012)
To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance force, mechanical stress and volume of globular animal cells under compression. We describe the AFM and cantilever setup, live cell culture in the AFM, how to ensure stability of AFM measurements during medium perfusion, integration of optical microscopy to measure parameters such as volume and track intracellular dynamics, and interpretation of the physical parameters measured. Although we use this protocol on trypsinized interphase and mitotic HeLa cells, it can also be applied to other cells with a relatively globular shape, especially animal cells in a low-adhesive environment. After a short setup phase, the protocol can be used to investigate approximately one cell per hour.

Alexander W. Bird, Axel Erler, Jun Fu, Jean-Karim Hériché, Marcello Maresca, Youming Zhang, Anthony A. Hyman, A Francis Stewart
High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.
Nat Methods, 9(1) 103-109 (2012)
Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.

Massimilano Scolz, Per Widlund, Silvano Piazza, Debora Rosa Bublik, Simone Reber, Leticia Y Peche, Yari Ciani, Nina Hubner, Mayumi Isokane, Martin Monte, Jan Ellenberg, Anthony Hyman, Claudio Schneider, Alexander W. Bird
GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.
PLoS ONE, 7(12) Art. No. e51259 (2012)
The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

Anthony A. Hyman
Whither systems biology.
Philos Trans R Soc Lond B Biol Sci, 366(1584) 3635-3637 (2011)
Cell biologists are interested in how complexity arises from the interaction of different molecules. However, cells are many orders of magnitude larger than the protein-binding interfaces. To bridge these vast difference in scales, biologists construct hierarchies of organization of cellular structures. I describe how systems biology provides an approach to bridge these different scales.

Simone Reber, Anthony A. Hyman
Samurai sword sets spindle size.
Cell, 147(6) 1224-1225 (2011)
Although the parts list is nearly complete for many cellular structures, mechanisms that control their size remain poorly understood. Loughlin and colleagues now show that phosphorylation of a single residue of katanin, a microtubule-severing protein, largely accounts for the difference in spindle length between two closely related frogs.

Anthony A. Hyman, Kai Simons
Beyond HeLa cells
Nature, 480(7375) 34-34 (2011)

Björn Hegemann✳︎, James R A Hutchins✳︎, Otto Hudecz, Maria Novatchkova, Jonathan Rameseder, Martina M Sykora, Shang-Yun Liu, Michael Mazanek, Péter Lénárt, Jean-Karim Hériché, Ina Poser, Norbert Kraut, Anthony A. Hyman, Michael B Yaffe, Karl Mechtler, Jan-Michael Peters
Systematic phosphorylation analysis of human mitotic protein complexes.
Sci Signal, 4(198) Art. No. rs12 (2011)
Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.

Antigoni Elefsinioti, Ömer Sinan Saraç, Anna Hegele, Conrad Plake, Nina C Hubner, Ina Poser, Mihail Sarov, Anthony A. Hyman, Matthias Mann, Michael Schroeder, Ulrich Stelzl, Andreas Beyer
Large-scale de novo prediction of physical protein-protein association.
Mol Cell Proteomics, 10(11) Art. No. M111.010629 (2011)
Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org.

Nathan Goehring, Philipp Khuc Trong, Justin Bois, Debanjan Chowdhury, Ernesto M Nicola, Anthony A. Hyman, Stephan W. Grill
Polarization of PAR Proteins by Advective Triggering of a Pattern-Forming System.
Science, 334(6059) 1137-1141 (2011)
In the Caenorhabditis elegans zygote, a conserved network of partitioning defective 4 (PAR) polarity proteins segregate into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.

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)
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.

Markus Decker, Steffen Jaensch, Andrei I. Pozniakovsky, Andrea Zinke, Kevin F O'Connell, Wolfgang Zachariae, Eugene Myers, Anthony A. Hyman
Limiting amounts of centrosome material set centrosome size in C. elegans embryos.
Curr Biol, 21(15) 1259-1267 (2011)
The ways in which cells set the size of intracellular structures is an important but largely unsolved problem [1]. Early embryonic divisions pose special problems in this regard. Many checkpoints common in somatic cells are missing from these divisions, which are characterized by rapid reductions in cell size and short cell cycles [2]. Embryonic cells must therefore possess simple and robust mechanisms that allow the size of many of their intracellular structures to rapidly scale with cell size.

Martin P Stewart, Yusuke Toyoda, Anthony A. Hyman, Daniel J. Müller
Force probing cell shape changes to molecular resolution.
Trends Biochem Sci, 36(8) 444-450 (2011)
Atomic force microscopy (AFM) is a force sensing nanoscopic tool that can be used to undertake a multiscale approach to understand the mechanisms that underlie cell shape change, ranging from the cellular to molecular scale. In this review paper, we discuss the use of AFM to characterize the dramatic shape changes of mitotic cells. AFM-based mechanical assays can be applied to measure the considerable rounding force and hydrostatic pressure generated by mitotic cells. A complementary AFM technique, single-molecule force spectroscopy, is able to quantify the interactions and mechanisms that functionally regulate individual proteins. Future developments of these nanomechanical methods, together with advances in light microscopy imaging and cell biological and genetic tools, should provide further insight into the biochemical, cellular and mechanical processes that govern mitosis and other cell shape change phenomena.

Anthony A. Hyman#, Clifford Brangwynne#
Beyond stereospecificity: liquids and mesoscale organization of cytoplasm.
Dev Cell, 21(1) 14-16 (2011)
The cytoplasm is not a homogenous solution but instead consists of large dynamic assemblies that arise from transient molecular interactions. Some of these structures have been shown to represent liquid droplets of concentrated protein and RNA. Liquid phase separation of cytoplasm may be a fundamental principle of cytoplasmic organization.

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)
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.

Rebecca A Green, Huey-Ling Kao, Anjon Audhya, Swathi Arur, Jonathan R Mayers, Heidi N Fridolfsson, Monty Schulman, Siegfried Schloissnig, Sherry Niessen, Kimberley Laband, Shaohe Wang, Daniel A Starr, Anthony A. Hyman, Tim Schedl, Arshad Desai, Fabio Piano, Kristin C. Gunsalus#, Karen Oegema#
A high-resolution C. elegans essential gene network based on phenotypic profiling of a complex tissue.
Cell, 145(3) 470-482 (2011)
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Lis Jakobsen, Katja Vanselow, Marie Skogs, Yusuke Toyoda, Emma Lundberg, Ina Poser, Lasse G Falkenby, Martin Bennetzen, Jens Westendorf, Erich A Nigg, Mathias Uhlen, Anthony A. Hyman, Jens S Andersen
Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods.
EMBO J, 30(8) 1520-1535 (2011)
Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP-SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non-specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.

Anthony A. Hyman
Q & A Tony Hyman
Curr Biol, 21(7) 240-242 (2011)

Clifford Brangwynne, Timothy J. Mitchison, Anthony A. Hyman
Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
Proc Natl Acad Sci U.S.A., 108(11) 4334-4339 (2011)
For most intracellular structures with larger than molecular dimensions, little is known about the connection between underlying molecular activities and higher order organization such as size and shape. Here, we show that both the size and shape of the amphibian oocyte nucleolus ultimately arise because nucleoli behave as liquid-like droplets of RNA and protein, exhibiting characteristic viscous fluid dynamics even on timescales of < 1 min. We use these dynamics to determine an apparent nucleolar viscosity, and we show that this viscosity is ATP-dependent, suggesting a role for active processes in fluidizing internal contents. Nucleolar surface tension and fluidity cause their restructuring into spherical droplets upon imposed mechanical deformations. Nucleoli exhibit a broad distribution of sizes with a characteristic power law, which we show is a consequence of spontaneous coalescence events. These results have implications for the function of nucleoli in ribosome subunit processing and provide a physical link between activity within a macromolecular assembly and its physical properties on larger length scales.

Julien Guizetti, Lothar Schermelleh, Jana Mäntler, Sandra Maar, Ina Poser, Heinrich Leonhardt, Thomas Müller-Reichert#, Daniel W Gerlich#
Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments.
Science, 331(6024) 1616-1620 (2011)
After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)-III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III-mediated fission of large membrane structures.

Stefanie Redemann, Siegfried Schloissnig, Susanne Ernst, Andrei I. Pozniakovsky, Swathi Ayloo, Anthony A. Hyman, Henrik Bringmann
Codon adaptation-based control of protein expression in C. elegans.
Nat Methods, 8(3) 250-252 (2011)
We present a method to control protein levels under native genetic regulation in Caenorhabditis elegans by using synthetic genes with adapted codons. We found that the force acting on the spindle in C. elegans embryos was related to the amount of the G-protein regulator GPR-1/2. Codon-adapted versions of any C. elegans gene can be designed using our web tool, C. elegans codon adapter.

Per Widlund, Jeffrey H. Stear, Andrei I. Pozniakovsky, Marija Zanic, Simone Reber, Gary J. Brouhard, Anthony A. Hyman#, Jonathon Howard#
XMAP215 polymerase activity is built by combining multiple tubulin-binding TOG domains and a basic lattice-binding region.
Proc Natl Acad Sci U.S.A., 108(7) 2741-2746 (2011)
XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.

Frank Schnütgen, Franziska Ehrmann, Ina Poser, Nina C Hubner, Jens Hansen, Thomas Floss, Ingrid deVries, Wolfgang Wurst, Anthony A. Hyman, Matthias Mann, Harald von Melchner
Resources for proteomics in mouse embryonic stem cells.
Nat Methods, 8(2) 103-104 (2011)

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)
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.

Wei Niu, Zhi John Lu, Mei Zhong, Mihail Sarov, James T Murray, Cathleen M Brdlik, Judith Janette, Chao Chen, Pedro Alves, Elicia A. Preston, Cindie Slightam, Lixia Jiang, Anthony A. Hyman, Stuart K Kim, Robert H Waterston, Mark Gerstein, Michael Snyder, Valerie Reinke
Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans.
Genome Res, 21(2) 245-254 (2011)
Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors-LIN-39, MAB-5, and EGL-5-indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.

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)
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.

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
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.

Christopher Gell, Claire Friel, Barbara Borgonovo, David N. Drechsel, Anthony A. Hyman, Jonathon Howard
Purification of tubulin from porcine brain.
Methods Mol Biol, 777 15-28 (2011)
Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain.

Andreas Ettinger, Michaela Wilsch-Bräuninger, Anne-Marie Marzesco, Marc Bickle, Annett Lohmann, Zoltan Maliga, Jana Karbanová, Denis Corbeil, Anthony A. Hyman, Wieland B. Huttner
Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour.
Nat Commun, 2 Art. No. 503 (2011)
The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation.

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)
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.

Steffen Jaensch
Time-Resolved Quantification of Centrosomes by Automated Image Analysis Suggests Limiting Component to Set Centrosome Size in C. Elegans Embryos
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Markus Decker
Setting centrosome size in the early caenorhabditis elegans embryo
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Tomoko Nishiyama, Rene Ladurner, Julia Schmitz, Emanuel Kreidl, Alexander Schleiffer, Venugopal Bhaskara, Masashige Bando, Katsuhiko Shirahige, Anthony A. Hyman, Karl Mechtler, Jan-Michael Peters
Sororin mediates sister chromatid cohesion by antagonizing Wapl.
Cell, 143(5) 737-749 (2010)
Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.

Nathan W. Goehring, Debanjan Chowdhury, Anthony A. Hyman, Stephan W. Grill
FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange
Biophys J, 99(8) 2443-2452 (2010)
Obtaining quantitative kinetic parameters from FRAP (Fluorescence Recovery After Photo-bleaching) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries, stripes and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry that creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by more accurately parameterizing the initial post-bleach state. This allows for "reconstruction" of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLCdelta1 in C. elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.

Michiel Vermeulen✳︎#, H Christian Eberl✳︎, Filomena Matarese✳︎, Hendrik Marks, Sergei Denissov, Falk Butter, Kenneth K Lee, Jesper V Olsen, Anthony A. Hyman, Hendrik G Stunnenberg#, Matthias Mann#
Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.
Cell, 142(6) 967-980 (2010)
Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these marks by high-accuracy, quantitative mass spectrometry. These chromatin "readers" are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.

Michael H A Schmitz, Michael Held, Veerle Janssens, James R A Hutchins, Otto Hudecz, Elitsa Ivanova, Jozef Goris, Laura Trinkle-Mulcahy, Angus I Lamond, Ina Poser, Anthony A. Hyman, Karl Mechtler, Jan-Michael Peters, Daniel W Gerlich
Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells.
Nat Cell Biol, 12(9) 886-893 (2010)
When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.

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)
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.

Stefanie Redemann✳︎#, Jacques Pecreaux✳︎#, Nathan W. Goehring, Khaled Khairy, Ernst H K Stelzer, Anthony A. Hyman#, Jonathon Howard#
Membrane invaginations reveal cortical sites that pull on mitotic spindles in one-cell C. elegans embryos.
PLoS ONE, 5(8) Art. No. e12301 (2010)
Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

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)
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.

Steffen Jaensch, Markus Decker, Anthony A. Hyman, Eugene Myers
Automated tracking and analysis of centrosomes in early Caenorhabditis elegans embryos
Bioinformatics, 26(12) 13-20 (2010)
Motivation: The centrosome is a dynamic structure in animal cells that serves as a microtubule organizing center during mitosis and also regulates cell-cycle progression and sets polarity cues. Automated and reliable tracking of centrosomes is essential for genetic screens that study the process of centrosome assembly and maturation in the nematode Caenorhabditis elegans.

Nina C. Hubner✳︎, Alexander W. Bird✳︎, Jürgen Cox, Bianca Splettstoesser, Peter Bandilla, Ina Poser, Anthony A. Hyman#, Matthias Mann#
Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions.
J Cell Biol, 189(4) 739-754 (2010)
Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC-green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.

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)
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.

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)
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.

James R A Hutchins✳︎, Yusuke Toyoda✳︎, Björn Hegemann✳︎, Ina Poser✳︎, Jean-Karim Hériché, Martina M Sykora, Martina Augsburg, Otto Hudecz, Bettina A Buschhorn, Jutta Bulkescher, Christian Conrad, David Comartin, Alexander Schleiffer, Mihail Sarov, Andrei I. Pozniakovsky, Mikolaj Slabicki, Siegfried Schloissnig, Ines Steinmacher, Marit Leuschner, Andrea Ssykor, Steffen Lawo, Laurence Pelletier, Holger Stark, Kim Nasmyth, Jan Ellenberg, Richard Durbin, Frank Buchholz, Karl Mechtler, Anthony A. Hyman#, Jan-Michael Peters#
Systematic analysis of human protein complexes identifies chromosome segregation proteins.
Science, 328(5978) 593-599 (2010)
Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

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)
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.

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)
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.

Mei Zhong, Wei Niu, Zhi John Lu, Mihail Sarov, James T Murray, Judith Janette, Debasish Raha, Karyn L Sheaffer, Hugo Y K Lam, Elicia A. Preston, Cindie Slightam, LaDeana W Hillier, Trisha Brock, Ashish Agarwal, Raymond Auerbach, Anthony A. Hyman, Mark Gerstein, Susan E Mango, Stuart K Kim, Robert H Waterston, Valerie Reinke#, Michael Snyder#
Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.
PLoS Genet, 6(2) Art. No. e1000848 (2010)
Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.

Alessia Errico✳︎, Krupa Deshmukh✳︎, Yoshimi Tanaka, Andrei I. Pozniakovsky, Tim Hunt
Identification of substrates for cyclin dependent kinases.
Adv Enzyme Regul, 50(1) 375-399 (2010)

Garrett Greenan, Clifford Brangwynne, Steffen Jaensch, Joebin Gharakhani, Frank Jülicher#, Anthony A. Hyman#
Centrosome size sets mitotic spindle length in Caenorhabditis elegans embryos.
Curr Biol, 20(4) 353-358 (2010)
Just as the size of an organism is carefully controlled, the size of intracellular structures must also be regulated. The mitotic spindle is a supramolecular machine that generates the forces which separate sister chromatids during mitosis. Although spindles show little size variation between cells of the same type, spindle length can vary at least 10-fold between different species. Recent experiments on spindle length showed that in embryonic systems spindle length varied with blastomere size. Furthermore, a comparison between two Xenopus species showed that spindle length was dependent on some cytoplasmic factor. These data point toward mechanisms to scale spindle length with cell size. Centrosomes play an important role in organizing microtubules during spindle assembly. Here we use Caenorhabditis elegans to study the role of centrosomes in setting spindle length. We show that spindle length correlates with centrosome size through development and that a reduction of centrosome size by molecular perturbation reduces spindle length. By systematically analyzing centrosome proteins, we show that spindle length does not depend on microtubule density at centrosomes. Rather, our data suggest that centrosome size sets mitotic spindle length by controlling the length scale of a TPXL-1 gradient along spindle microtubules.

Garrett Greenan
Setting mitotic spindle length in the early Caenorhabditis elegans embryo
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)

Jonathon Howard, Anthony A. Hyman
Growth, fluctuation and switching at microtubule plus ends.
Nat Rev Mol Cell Biol, 10(8) 569-574 (2009)
Recent experiments suggest that microtubules do not grow steadily but instead elongate at a rate that varies in time. We argue that this variation might arise from fluctuations in the length of a dynamic GTP-tubulin cap at the microtubule end. We propose that these fluctuations can lead to a switch in the dynamics of a microtubule end between growth and shrinkage, and provide insight into how the sensitivity of this switch can be changed by microtubule polymerases, such as XMAP215, and tensile forces, through the stabilization of initial contacts in the cap.

Clifford Brangwynne, Christian R. Eckmann, David S Courson, Agata Rybarska, Carsten Hoege, Joebin Gharakhani, Frank Jülicher, Anthony A. Hyman
Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
Science, 324(5935) 1729-1732 (2009)
In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.

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)
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.

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)

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)
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.

Zdenek Petrásek✳︎, Carsten Hoege✳︎, Alireza Mashaghi, Thomas Ohrt, Anthony A. Hyman, Petra Schwille
Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy.
Biophys J, 95(11) 5476-5486 (2008)
The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

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)
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.

Mike Boxem#, Zoltan Maliga, Niels Klitgord, Na Li, Irma Lemmens, Miyeko Mana, Lorenzo de Lichtervelde, Joram D. Mul, Diederik van de Peut, Maxime Devos, Nicolas Simonis, Muhammed A. Yildirim, Murat Cokol, Huey-Ling Kao, Anne-Sophie de Smet, Anne-Lore Schlaitz, Haidong Wang, Tong Hao, Stuart Milstein, Changyu Fan, Mike Tipsword, Kevin Drew, Matilde Galli, Kahn Rhrissorrakrai, David N. Drechsel, Daphne Koller, Frederick P. Roth, Lilia M. Iakoucheva, A. Keith Dunker, Richard Bonneau, Kristin C. Gunsalus, David E. Hill, Fabio Piano, Jan Tavernier, Sander van den Heuvel, Anthony A. Hyman#, Marc Vidal#
A protein domain-based interactome network for C. elegans early embryogenesis.
Cell, 134(3) 534-545 (2008)
Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.

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)
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.

Ina Poser, Mihail Sarov, James R A Hutchins, Jean-Karim Hériché, Yusuke Toyoda, Andrei I. Pozniakovsky, Daniela Weigl, Anja Nitzsche, Björn Hegemann, Alexander W. Bird, Laurence Pelletier, Ralf Kittler, Sujun Hua, Ronald Naumann, Martina Augsburg, Martina M Sykora, Helmut Hofemeister, Youming Zhang, Kim Nasmyth, Kevin P White, Steffen Dietzel, Karl Mechtler, Richard Durbin, A. Francis Stewart, Jan-Michael Peters, Frank Buchholz, Anthony A. Hyman
BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
Nat Methods, 5(5) 409-415 (2008)
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Gary J. Brouhard✳︎, Jeffrey H. Stear✳︎, Tim L. Noetzel, Jawdat Al-Bassam, Kazuhisa Kinoshita, Stephen C. Harrison, Jonathon Howard#, Anthony A. Hyman#
XMAP215 is a processive microtubule polymerase.
Cell, 132(1) 79-88 (2008)
Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.

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)
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.

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
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.

Victor F Lundin, Martin Srayko, Anthony A. Hyman, Michel R Leroux
Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans.
Dev Biol, 313(1) 320-334 (2008)
The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in alpha-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.

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)
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.

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)
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.

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)
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.

Peter C Stirling, Martin Srayko, Karam S Takhar, Andrei I. Pozniakovsky, Anthony A. Hyman, Michel R Leroux
Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression.
Mol Biol Cell, 18(6) 2336-2345 (2007)
The Chaperonin Containing Tcp1 (CCT) maintains cellular protein folding homeostasis in the eukaryotic cytosol by assisting the biogenesis of many proteins, including actins, tubulins, and regulators of the cell cycle. Here, we demonstrate that the essential and conserved eukaryotic phosducin-like protein 2 (PhLP2/PLP2) physically interacts with CCT and modulates its folding activity. Consistent with this functional interaction, temperature-sensitive alleles of Saccharomyces cerevisiae PLP2 exhibit cytoskeletal and cell cycle defects. We uncovered several high-copy suppressors of the plp2 alleles, all of which are associated with G1/S cell cycle progression but which do not appreciably affect cytoskeletal protein function or fully rescue the growth defects. Our data support a model in which Plp2p modulates the biogenesis of several CCT substrates relating to cell cycle and cytoskeletal function, which together contribute to the essential function of PLP2.

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)
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.

Jawdat Al-Bassam, Nicholas A. Larsen, Anthony A. Hyman, Stephen C. Harrison
Crystal structure of a TOG domain: conserved features of XMAP215/Dis1-family TOG domains and implications for tubulin binding.
Structure, 15(3) 355-362 (2007)
Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.

Carrie R. Cowan, Anthony A. Hyman
Acto-myosin reorganization and PAR polarity in C. elegans.
Development, 134(6) 1035-1043 (2007)
The symmetry-breaking event during polarization of C. elegans embryos is an asymmetric rearrangement of the acto-myosin network, which dictates cell polarity through the differential recruitment of PAR proteins. The sperm-supplied centrosomes are required to initiate this cortical reorganization. Several questions about this event remain unanswered: how is the acto-myosin network regulated during polarization and how does acto-myosin reorganization lead to asymmetric PAR protein distribution? As we discuss, recent studies show that C. elegans embryos use two GTPases, RHO-1 and CDC-42, to regulate these two steps in polarity establishment. Although RHO-1 and CDC-42 control distinct aspects of polarization, they function interdependently to regulate polarity establishment in C. elegans embryos.

Alexander Zumdieck, Karsten Kruse, Henrik Bringmann, Anthony A. Hyman, Frank Jülicher
Stress generation and filament turnover during actin ring constriction.
PLoS ONE, 2(1) 696-696 (2007)
We present a physical analysis of the dynamics and mechanics of contractile actin rings. In particular, we analyze the dynamics of ring contraction during cytokinesis in the Caenorhabditis elegans embryo. We present a general analysis of force balances and material exchange and estimate the relevant parameter values. We show that on a microscopic level contractile stresses can result from both the action of motor proteins, which cross-link filaments, and from the polymerization and depolymerization of filaments in the presence of end-tracking cross-linkers.

Jonathon Howard, Anthony A. Hyman
Microtubule polymerases and depolymerases.
Curr Opin Cell Biol, 19(1) 31-35 (2007)
The variety of shapes and sizes of the microtubule cytoskeleton is as great as the number of different cell types. This large variety is a consequence of the dynamic properties of microtubules, which allow them to adopt distributions of arbitrary size and form. How is the distribution of microtubule lengths controlled? Recent work suggests that the length distribution is controlled, at least in part, by the activity of microtubule polymerases and depolymerases, which accelerate microtubule growth and shrinkage. Specifically, biochemical and single-molecule studies have shown how MCAK (kinesin-13) and Kip3p (kinesin-8) accelerate depolymerization and how XMAP215 may accelerate growth. Studies on the yeast Dam1 complex have shown how proteins can couple a cellular structure, the kinetochore, to the ends of polymerizing and depolymerizing microtubules.

Daisuke Mori, Yoshihisa Yano, Kazuhito Toyo-oka, Noriyuki Yoshida, Masami Yamada, Masami Muramatsu, Dongwei Zhang, Hideyuki Saya, Yoko Y Toyoshima, Kazuhisa Kinoshita, Anthony Wynshaw-Boris, Shinji Hirotsune
NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment.
Mol Cell Biol, 27(1) 352-367 (2007)
NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.

David A. Cisneros
Molecular assemblies observed by atomic force microscopy
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2007)
Atomic force microscopy (AFM) is a powerful technique that enables for

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)

Henrik Bringmann, Carrie R. Cowan, Jun Kong, Anthony A. Hyman
LET-99, GOA-1/GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis.
Curr Biol, 17(2) 185-191 (2007)
At anaphase, the mitotic spindle positions the cytokinesis furrow [1]. Two populations of spindle microtubules are implicated in cytokinesis: radial microtubule arrays called asters and bundled nonkinetochore microtubules called the spindle midzone [2-4]. In C. elegans embryos, these two populations of microtubules provide two consecutive signals that position the cytokinesis furrow: The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone [5]. Evidence for two cytokinesis signals came from the identification of molecules that block midzone-positioned cytokinesis [5-7]. However, no molecules that are only required for, and thus define, the molecular pathway of aster-positioned cytokinesis have been identified. With RNAi screening, we identify LET-99 and the heterotrimeric G proteins GOA-1/GPA-16 and their regulator GPR-1/2 [10-12] in aster-positioned cytokinesis. By using mechanical spindle displacement, we show that the anaphase spindle positions cortical LET-99, at the site of the presumptive cytokinesis furrow. LET-99 enrichment at the furrow depends on the G proteins. GPR-1 is locally reduced at the site of cytokinesis-furrow formation by LET-99, which prevents accumulation of GPR-1 at this site. We conclude that LET-99 and the G proteins define a molecular pathway required for aster-positioned cytokinesis.

Henrik Bringmann
Experiments concerning the mechanisms of cytokinesis in Caenorhabditis elegans embryos
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2007)
In my thesis I aimed to contribute to the understanding of the

Carrie R. Cowan, Anthony A. Hyman
Cyclin E-Cdk2 temporally regulates centrosome assembly and establishment of polarity in Caenorhabditis elegans embryos.
Nat Cell Biol, 8(12) 1441-1447 (2006)
Establishment of polarity in C. elegans embryos is dependent on the centrosome. The sperm contributes a pair of centrioles to the egg and these centrioles remain incapable of polarizing the cortex while the egg completes meiosis. Coincident with the establishment of polarity, the centrioles recruit centrosomal proteins, several of which are required for polarity, suggesting that the temporal regulation of centrosome assembly may control the initiation of polarization. We found that cyclin E-Cdk2 is required for the establishment of polarity. Cyclin E-Cdk2 controls the recruitment of centrosomal proteins specifically at the time of polarity establishment. Cyclin E is required for several examples of asymmetric cell division and fate determination in C. elegans and Drosophila. Here, we suggest a possible mechanism for cyclin E-Cdk2-dependent differentiation: the establishment of cortical polarity by the centrosome.

Sharsti Sandall, Fedor F. Severin, Ian X. McLeod, John R. Yates, Karen Oegema, Anthony A. Hyman, Arshad Desai
A Bir1-Sli15 complex connects centromeres to microtubules and is required to sense kinetochore tension.
Cell, 127(6) 1179-1191 (2006)
Proper connections between centromeres and spindle microtubules are of critical importance in ensuring accurate segregation of the genome during cell division. Using an in vitro approach based on the sequence-specific budding yeast centromere, we identified a complex of the chromosomal passenger proteins Bir1 and Sli15 (Survivin and INCENP) that links centromeres to microtubules. This linkage does not require Ipl1/Aurora B kinase, whose targeting and activation are controlled by Bir1 and Sli15. Ipl1 is the tension-dependent regulator of centromere-microtubule interactions that ensures chromosome biorientation on the spindle. Elimination of the linkage between centromeres and microtubules mediated by Bir1-Sli15 phenocopies mutations that selectively cripple Ipl1 kinase activation. These findings lead us to propose that the Bir1-Sli15-mediated linkage, which bridges centromeres and microtubules and includes the Aurora kinase-activating domain of INCENP family proteins, is the tension sensor that relays the mechanical state of centromere-microtubule attachments into local control of Ipl1 kinase activity.

Jacques Pecreaux✳︎, Jens-Christian Röper✳︎, Karsten Kruse, Frank Jülicher, Anthony A. Hyman, Stephan W. Grill, Jonathon Howard
Spindle oscillations during asymmetric cell division require a threshold number of active cortical force generators.
Curr Biol, 16(21) 2111-2122 (2006)
BACKGROUND: Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. These motions are thought to be driven by a force-generating complex-possibly containing the motor protein cytoplasmic dynein-that is located at the cell cortex and pulls on microtubules growing out from the spindle poles. A theoretical analysis indicates that the oscillations might arise from mechanical coordination of the force-generating motors, and this coordination is mediated by the load dependence of the motors' detachment from the microtubules. The model predicts that the motor activity must exceed a threshold for oscillations to occur. RESULTS: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of dynein light intermediate chain as well as GPR-1 and GPR-2 that are involved in the G protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the motor activity dropped below a threshold. Furthermore, we can account for the complex choreography of the mitotic spindle-the precise temporal coordination of the buildup and die-down of the transverse oscillations with the posterior displacement-by a gradual increase in the processivity of a single type of motor machinery during metaphase and anaphase. CONCLUSIONS: The agreement between our results and modeling suggests that the force generators themselves have the intrinsic capability of generating oscillations when opposing forces exceed a threshold.

Laurence Pelletier, Eileen T. O'Toole, Anne Schwager, Anthony A. Hyman, Thomas Müller-Reichert
Centriole assembly in Caenorhabditis elegans.
Nature, 444(7119) 619-623 (2006)
Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.

Mihail Sarov, Susan Schneider, Andrei I. Pozniakovsky, Assen Roguev, Susanne Ernst, Youming Zhang, Anthony A. Hyman, A. Francis Stewart
A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.
Nat Methods, 3(10) 839-844 (2006)
We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Vladimir Varga, Jonne H. Helenius, Kozo Tanaka, Anthony A. Hyman, Tomoyuki U Tanaka, Jonathon Howard
Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner.
Nat Cell Biol, 8(9) 957-962 (2006)
The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.

Stephanie Schonegg, Anthony A. Hyman
CDC-42 and RHO-1 coordinate acto-myosin contractility and PAR protein localization during polarity establishment in C. elegans embryos.
Development, 133(18) 3507-3516 (2006)
In C. elegans one-cell embryos, polarity is conventionally defined along the anteroposterior axis by the segregation of partitioning-defective (PAR) proteins into anterior (PAR-3, PAR-6) and posterior (PAR-1, PAR-2) cortical domains. The establishment of PAR asymmetry is coupled with acto-myosin cytoskeleton rearrangements. The small GTPases RHO-1 and CDC-42 are key players in cytoskeletal remodeling and cell polarity in a number of different systems. We investigated the roles of these two GTPases and the RhoGEF ECT-2 in polarity establishment in C. elegans embryos. We show that CDC-42 is required to remove PAR-2 from the cortex at the end of meiosis and to localize PAR-6 to the cortex. By contrast, RHO-1 activity is required to facilitate the segregation of CDC-42 and PAR-6 to the anterior. Loss of RHO-1 activity causes defects in the early organization of the myosin cytoskeleton but does not inhibit segregation of myosin to the anterior. We therefore propose that RHO-1 couples the polarization of the acto-myosin cytoskeleton with the proper segregation of CDC-42, which, in turn, localizes PAR-6 to the anterior cortex.

Jacob W. J. Kerssemakers, Emilia-Laura Munteanu, Liedewij Laan, Tim L. Noetzel, Marcel E Janson, Marileen Dogterom
Assembly dynamics of microtubules at molecular resolution.
Nature, 442(7103) 709-712 (2006)
Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.

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)

Jawdat Al-Bassam✳︎, Mark van Breugel✳︎, Stephen C. Harrison, Anthony A. Hyman
Stu2p binds tubulin and undergoes an open-to-closed conformational change.
J Cell Biol, 172(7) 1009-1022 (2006)
Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat-containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Cerasela Z. Dinu
Leveraging the motor protein kinesin to manipulate DNA molecules in synthetic environment
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2006)

Svyatoslav Sokolov, Dmitry A. Knorre, Ekaterina Smirnova, Olga V. Markova, Andrei I. Pozniakovsky, Vladimir P. Skulachev, Fedor F. Severin
Ysp2 mediates death of yeast induced by amiodarone or intracellular acidification.
Biochim Biophys Acta, 1757(9-10) 1366-1370 (2006)
Recently we have found that the drug amiodarone induces apoptosis in yeast, which is mediated by reactive oxygen species (ROS). Here we have used this finding as a tool to screen for genes involved in the death program. We have described a novel mitochondrial protein, Ysp2, acting in the amiodarone-induced death cascade. After amiodarone addition both the control and amiodarone-resistant ysp2-deleted cells formed ROS, but the mutant (unlike the control) did not undergo the mitochondrial thread-to-grain transition. To test whether the action of Ysp2 is amiodarone-specific we tried to induce PCD by other agents. We have found that acetic acid-induced PCD also depends on Ysp2. We also demonstrate that, like acetic acid, propionic acid or nigericin triggered intracellular acidification causing ROS-dependent death. We suggest that intracellular acidification results in the protonation of superoxide anion (O2-*) to form HO2, one of the most aggressive ROS, which in turn induces Ysp2-mediated PCD.

Kazuhisa Kinoshita, Tim L. Noetzel, Isabelle Arnal, David N. Drechsel, Anthony A. Hyman
Global and local control of microtubule destabilization promoted by a catastrophe kinesin MCAK/XKCM1.
J Muscle Res Cell Motil, 27(2) 107-114 (2006)
Traditionally, kinesins have been identified as proteins that use the energy of ATP to translocate along microtubules. However, in the last decade some kinesin-like proteins were found to destabilize microtubule ends. The kinesins that destabilize microtubules are known as "catastrophe kinesins". Analyses of a Xenopus member of the catastrophe kinesins called MCAK/XKCM1 have shown that, in fact, catastrophe kinesins are essential for controlling the distribution of microtubules by inducing their depolymerization. Therefore, unraveling the mechanisms of how microtubule destabilization promoted by these catastrophe kinesins is controlled is essential for understanding how microtubules in a cell are distributed. Here we give an overview of the studies that have focused on the global and local control of microtubule destabilization promoted by MCAK/XKCM1.

Svyatoslav Sokolov, Andrei I. Pozniakovsky, Natalia Bocharova, Dmitry A. Knorre, Fedor F. Severin
Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers.
Biochim Biophys Acta, 1757(5-6) 660-666 (2006)
Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.

K. Tanuj Sapra, Hüseyin Besir, Dieter Oesterhelt, Daniel J. Müller
Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy.
J Mol Biol, 355(4) 640-650 (2006)
Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.

Karen Oegema#, Anthony A. Hyman#
Cell division.
WormBook : the online review of C. elegans biology, Jan 19 1-40 (2006)
The C. elegans embryo is a powerful model system for studying the mechanics of metazoan cell division. Its primary advantage is that the architecture of the syncytial gonad makes it possible to use RNAi to generate oocytes whose cytoplasm is reproducibly (typically >95%) depleted of targeted essential gene products via a process that does not depend exclusively on intrinsic protein turnover. The depleted oocytes can then be analyzed as they attempt their first mitotic division following fertilization. Here we outline the characteristics that contribute to the usefulness of the C. elegans embryo for cell division studies. We provide a timeline for the first embryonic mitosis and highlight some of its key features. We also summarize some of the recent discoveries made using this system, particularly in the areas of nuclear envelope assembly/dissassembly, centrosome dynamics, formation of the mitotic spindle, kinetochore assembly, chromosome segregation, and cytokinesis.

Henrik Bringmann
Cytokinesis and the spindle midzone.
Cell Cycle, 4(12) 1709-1712 (2005)
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.

Stephanie Schonegg
Rho GTPase family members in establishment of polarity in C.elegans embryos
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2005)

Kazuhisa Kinoshita, Tim L. Noetzel, Laurence Pelletier, Karl Mechtler, David N. Drechsel, Anne Schwager, Mike Lee, Jordan W Raff, Anthony A. Hyman
Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis.
J Cell Biol, 170(7) 1047-1055 (2005)
Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

Teresa P. Barros, Kazuhisa Kinoshita, Anthony A. Hyman, Jordan W Raff
Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules.
J Cell Biol, 170(7) 1039-1046 (2005)
Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.

Kristin C. Gunsalus✳︎, Hui Ge✳︎, Aaron J Schetter✳︎, Debra S Goldberg✳︎, Jing-Dong J Han, Tong Hao, Gabriel F Berriz, Nicolas Bertin, Jerry Huang, Ling-Shiang Chuang, Ning Li, Ramamurthy Mani, Anthony A. Hyman, Birte Sönnichsen, Christophe J. Echeverri, Frederick P. Roth, Marc Vidal, Fabio Piano
Predictive models of molecular machines involved in Caenorhabditis elegans early embryogenesis.
Nature, 436(7052) 861-865 (2005)
Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

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)
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)
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.

Henrik Bringmann, Anthony A. Hyman
A cytokinesis furrow is positioned by two consecutive signals.
Nature, 436(7051) 731-734 (2005)
The position of the cytokinesis furrow in a cell determines the relative sizes of its two daughter cells as well as the distribution of their contents. In animal cells, the position of the cytokinesis furrow is specified by the position of the mitotic spindle. The cytokinesis furrow bisects the spindle midway between the microtubule asters, at the site of the microtubule-based midzone, producing two daughter cells. Experiments in some cell types have suggested that the midzone positions the furrow, but experiments in other cells have suggested that the asters position the furrow. One possibility is that different organisms and cell types use different mechanisms to position the cytokinesis furrow. An alternative possibility is that both asters and the midzone contribute to furrow positioning. Recent work in C. elegans has suggested that centrosome separation and the midzone are implicated in cytokinesis. Here we examine the relative contributions of different parts of the mitotic spindle to positioning of the cytokinesis furrow in the C. elegans zygote. By spatially separating the spindle midzone from one of the asters using an ultraviolet laser, we show that the cytokinesis furrow is first positioned by a signal determined by microtubule asters, and then by a second signal that is derived from the spindle midzone. Thus, the position of the cytokinesis furrow is specified by two consecutive furrowing activities.

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)
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.

Stephan W. Grill, Anthony A. Hyman
Spindle positioning by cortical pulling forces.
Dev Cell, 8(4) 461-465 (2005)
Proper spatial control of the cell division plane is essential to any developing organism. In most cell types, the relative size of the two daughter cells is determined by the position of the mitotic spindle within the geometry of the mother cell. We review the underlying mechanisms responsible for positioning of the mitotic spindle, both in cases where the spindle is placed in the center of the cell and in cases where the spindle is placed away from the center of the cell. We discuss the idea that cortical pulling forces are sufficient to provide a general mechanism for spindle positioning within symmetrically and asymmetrically dividing cells.

Kozo Tanaka, Naomi Mukae, Hilary Dewar, Mark van Breugel, Euan K James, Alan R Prescott, Claude Antony, Tomoyuki U Tanaka
Molecular mechanisms of kinetochore capture by spindle microtubules.
Nature, 434(7036) 987-994 (2005)
For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial kinetochore capture have remained elusive. Here we visualized individual kinetochore-microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct kinetochore components are used for kinetochore capture by microtubules and for ensuring subsequent sister kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured kinetochores along microtubules. During such transport, kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured kinetochores to the plus ends of microtubules.

Birte Sönnichsen, L B Koski, A Walsh, P Marschall, B Neumann, Michael Brehm, Anne-Marie Alleaume, J. Artelt, P Bettencourt, E Cassin, M Hewitson, Christian Holz, M Khan, S Lazik, Claudia Martin, Bert Nitzsche, Martine Ruer, Joanne Stamford, Maria Winzi, R Heinkel, M Röder, J Finell, H Häntsch, S J M Jones, M Jones, Fabio Piano, Kristin C. Gunsalus, Karen Oegema, Pierre Gönczy, A Coulson, Anthony A. Hyman, C J Echeverri
Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.
Nature, 434(7032) 462-469 (2005)
A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Anthony A. Hyman
Boveri revisited.
EMBO J, 24(6) 1104-1110 (2005)

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)
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.

Ralf Kittler, Laurence Pelletier, Chunling Ma, Ina Poser, Steffi Fischer, Anthony A. Hyman, Frank Buchholz
RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.
Proc Natl Acad Sci U.S.A., 102(7) 2396-2401 (2005)
RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

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)
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.

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)

Anthony A. Hyman✳︎, Jonathon Howard✳︎
Cell structure and dynamics
Curr Opin Cell Biol, 17 1-2 (2005)

Henry Hess, Jens-Christian Röper, Stephan W. Grill, Michael R Koelle
RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans.
Cell, 119(2) 209-218 (2004)
Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. We found that mutating a regulator of G protein signaling, RGS-7, resulted in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galphao-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, we found that RIC-8 stimulates GTP binding by Galphao and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galphao, demonstrating that Galphao passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector.

Carrie R. Cowan, Anthony A. Hyman
Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos.
Nature, 431(7004) 92-96 (2004)
Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.

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)
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.

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)
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.

Henrik Bringmann, Georgios Skiniotis, Annina Spilker, Stefanie Kandels-Lewis, Isabelle Vernos, Thomas Surrey
A kinesin-like motor inhibits microtubule dynamic instability.
Science, 303(5663) 1519-1522 (2004)
The motility of molecular motors and the dynamic instability of microtubules are key dynamic processes for mitotic spindle assembly and function. We report here that one of the mitotic kinesins that localizes to chromosomes, Xklp1 from Xenopus laevis, could inhibit microtubule growth and shrinkage. This effect appeared to be mediated by a structural change in the microtubule lattice. We also found that Xklp1 could act as a fast, nonprocessive, plus end-directed molecular motor. The integration of the two properties, motility and inhibition of microtubule dynamics, in one molecule emphasizes the versatile properties of kinesin family members.

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)
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.

Carrie R. Cowan, Anthony A. Hyman
Asymmetric cell division in C. elegans: cortical polarity and spindle positioning.
Annu Rev Cell Dev Biol, 20 427-453 (2004)
The one-cell Caenorhabditis elegans embryo divides asymmetrically into a larger and smaller blastomere, each with a different fate. How does such asymmetry arise? The sperm-supplied centrosome establishes an axis of polarity in the embryo that is transduced into the establishment of anterior and posterior cortical domains. These cortical domains define the polarity of the embryo, acting upstream of the PAR proteins. The PAR proteins, in turn, determine the subsequent segregation of fate determinants and the plane of cell division. We address how cortical asymmetry could be established, relying on data from C. elegans and other polarized cells, as well as from applicable models. We discuss how cortical polarity influences spindle position to accomplish an asymmetric division, presenting the current models of spindle orientation and anaphase spindle displacement. We focus on asymmetric cell division as a function of the actin and microtubule cytoskeletons, emphasizing the cell biology of polarity.

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)
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.

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)
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.

Martin Srayko, Sophie Quintin, Anne Schwager, Anthony A. Hyman
Caenorhabditis elegans TAC-1 and ZYG-9 form a complex that is essential for long astral and spindle microtubules.
Curr Biol, 13(17) 1506-1511 (2003)
TACC (transforming acidic coiled-coil) proteins were first identified by their ability to transform cell lines [1], and links between human cancer and the overexpression of TACC proteins highlight the importance of understanding the biological function of this family of proteins. Herein, we describe the characterization of a new member of the TACC family of proteins in Caenorhabditis elegans, TAC-1. In other systems, TACC proteins associate with the XMAP215 family of microtubule-stabilizing proteins; however, it is unclear whether TACC proteins have microtubule-based functions distinct from XMAP215. We depleted both the XMAP215 ortholog ZYG-9 and TAC-1 via dsRNA-mediated interference (RNAi). We found that tac-1(RNAi) resulted in microtubule-based defects that were very similar to zyg-9(RNAi). Furthermore, TAC-1 and ZYG-9 are required for long astral microtubules in general and long spindle microtubules during spindle assembly. Loss of either protein did not affect the alpha-tubulin immunofluorescence intensity near centrosomes; this finding suggests that microtubule nucleation was not compromised. Both proteins localize to centrosomes and the kinetochore/microtubule region of chromosomes in metaphase and early anaphase. Furthermore, we found that ZYG-9 and TAC-1 physically interact in vivo, and this interaction is important for the efficient localization of the ZYG-9/TAC-1 complex to centrosomes.

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)
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.

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)
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.

Jonathon Howard, Anthony A. Hyman
Dynamics and mechanics of the microtubule plus end.
Nature, 422(6933) 753-758 (2003)
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)
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.

Matthew Kirkham✳︎, Thomas Müller-Reichert✳︎, Karen Oegema✳︎, Stephan W. Grill, Anthony A. Hyman
SAS-4 is a C. elegans centriolar protein that controls centrosome size.
Cell, 112(4) 575-587 (2003)
Centrosomes consist of a centriole pair surrounded by pericentriolar material (PCM). Previous work suggested that centrioles are required to organize PCM to form a structurally stable organelle. Here, we characterize SAS-4, a centriole component in Caenorhabditis elegans. Like tubulin, SAS-4 is incorporated into centrioles during their duplication and remains stably associated thereafter. In the absence of SAS-4, centriole duplication fails. Partial depletion of SAS-4 results in structurally defective centrioles that contain reduced levels of SAS-4 and organize proportionally less PCM. Thus, SAS-4 is a centriole-associated component whose amount dictates centrosome size. These results provide novel insight into the poorly understood role of centrioles as centrosomal organizers.

Manuel Mendoza, Anthony A. Hyman, Michael Glotzer
GTP binding induces filament assembly of a recombinant septin.
Curr Biol, 12(21) 1858-1863 (2002)
The septins are a family of GTPases involved in cytokinesis in budding yeast, Drosophila, and vertebrates (see for review). Septins are associated with a system of 10 nm filaments at the S. cerevisiae bud neck, and heteromultimeric septin complexes have been isolated from cell extracts in a filamentous state. A number of septins have been shown to bind and hydrolyze guanine nucleotide. However, the role of GTP binding and hydrolysis in filament formation has not been elucidated. Furthermore, several lines of evidence suggest that not all the subunits of the septin complex are required for all aspects of septin function. To address these questions, we have reconstituted filament assembly in vitro by using a recombinant Xenopus septin, Xl Sept2. Filament assembly is GTP dependent; moreover, the coiled-coil domain common to most septins is not essential for filament formation. Septin polymerization is preceded by a lag phase, suggesting a cooperative assembly mechanism. The slowly hydrolyzable GTP analog, GTP-gamma-S, also induces polymerization, indicating that polymerization does not require GTP hydrolysis. If the properties of Xl Sept2 filaments reflect those of native septin complexes, these results imply that the growth or stability of septin filaments, or both, is regulated by the state of bound nucleotide.

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)
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.

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)
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.

Eva Hannak, Matthew Kirkham, Anthony A. Hyman, Karen Oegema
Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.
J Cell Biol, 155(7) 1109-1116 (2001)
Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.

Pierre Gönczy✳︎, Jean-Michel Bellanger✳︎, Matthew Kirkham, Andrei Pozniakovski, Karine Baumer, Jennifer B. Phillips, Anthony A. Hyman
zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly.
Dev Cell, 1(3) 363-375 (2001)
Proper spindle positioning is essential for spatial control of cell division. Here, we show that zyg-8 plays a key role in spindle positioning during asymmetric division of one-cell stage C. elegans embryos by promoting microtubule assembly during anaphase. ZYG-8 harbors a kinase domain and a domain related to Doublecortin, a microtubule-associated protein (MAP) affected in patients with neuronal migration disorders. Sequencing of zyg-8 mutant alleles demonstrates that both domains are essential for function. ZYG-8 binds to microtubules in vitro, colocalizes with microtubules in vivo, and promotes stabilization of microtubules to drug or cold depolymerization in COS-7 cells. Our findings demonstrate that ZYG-8 is a MAP crucial for proper spindle positioning in C. elegans, and indicate that the function of the Doublecortin domain in modulating microtubule dynamics is conserved across metazoan evolution.

Karen Oegema✳︎, Arshad Desai✳︎, Sonja Rybina, Matthew Kirkham, Anthony A. Hyman
Functional analysis of kinetochore assembly in Caenorhabditis elegans.
J Cell Biol, 153(6) 1209-1226 (2001)
In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore null" phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

Andrei V. Popov, Andrei I. Pozniakovsky, Isabelle Arnal, Claude Antony, Anthony J. Ashford, Kazuhisa Kinoshita, Regis Tournebize, Anthony A. Hyman, Eric Karsenti
XMAP215 regulates microtubule dynamics through two distinct domains.
EMBO J, 20(3) 397-410 (2001)
XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity.

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)
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.

Torsten Wittmann, Anthony A. Hyman, Arshad Desai
The spindle: a dynamic assembly of microtubules and motors.
Nat Cell Biol, 3(1) 28-34 (2001)
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.

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)
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.

Erik Nielsen, Fedor F. Severin, Anthony A. Hyman, Marino Zerial
In vitro reconstitution of endosome motility along microtubules
In: Kinesin protocols. (Eds.) Isabelle Vernos Methods in molecular biology ; 164.,Totowa, USA,Humana Press (2001),133-146 Ch. 12

Pierre Gönczy, Christophe J. Echeverri, Karen Oegema, A Coulson, Steven J. M. Jones, Richard R. Copley, John Duperon, Jeffrey Oegema, Michael Brehm, E Cassin, Eva Hannak, Matthew Kirkham, Silke Pichler, Kathrin Flohrs, A Goessen, Sebastian Leidel, Anne-Marie Alleaume, Cecilie Martin, Nurhan Ozlü, Peer Bork, Anthony A. Hyman
Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.
Nature, 408(6810) 331-336 (2000)
Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.

V Jantsch-Plunger, Pierre Gönczy, Alper Romano, Heinke Schnabel, Danielle Hamill, Ralf Schnabel, Anthony A. Hyman, Michael Glotzer
CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis.
J Cell Biol, 149(7) 1391-1404 (2000)
During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Isabelle Arnal, Eric Karsenti, Anthony A. Hyman
Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts.
J Cell Biol, 149(4) 767-774 (2000)
Microtubules are dynamically unstable polymers that interconvert stochastically between growing and shrinking states by the addition and loss of subunits from their ends. However, there is little experimental data on the relationship between microtubule end structure and the regulation of dynamic instability. To investigate this relationship, we have modulated dynamic instability in Xenopus egg extracts by adding a catastrophe-promoting factor, Op18/stathmin. Using electron cryomicroscopy, we find that microtubules in cytoplasmic extracts grow by the extension of a two- dimensional sheet of protofilaments, which later closes into a tube. Increasing the catastrophe frequency by the addition of Op18/stathmin decreases both the length and frequency of the occurrence of sheets and increases the number of frayed ends. Interestingly, we also find that more dynamic populations contain more blunt ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. Our results demonstrate for the first time that microtubule assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of catastrophes is directly correlated with the structural state of microtubule ends.

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)
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.

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)
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.

Anthony A. Hyman
Centrosomes: Sic transit gloria centri.
Curr Biol, 10(7) 276-278 (2000)
Centrosomes are thought to ensure spindle bipolarity and thus correct chromosome segregation during mitosis, but recent studies indicate that somatic cells have an alternative mechanism that enables them to form a bipolar spindle and segregate chromosomes independently of centrosomes.

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)
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.

Arshad Desai, Anthony A. Hyman
Microtubule cytoskeleton: No longer an also Ran.
Curr Biol, 9(18) 704-707 (1999)
The Ran GTPase cycle has been extensively studied in the context of nuclear transport. Recent work indicates that this GTPase cycle also plays an important role in regulating the microtubule cytoskeleton.

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)
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.

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

Fedor F. Severin, Ken Kaplan, Peter Sorger, Anthony A. Hyman
In vitro assays for studying Saccharomyces cerevisiae kinetochore activity
In: Mitosis and meiosis. (Eds.) Conly L. Rieder Methods in cell biology ; 61.,Amsterdam, Netherlands,Academic Press (1999),145-153 Ch. 8