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Most Recent Publications
Felipe Mora-Bermúdez, Philipp Kanis, Dominik Macak, Jula Peters, Ronald Naumann, Lei Xing, Mihail Sarov, Sylke Winkler, Christina Eugster Oegema, Christiane Haffner, Pauline Wimberger, Stephan Riesenberg, Tomislav Maricic, Wieland Huttner, Svante Pääbo Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development. Sci Adv, 8(30) Art. No. eabn7702 (2022)
Open Access DOI
Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.
Hugo van den Hoek✳︎, Nikolai Klena✳︎, Mareike A Jordan, Gonzalo Alvarez Viar, Ricardo D Righetto, Miroslava Schaffer, Philipp S Erdmann, William Wan, Stefan Geimer, Jürgen M Plitzko, Wolfgang Baumeister, Gaia Pigino#, Virginie Hamel#, Paul Guichard#, Benjamin D Engel# In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains. Science, 377(6605) 543-548 (2022) DOI
The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.
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.
Mateusz Susik, Holger Schönborn, Ivo F. Sbalzarini Hamiltonian Monte Carlo with strict convergence criteria reduces run-to-run variability in forensic DNA mixture deconvolution. FSI Genetics, 60 Art. No. 102744 (2022)
Motivation: Analysing mixed DNA profiles is a common task in forensic genetics. Due to the complexity of the data, such analysis is often performed using Markov Chain Monte Carlo (MCMC)-based genotyping algorithms. These trade off precision against execution time. When default settings (including default chain lengths) are used, as large as a 10-fold changes in inferred log-likelihood ratios (LR) are observed when the software is run twice on the same case. So far, this uncertainty has been attributed to the stochasticity of MCMC algorithms. Since LRs translate directly to strength of the evidence in a criminal trial, forensic laboratories desire LR with small run-to-run variability.
Results: We present the use of a Hamiltonian Monte Carlo (HMC) algorithm that reduces run-to-run variability in forensic DNA mixture deconvolution by around an order of magnitude without increased runtime. We achieve this by enforcing strict convergence criteria. We show that the choice of convergence metric strongly influences precision. We validate our method by reproducing previously published results for benchmark DNA mixtures (MIX05, MIX13, and ProvedIt). We also present a complete software implementation of our algorithm that is able to leverage GPU acceleration for the inference process. In the benchmark mixtures, on consumer-grade hardware, the runtime is less than 7 min for 3 contributors, less than 35 min for 4 contributors, and less than an hour for 5 contributors with one known contributor.
Maren Rudolph✳︎, Yuting Wang✳︎, Theresa Simolka, Emilie Huc-Claustre, Lingyun Dai, Gijsbert Grotenbreg, Gurdyal Besra, Anna Shevchenko, Andrej Shevchenko, Sebastian Zeissig Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids. Front Immunol, 13 Art. No. 897873 (2022)
Open Access DOI
CD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids through its two hydrophobic pockets. Purification of the transmembrane protein CD1d for the analysis of bound lipids is technically challenging as the use of detergents releases CD1d-bound lipids. To address these challenges, we have developed a novel approach based on Sortase A-dependent enzymatic release of CD1d at the cell surface of live mammalian cells, which allows for single step release and affinity tagging of CD1d for shotgun lipidomics. Using this system, we demonstrate that CD1d carrying the Sortase A recognition motif shows unimpaired subcellular trafficking through the secretory and endolysosomal pathway and is able to load lipids in these compartments and present them to NKT cells. Comprehensive shotgun lipidomics demonstrated that the spectrum and abundance of CD1d-associated lipids is not representative of the total cellular lipidome but rather characterized by preferential binding to long chain sphingolipids and glycerophospholipids. As such, sphingomyelin species recently identified as critical negative regulators of NKT cell activation, represented the vast majority of endogenous CD1d-associated lipids. Moreover, we observed that inhibition of endolysosomal trafficking of CD1d surprisingly did not affect the spectrum of CD1d-bound lipids, suggesting that the majority of endogenous CD1d-associated lipids load onto CD1d in the secretory rather than the endolysosomal pathway. In conclusion, we present a novel system for the analysis of CD1d-bound lipids in mammalian cells and provide new insight into the spectrum of CD1d-associated lipids, with important functional implications for NKT cell activation.
Adrian Zambrano✳︎, Giorgio Fracasso✳︎, Mengfei Gao✳︎, Martina Ugrinic, Dishi Wang, Dietmar Appelhans, Andrew deMello, T-Y Dora Tang Programmable synthetic cell networks regulated by tuneable reaction rates. Nat Commun, 13(1) Art. No. 3885 (2022)
Open Access DOI
Coupled compartmentalised information processing and communication via molecular diffusion underpin network based population dynamics as observed in biological systems. Understanding how both compartmentalisation and communication can regulate information processes is key to rational design and control of compartmentalised reaction networks. Here, we integrate PEN DNA reactions into semi-permeable proteinosomes and characterise the effect of compartmentalisation on autocatalytic PEN DNA reactions. We observe unique behaviours in the compartmentalised systems which are not accessible under bulk conditions; for example, rates of reaction increase by an order of magnitude and reaction kinetics are more readily tuneable by enzyme concentrations in proteinosomes compared to buffer solution. We exploit these properties to regulate the reaction kinetics in two node compartmentalised reaction networks comprised of linear and autocatalytic reactions which we establish by bottom-up synthetic biology approaches.
Maike R Pollmanns, Judith Beer, Ines Rosignol, Natalia Rodriguez-Muela, Björn H Falkenburger, Elisabeth Dinter Activated Endolysosomal Cation Channel TRPML1 Facilitates Maturation of α-Synuclein-Containing Autophagosomes. Front Cell Neurosci, 16 Art. No. 861202 (2022)
Open Access DOI
Background: Protein aggregates are degraded via the autophagy-lysosome pathway and alterations in the lysosomal system leading to the accumulation of pathogenic proteins, including aggregates of α-synuclein in Parkinson's disease (PD). The importance of the endolysosomal transient receptor potential cation channel, mucolipin subfamily 1 (TRPML1) for the lysosomal function is highlighted by the fact that TRPML1 mutations cause the lysosomal storage disease mucolipidosis type IV. In this study, we investigated the mechanism by which activation of TRPML1 affects the degradation of α-synuclein. Methods: As a model of α-synuclein pathology, we expressed the pathogenic A53Tα-synuclein mutant in HEK293T cells. These cells were treated with the synthetic TRPML1 agonist ML-SA1. The amount of α-synuclein protein was determined by immunoblots. The abundance of aggregates and autolysosomal vesicles was determined by fluorescence microscopy and immunocytochemistry. Findings were confirmed by life-cell imaging and by application of ML-SA1 and the TRPML1 antagonist ML-SI3 to human dopaminergic neurons and human stem cell-derived neurons. Results: ML-SA1 reduced the percentage of HEK293T cells with α-synuclein aggregates and the amount of α-synuclein protein. The effect of ML-SA1 was blocked by pharmacological and genetic inhibition of autophagy. Consistent with TRPML function, it required the membrane lipid PI(3,5)P2, and cytosolic calcium. ML-SA1 shifted the composition of autophagosomes towards a higher fraction of mature autolysosomes, also in presence of α-synuclein. In neurons, inhibition of TRPML1 by its antagonist ML-SI3 blocked autophagosomal clearance, whereas the agonist ML-SA1 shifted the composition of a-synuclein particles towards a higher fraction of acidified particles. ML-SA1 was able to override the effect of Bafilomycin A1, which blocks the fusion of the autophagosome and lysosome and its acidification. Conclusion: These findings suggest, that activating TRPML1 with ML-SA1 facilitates clearance of α-synuclein aggregates primarily by affecting the late steps of the autophagy, i.e., by promoting autophagosome maturation. In agreement with recent work by others, our findings indicate that TRPML1 might constitute a plausible therapeutic target for PD, that warrants further validation in rodent models of α-synuclein pathology.
Anubha Seth, Marius Landau, Andrej Shevchenko, Sofia Traikov, Anita Schultz, Sherif Elsabbagh, Joachim E Schultz Distinct glycerophospholipids potentiate Gsα-activated adenylyl cyclase activity. Cell Signal, 97 Art. No. 110396 (2022)
Open Access DOI
Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains, which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (https://doi.org/10.7554/eLife.13098; https://doi.org/10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gsα-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gsα-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gsα-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.
Fereshteh R Najafabadi✳︎, Mark Leaver✳︎, Stephan W. Grill Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis. Mol Biol Cell, 33(8) Art. No. ar74 (2022) DOI
Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.
Rashmiparvathi Keshara#, Yung Hae Kim#, Anne Grapin-Botton# Organoid Imaging: Seeing Development and Function. Annu Rev Cell Dev Biol, Art. No. doi: 10.1146/annurev-cellbio-120320-035146 (2022) DOI
Organoids are miniaturized and simplified versions of an organ produced in vitro from stem or progenitor cells. They are used as a model system consisting of multiple cell types forming an architecture relevant to the organ and carrying out the function of the organ. They are a useful tool to study development, homeostasis, regeneration, and disease. The imaging of organoids has become a pivotal method to visualize and understand their self-organization, symmetry breaking, growth, differentiation, and function. In this review, we discuss imaging methods, how to analyze these images, and challenges in organoid research. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.