* joined first author # joined corresponding author

2022
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.


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, Art. No. doi: 10.1111/evo.14520 (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. This article is protected by copyright. All rights reserved.


Tina Wiegand#, Arjun Narayanan#
Caught by a cytoskeletal web.
Nat Phys, 18(5) 483-484 (2022)
DOI
Biomolecular condensates grow in busy cellular environments. Statistical image analysis of heterogeneous structures now enables quantification of macromolecular interactions between condensates and cytoskeletal filaments.


Tzer Han Tan✳︎, Jifeng Liu✳︎, Anne Grapin-Botton
Mapping and exploring the organoid state space using synthetic biology.
Semin Cell Dev Biol, Art. No. doi: 10.1016/j.semcdb.2022.04.015 (2022)
DOI
The functional relevance of an organoid is dependent on the differentiation, morphology, cell arrangement and biophysical properties, which collectively define the state of an organoid. For an organoid culture, an individual organoid or the cells that compose it, these state variables can be characterised, most easily by transcriptomics and by high-content image analysis. Their states can be compared to their in vivo counterparts. Current evidence suggests that organoids explore a wider state space than organs in vivo due to the lack of niche signalling and the variability of boundary conditions in vitro. Using data-driven state inference and in silico modelling, phase diagrams can be constructed to systematically sort organoids along biochemical or biophysical axes. These phase diagrams allow us to identify control strategies to modulate organoid state. To do so, the biochemical and biophysical environment, as well as the cells that seed organoids, can be manipulated.


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.


Timothy J Welsh✳︎, Georg Krainer✳︎, Jorge R Espinosa✳︎, Jerelle A Joseph, Akshay Sridhar, Marcus Jahnel, William E Arter, Kathrin Saar, Simon Alberti#, Rosana Collepardo-Guevara#, Tuomas P J Knowles#
Surface Electrostatics Govern the Emulsion Stability of Biomolecular Condensates.
Nano Lett, 22(2) 612-621 (2022)
DOI
Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions that in general have a propensity to fuse and coalesce; however, many condensates persist as independent droplets in the test tube and inside cells. This stability is crucial for their function, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modeling, we investigate how the nanoscale forces that sustain condensates impact their stability against fusion. By comparing peptide-RNA (PR25:PolyU) and proteinaceous (FUS) condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity. Moreover, measurements of single condensate zeta potentials reveal that such systems can constitute classically stable emulsions. Taken together, these results highlight the role of passive stabilization mechanisms in protecting biomolecular condensates against coalescence.


2021
Judit Clopes, Jaeoh Shin, Marcus Jahnel, Stephan W. Grill, Vasily Zaburdaev
Thermal fluctuations assist mechanical signal propagation in coiled-coil proteins.
Phys Rev E, 104(5) Art. No. 054403 (2021)
Open Access DOI
Recently, it has been shown that the long coiled-coil membrane tether protein early endosome antigen 1 (EEA1) switches from a rigid to a flexible conformation upon binding of a signaling protein to its free end. This flexibility switch represents a motorlike activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. It was hypothesized that the binding-induced signal could propagate along the coiled coil and lead to conformational changes through the localized domains of the protein chain that deviate from a perfect coiled-coil structure. To elucidate, if upon binding of a single protein the corresponding mechanical signal could propagate through the whole 200-nm-long chain, we propose a simplified description of the coiled coil as a one-dimensional Frenkel-Kontorova chain. Using numerical simulations, we find that an initial perturbation of the chain can propagate along its whole length in the presence of thermal fluctuations. This may enable the change of the configuration of the entire molecule and thereby affect its stiffness. Our work sheds light on intramolecular communication and force generation in long coiled-coil proteins.


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)
DOI
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.


Thomas Quail, Stefan Golfier, Maria Elsner, Keisuke Ishihara, Vasanthanarayan Murugesan, Roman Renger, Frank Jülicher#, Jan Brugués#
Force generation by protein-DNA co-condensation.
Nat Phys, 17(9) 1007-1012 (2021)
Open Access DOI
Interactions between liquids and surfaces generate forces(1,2) that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water(3), modulation of the material properties of spider silk(4) and self-assembly of microstructures(5). Recent studies have shown that cells assemble biomolecular condensates via phase separation(6). In the nucleus, these condensates are thought to drive transcription(7), heterochromatin formation(8), nucleolus assembly(9) and DNA repair(10). Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein-DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.


Teije C Middelkoop, Júlia Garcia-Baucells, Porfirio Quintero-Cadena, Lokesh G Pimpale, Shahrzad Yazdi, Paul W Sternberg, Peter Gross, Stephan W. Grill
CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking.
Proc Natl Acad Sci U.S.A., 118(20) Art. No. e2021814118 (2021)
Open Access DOI
Proper left-right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left-right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left-right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin-dependent manner. Altogether, we conclude that CYK-1/Formin-dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left-right symmetry breaking in the nematode worm.


Claudio Durán✳︎, Sara Ciucci✳︎, Alessandra Palladini✳︎, Umer Z Ijaz, Antonio Giuliano Zippo, Francesco Paroni Sterbini, Luca Masucci, Giovanni Cammarota, Gianluca Ianiro, Pirjo Spuul, Michael Schroeder, Stephan W. Grill, Bryony N Parsons, D Mark Pritchard, Brunella Posteraro, Maurizio Sanguinetti, Giovanni Gasbarrini, Antonio Gasbarrini, Carlo Vittorio Cannistraci
Nonlinear machine learning pattern recognition and bacteria-metabolite multilayer network analysis of perturbed gastric microbiome.
Nat Commun, 12(1) Art. No. 1926 (2021)
Open Access DOI
The stomach is inhabited by diverse microbial communities, co-existing in a dynamic balance. Long-term use of drugs such as proton pump inhibitors (PPIs), or bacterial infection such as Helicobacter pylori, cause significant microbial alterations. Yet, studies revealing how the commensal bacteria re-organize, due to these perturbations of the gastric environment, are in early phase and rely principally on linear techniques for multivariate analysis. Here we disclose the importance of complementing linear dimensionality reduction techniques with nonlinear ones to unveil hidden patterns that remain unseen by linear embedding. Then, we prove the advantages to complete multivariate pattern analysis with differential network analysis, to reveal mechanisms of bacterial network re-organizations which emerge from perturbations induced by a medical treatment (PPIs) or an infectious state (H. pylori). Finally, we show how to build bacteria-metabolite multilayer networks that can deepen our understanding of the metabolite pathways significantly associated to the perturbed microbial communities.


Georgia R Squyres, Matthew J Holmes, Sarah R Barger, Betheney R Pennycook, Joel Ryan, Victoria T Yan, Ethan C Garner
Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis.
Nat Microbiol, 6(5) 553-562 (2021)
DOI
Although many components of the cell division machinery in bacteria have been identified1,2, the mechanisms by which they work together to divide the cell remain poorly understood. Key among these components is the tubulin FtsZ, which forms a Z ring at the midcell. FtsZ recruits the other cell division proteins, collectively called the divisome, and the Z ring constricts as the cell divides. We applied live-cell single-molecule imaging to describe the dynamics of the divisome in detail, and to evaluate the individual roles of FtsZ-binding proteins (ZBPs), specifically FtsA and the ZBPs EzrA, SepF and ZapA, in cytokinesis. We show that the divisome comprises two subcomplexes that move differently: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a moving complex that includes cell wall synthases. Our imaging analyses reveal that ZBPs bundle FtsZ filaments together and condense them into Z rings, and that this condensation is necessary for cytokinesis.


Madeline M Keenen, David Brown, Lucy D Brennan, Roman Renger, Harrison Khoo, Christopher R Carlson, Bo Huang, Stephan W. Grill, Geeta J Narlikar#, Sy Redding#
HP1 proteins compact DNA into mechanically and positionally stable phase separated domains.
Elife, 10 Art. No. e64563 (2021)
Open Access DOI
In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog's DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.


Nicolas T Chartier✳︎, Arghyadip Mukherjee✳︎, Julia Pfanzelter✳︎, Sebastian Fürthauer, Ben T Larson, Anatol Fritsch, Rana Amini, Moritz Kreysing, Frank Jülicher#, Stephan W. Grill#
A hydraulic instability drives the cell death decision in the nematode germline.
Nat Phys, 17(8) 920-925 (2021)
Open Access PDF DOI
Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow-typically at the expense of others that undergo cell death2-4. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability5. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping6. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.


2020
Akanksha Jain, Vladimir Ulman, Arghyadip Mukherjee, Mangal Prakash, Marina B Cuenca, Lokesh G Pimpale, Stefan Münster, Robert Haase, Kristen A Panfilio, Florian Jug, Stephan W. Grill, Pavel Tomancak#, Anastasios Pavlopoulos#
Regionalized tissue fluidization is required for epithelial gap closure during insect gastrulation.
Nat Commun, 11(1) Art. No. 5604 (2020)
Open Access DOI
Many animal embryos pull and close an epithelial sheet around the ellipsoidal egg surface during a gastrulation process known as epiboly. The ovoidal geometry dictates that the epithelial sheet first expands and subsequently compacts. Moreover, the spreading epithelium is mechanically stressed and this stress needs to be released. Here we show that during extraembryonic tissue (serosa) epiboly in the insect Tribolium castaneum, the non-proliferative serosa becomes regionalized into a solid-like dorsal region with larger non-rearranging cells, and a more fluid-like ventral region surrounding the leading edge with smaller cells undergoing intercalations. Our results suggest that a heterogeneous actomyosin cable contributes to the fluidization of the leading edge by driving sequential eviction and intercalation of individual cells away from the serosa margin. Since this developmental solution utilized during epiboly resembles the mechanism of wound healing, we propose actomyosin cable-driven local tissue fluidization as a conserved morphogenetic module for closure of epithelial gaps.


Lokesh G Pimpale, Teije C Middelkoop, Alexander Mietke, Stephan W. Grill
Cell lineage-dependent chiral actomyosin flows drive cellular rearrangements in early C. elegans development.
Elife, 9 Art. No. e54930 (2020)
Open Access DOI
Proper positioning of cells is essential for many aspects of development. Daughter cell positions can be specified via orienting the cell division axis during cytokinesis. Rotatory actomyosin flows during division have been implied in specifying and reorienting the cell division axis, but how general such reorientation events are, and how they are controlled, remains unclear. We followed the first nine divisions of Caenorhabditis elegans embryo development and demonstrate that chiral counter-rotating flows arise systematically in early AB lineage, but not in early P/EMS lineage cell divisions. Combining our experiments with thin film active chiral fluid theory we identify a mechanism by which chiral counter-rotating actomyosin flows arise in the AB lineage only, and show that they drive lineage-specific spindle skew and cell reorientation events. In conclusion, our work sheds light on the physical processes that underlie chiral morphogenesis in early development.


2019
Lorna M Y Mitchison-Field, José M Vargas-Muñiz, Benjamin M Stormo, Ellysa J D Vogt, Sarah Van Dierdonck, James F. Pelletier, Christoph Ehrlich, Daniel J Lew, Christine M Field#, Amy Gladfelter#
Unconventional Cell Division Cycles from Marine-Derived Yeasts.
Curr Biol, 29(20) 3439-3456 (2019)
DOI
Fungi have been found in every marine habitat that has been explored; however, the diversity and functions of fungi in the ocean are poorly understood. In this study, fungi were cultured from the marine environment in the vicinity of Woods Hole, MA, USA, including from plankton, sponge, and coral. Our sampling resulted in 35 unique species across 20 genera. We observed many isolates by time-lapse, differential interference contrast (DIC) microscopy and analyzed modes of growth and division. Several black yeasts displayed highly unconventional cell division cycles compared to those of traditional model yeast systems. Black yeasts have been found in habitats inhospitable to other life and are known for halotolerance, virulence, and stress resistance. We find that this group of yeasts also shows remarkable plasticity in terms of cell size control, modes of cell division, and cell polarity. Unexpected behaviors include division through a combination of fission and budding, production of multiple simultaneous buds, and cell division by sequential orthogonal septations. These marine-derived yeasts reveal alternative mechanisms for cell division cycles that seem likely to expand the repertoire of rules established from classic model system yeasts.


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)
DOI
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.


Stefan Münster, Akanksha Jain, Alexander Mietke, Anastasios Pavlopoulos, Stephan W. Grill#, Pavel Tomancak#
Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.
Nature, 568(7752) 395-399 (2019)
DOI
During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the gastrulating tissue is surrounded by a rigid protective shell4. Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility5,6, it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle (Tribolium castaneum) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly (Drosophila melanogaster) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects.


Kerstin Klinkert, Nicolas Levernier, Peter Gross, Christian Gentili, Lukas von Tobel, Marie Pierron, Coralie Busso, Sarah Herrman, Stephan W. Grill, Karsten Kruse, Pierre Gönczy
Aurora A depletion reveals centrosome-independent polarization mechanism in C. elegans.
Elife, 8 Art. No. e44552 (2019)
Open Access DOI
How living systems break symmetry in an organized manner is a fundamental question in biology. In wild type Caenorhabditis elegans zygotes, symmetry breaking during anterior-posterior axis specification is guided by centrosomes, resulting in anterior-directed cortical flows and a single posterior PAR-2 domain. We uncover that C. elegans zygotes depleted of the Aurora A kinase AIR-1 or lacking centrosomes entirely usually establish two posterior PAR-2 domains, one at each pole. We demonstrate that AIR-1 prevents symmetry breaking early in the cell cycle, whereas centrosomal AIR-1 instructs polarity initiation thereafter. Using triangular microfabricated chambers, we establish that bipolarity of air-1(RNAi) embryos occurs effectively in a cell-shape and curvature-dependent manner. Furthermore, we develop an integrated physical description of symmetry breaking, wherein local PAR-2-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for spontaneous symmetry breaking without centrosomes.


2018
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)
DOI
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.


Sundar Ram Naganathan#, Sebastian Fürthauer, Josana Rodriguez, Bruno Thomas Fievet, Frank Jülicher, Julie Ahringer, Carlo Vittorio Cannistraci, Stephan W. Grill#
Morphogenetic degeneracies in the actomyosin cortex.
Elife, 7 Art. No. e37677 (2018)
Open Access DOI
One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.


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

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)
DOI
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.


Matthäus Mittasch, Peter Gross, Michael Nestler, Anatol W Fritsch, Christiane Iserman, Mrityunjoy Kar, Matthias Munder, Axel Voigt, Simon Alberti, Stephan W. Grill, Moritz Kreysing
Non-invasive perturbations of intracellular flow reveal physical principles of cell organization.
Nat Cell Biol, 20(3) 344-351 (2018)
DOI
Recent advances in cell biology enable precise molecular perturbations. The spatiotemporal organization of cells and organisms, however, also depends on physical processes such as diffusion or cytoplasmic flows, and strategies to perturb physical transport inside cells are not yet available. Here, we demonstrate focused-light-induced cytoplasmic streaming (FLUCS). FLUCS is local, directional, dynamic, probe-free, physiological, and is even applicable through rigid egg shells or cell walls. We explain FLUCS via time-dependent modelling of thermoviscous flows. Using FLUCS, we demonstrate that cytoplasmic flows drive partitioning-defective protein (PAR) polarization in Caenorhabditis elegans zygotes, and that cortical flows are sufficient to transport PAR domains and invert PAR polarity. In addition, we find that asymmetric cell division is a binary decision based on gradually varying PAR polarization states. Furthermore, the use of FLUCS for active microrheology revealed a metabolically induced fluid-to-solid transition of the yeast cytoplasm. Our findings establish how a wide range of transport-dependent models of cellular organization become testable by FLUCS.


Matthäus Mittasch, Peter Gross, Michael Nestler, Anatol W Fritsch, Christiane Iserman, Mrityunjoy Kar, Matthias Munder, Axel Voigt, Simon Alberti, Stephan W. Grill, Moritz Kreysing
How to apply FLUCS in single cells and living embryos.
Protocol Exchange, Art. No. doi:10.1038/protex.2017.157 (2018)
Open Access PDF DOI

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.


2017
Cordula Reuther, Matthäus Mittasch, Sundar Naganathan, Stephan W. Grill, Stefan Diez
Highly-Efficient Guiding of Motile Microtubules on Non-Topographical Motor Patterns.
Nano Lett, 17(9) 5699-5705 (2017)
DOI
Molecular motors, highly efficient biological nanomachines, hold the potential to be employed for a wide range of nanotechnological applications. Toward this end, kinesin, dynein, or myosin motor proteins are commonly surface-immobilized within engineered environments in order to transport cargo attached to cytoskeletal filaments. Being able to flexibly control the direction of filament motion, and in particular on planar, non-topographical surfaces, has, however, remained challenging. Here, we demonstrate the applicability of a UV-laser-based ablation technique to programmably generate highly localized patterns of functional kinesin-1 motors with different shapes and sizes on PLL-g-PEG-coated polystyrene surfaces. Straight and curved motor tracks with widths of less than 500 nm could be generated in a highly reproducible manner and proved to reliably guide gliding microtubules. Though dependent on track curvature, the characteristic travel lengths of the microtubules on the tracks significantly exceeded earlier predictions. Moreover, we experimentally verified the performance of complex kinesin-1 patterns, recently designed by evolutionary algorithms for controlling the global directionality of microtubule motion on large-area substrates.


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

Peter Gross, K Vijay Kumar, Stephan W. Grill
How Active Mechanics and Regulatory Biochemistry Combine to Form Patterns in Development.
Ann Rev Biophys, 46 337-356 (2017)
DOI
The development of organisms starting from their zygotic state involves a tight integration of the myriad biochemical signaling interactions with the mechanical forces that eventually pattern and shape the resulting embryo. In the past decade, it has become increasingly evident that several important developmental processes involve mechanical forces in an essential manner. In this review, we highlight the multifaceted role of mechanics in pattern formation, from protein and cell sorting to the generation of tissue shape. We then review the ways in which the active cellular cytoskeleton self-organizes to form dynamic patterns. Finally, we focus on mechanochemical feedback, where signaling proteins can establish patterns via coupling to the activity of the cytoskeleton. Throughout the review, we focus on the generic physical principles of the establishment of active mechanochemical patterns and point toward future directions in studying how the principles of mechanics and chemistry combine to drive morphogenetic pattern formation.


Bipul R Acharya, Selwin K Wu, Zi Zhao Lieu, Robert G. Parton, Stephan W. Grill, Alexander D Bershadsky, Guillermo A Gomez, Alpha S Yap
Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility.
Cell Rep, 18(12) 2854-2867 (2017)
Open Access PDF DOI
Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across αE-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia.


Ana Lisica, Stephan W. Grill
Optical tweezers studies of transcription by eukaryotic RNA polymerases.
Biomol Concepts, 8(1) 1-11 (2017)
DOI
Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.


Masatoshi Nishikawa, Sundar Naganathan, Frank Jülicher, Stephan W. Grill
Controlling contractile instabilities in the actomyosin cortex.
Elife, 6 Art. No. e19595 (2017)
Open Access DOI
The actomyosin cell cortex is an active contractile material for driving cell- and tissue morphogenesis. The cortex has a tendency to form a pattern of myosin foci, which is a signature of potentially unstable behavior. How a system that is prone to such instabilities can rveliably drive morphogenesis remains an outstanding question. Here, we report that in the Caenorhabditis elegans zygote, feedback between active RhoA and myosin induces a contractile instability in the cortex. We discover that an independent RhoA pacemaking oscillator controls this instability, generating a pulsatory pattern of myosin foci and preventing the collapse of cortical material into a few dynamic contracting regions. Our work reveals how contractile instabilities that are natural to occur in mechanically active media can be biochemically controlled to robustly drive morphogenetic events.


2016
Veronika Fitz, Jaeoh Shin, Christoph Ehrlich, Lucas Farnung, Patrick Cramer, Vasily Zaburdaev, Stephan W. Grill
Nucleosomal arrangement affects single-molecule transcription dynamics.
Proc Natl Acad Sci U.S.A., 113(45) 12733-12738 (2016)
DOI
In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, χ, which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, χ can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes χ in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.


Anne-Cecile Reymann, Fabio Staniscia, Anna Erzberger, Guillaume Salbreux, Stephan W. Grill
Cortical flow aligns actin filaments to form a furrow.
Elife, 5 Art. No. e17807 (2016)
Open Access PDF DOI
Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture.


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)
DOI
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.


David Murray, Marcus Jahnel, Janelle Lauer, Mario Avellaneda, Nicolas Brouilly, Alice Cezanne, Hernán Morales-Navarrete, Enrico Perini, Charles Ferguson, Andrei N Lupas, Yannis Kalaidzidis, Robert G. Parton, Stephan W. Grill, Marino Zerial
An endosomal tether undergoes an entropic collapse to bring vesicles together.
Nature, 537(7618) 107-111 (2016)
DOI
An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.


Édgar Roldán, Ana Lisica, Daniel Sánchez-Taltavull, Stephan W. Grill
Stochastic resetting in backtrack recovery by RNA polymerases.
Phys Rev E, 93(6-1) Art. No. 062411 (2016)
DOI
Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.


Arnab Saha, Masatoshi Nishikawa, Martin Behrndt, Carl-Philipp Heisenberg, Frank Jülicher, Stephan W. Grill
Determining Physical Properties of the Cell Cortex.
Biophys J, 110(6) 1421-1429 (2016)
Open Access PDF DOI
Actin and myosin assemble into a thin layer of a highly dynamic network underneath the membrane of eukaryotic cells. This network generates the forces that drive cell- and tissue-scale morphogenetic processes. The effective material properties of this active network determine large-scale deformations and other morphogenetic events. For example, the characteristic time of stress relaxation (the Maxwell time τM) in the actomyosin sets the timescale of large-scale deformation of the cortex. Similarly, the characteristic length of stress propagation (the hydrodynamic length λ) sets the length scale of slow deformations, and a large hydrodynamic length is a prerequisite for long-ranged cortical flows. Here we introduce a method to determine physical parameters of the actomyosin cortical layer in vivo directly from laser ablation experiments. For this we investigate the cortical response to laser ablation in the one-cell-stage Caenorhabditis elegans embryo and in the gastrulating zebrafish embryo. These responses can be interpreted using a coarse-grained physical description of the cortex in terms of a two-dimensional thin film of an active viscoelastic gel. To determine the Maxwell time τM, the hydrodynamic length λ, the ratio of active stress ζΔμ, and per-area friction γ, we evaluated the response to laser ablation in two different ways: by quantifying flow and density fields as a function of space and time, and by determining the time evolution of the shape of the ablated region. Importantly, both methods provide best-fit physical parameters that are in close agreement with each other and that are similar to previous estimates in the two systems. Our method provides an accurate and robust means for measuring physical parameters of the actomyosin cortical layer. It can be useful for investigations of actomyosin mechanics at the cellular-scale, but also for providing insights into the active mechanics processes that govern tissue-scale morphogenesis.


Ana Lisica, Christoph Engel, Marcus Jahnel, Édgar Roldán, Eric A. Galburt, Patrick Cramer, Stephan W. Grill
Mechanisms of backtrack recovery by RNA polymerases I and II.
Proc Natl Acad Sci U.S.A., 113(11) 2946-2951 (2016)
Open Access PDF DOI
During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes.


Sundar Naganathan, Teije C Middelkoop, Sebastian Fürthauer, Stephan W. Grill
Actomyosin-driven left-right asymmetry: from molecular torques to chiral self organization.
Curr Opin Cell Biol, 38 24-30 (2016)
DOI
Chirality or mirror asymmetry is a common theme in biology found in organismal body plans, tissue patterns and even in individual cells. In many cases the emergence of chirality is driven by actin cytoskeletal dynamics. Although it is well established that the actin cytoskeleton generates rotational forces at the molecular level, we are only beginning to understand how this can result in chiral behavior of the entire actin network in vivo. In this review, we will give an overview of actin driven chiralities across different length scales known until today. Moreover, we evaluate recent quantitative models demonstrating that chiral symmetry breaking of cells can be achieved by properly aligning molecular-scale torque generation processes in the actomyosin cytoskeleton.


2015
Veronika Fitz
Chromatin transcription by RNA polymerase II - nucleosomal geometry matters
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2015)


Avinash Patel, Hyun-Ok Kate Lee, Louise Jawerth, Shovamayee Maharana, Marcus Jahnel, Marco Y Hein, Stoyno Stoynov, Julia Mahamid, Shambaditya Saha, Titus Franzmann, Andrei Pozniakovski, Ina Poser, Nicola Maghelli, Loic Royer, Martin Weigert, Eugene W Myers, Stephan W. Grill, David N. Drechsel, Anthony Hyman, Simon Alberti
A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.
Cell, 162(5) 1066-1077 (2015)
DOI
Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases. VIDEO ABSTRACT.


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.


2014
Bi-Chang Chen, Wesley R Legant, Kai Wang, Lin Shao, Daniel E Milkie, Michael W Davidson, Chris Janetopoulos, Xufeng S Wu, John A Hammer, Zhe Liu, Brian P English, Yuko Mimori-Kiyosue, Daniel P Romero, Alex T Ritter, Jennifer Lippincott-Schwartz, Lillian Fritz-Laylin, R Dyche Mullins, Diana M Mitchell, Joshua N Bembenek, Anne-Cecile Reymann, Ralph Böhme, Stephan W. Grill, Jennifer T Wang, Geraldine Seydoux, U Serdar Tulu, Daniel P Kiehart, Eric Betzig
Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.
Science, 346(6208) Art. No. 1257998 (2014)
DOI
Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.


Philipp Khuc Trong, Ernesto M Nicola, Nathan W. Goehring, K Vijay Kumar, Stephan W. Grill
Parameter-space topology of models for cell polarity
New J Phys, 16 Art. No. 065009 (2014)
Open Access DOI

K Vijay Kumar, Justin Bois, Frank Jülicher, Stephan W. Grill
Pulsatory Patterns in Active Fluids
Phys Rev Lett, 112 Art. No. 208101 (2014)
DOI

Manuela Weitkunat, Aynur Kaya-Çopur, Stephan W. Grill, Frank Schnorrer
Tension and force-resistant attachment are essential for myofibrillogenesis in Drosophila flight muscle.
Curr Biol, 24(7) 705-716 (2014)
DOI
Higher animals generate an elaborate muscle-tendon network to perform their movements. To build a functional network, developing muscles must establish stable connections with tendons and assemble their contractile apparatuses. Current myofibril assembly models do not consider the impact of muscle-tendon attachment on myofibrillogenesis. However, if attachment and myofibrillogenesis are not properly coordinated, premature muscle contractions can destroy an unstable myotendinous system, leading to severe myopathies.


Evan Heller, K Vijay Kumar, Stephan W. Grill, Elaine Fuchs
Forces generated by cell intercalation tow epidermal sheets in Mammalian tissue morphogenesis.
Dev Cell, 28(6) 617-632 (2014)
DOI
While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting, and cell-cycle inhibitors reveal that closure does not require overlying periderm, proliferation, or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA- and α5β1 integrin/fibronectin-mediated migration and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue.


Sundar Naganathan, Sebastian Fürthauer, Masatoshi Nishikawa, Frank Jülicher, Stephan W. Grill
Active torque generation by the actomyosin cell cortex drives left-right symmetry breaking.
Elife, 3 Art. No. e04165 (2014)
Open Access PDF DOI
Many developmental processes break left-right (LR) symmetry with a consistent handedness. LR asymmetry emerges early in development, and in many species the primary determinant of this asymmetry has been linked to the cytoskeleton. However, the nature of the underlying chirally asymmetric cytoskeletal processes has remained elusive. In this study, we combine thin-film active chiral fluid theory with experimental analysis of the C. elegans embryo to show that the actomyosin cortex generates active chiral torques to facilitate chiral symmetry breaking. Active torques drive chiral counter-rotating cortical flow in the zygote, depend on myosin activity, and can be altered through mild changes in Rho signaling. Notably, they also execute the chiral skew event at the 4-cell stage to establish the C. elegans LR body axis. Taken together, our results uncover a novel, large-scale physical activity of the actomyosin cytoskeleton that provides a fundamental mechanism for chiral morphogenesis in development.


2013
Martin Depken, Juan Parrondo, Stephan W. Grill
Intermittent transcription dynamics for the rapid production of long transcripts of high fidelity.
Cell Rep, 5(2) 521-530 (2013)
Open Access DOI
Normal cellular function relies on the efficient and accurate readout of the genetic code. Single-molecule experiments show that transcription and replication are highly intermittent processes that are frequently interrupted by polymerases pausing and reversing directions. Although intermittent dynamics in replication are known to result from proofreading, their origin and significance during transcription remain controversial. Here, we theoretically investigate transcriptional fidelity and show that the kinetic scheme provided by the RNA-polymerase backtracking and transcript-cleavage pathway can account for measured error rates. Importantly, we find that intermittent dynamics provide an enormous increase in the rate of producing long transcripts of high fidelity. Our results imply that intermittent dynamics during transcription may have evolved as a way to mitigate the competing demands of speed and fidelity in the transcription of extended sequences.


Sundar Naganathan
Regulation of mesoscale physical properties of the C.elegans actomyosin cortex
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2013)


Nathan Goehring#, Stephan W. Grill#
Cell polarity: mechanochemical patterning.
Trends Cell Biol, 23(2) 72-80 (2013)
DOI
Nearly every cell type exhibits some form of polarity, yet the molecular mechanisms vary widely. Here we examine what we term 'chemical systems' where cell polarization arises through biochemical interactions in signaling pathways, 'mechanical systems' where cells polarize due to forces, stresses and transport, and 'mechanochemical systems' where polarization results from interplay between mechanics and chemical signaling. To reveal potentially unifying principles, we discuss mathematical conceptualizations of several prototypical examples. We suggest that the concept of local activation and global inhibition - originally developed to explain spatial patterning in reaction-diffusion systems - provides a framework for understanding many cases of cell polarity. Importantly, we find that the core ingredients in this framework - symmetry breaking, self-amplifying feedback, and long-range inhibition - involve processes that can be chemical, mechanical, or even mechanochemical in nature.


Sebastian Fürthauer, M. Strempel, Stephan W. Grill, Frank Jülicher
Active Chiral Processes in Thin Films
Phys Rev Lett, 110(4) Art. No. 048103 (2013)
DOI

Bruno Thomas Fievet, Josana Rodriguez, Sundar Naganathan, Christine Lee, Eva Zeiser, Takao Ishidate, Masaki Shirayama, Stephan W. Grill, Julie Ahringer
Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy.
Nat Cell Biol, 15(1) 103-112 (2013)
DOI
Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.


2012
Martin Behrndt✳︎, Guillaume Salbreux✳︎, Pedro Campinho, Robert Hauschild, Felix Oswald, Julia Roensch, Stephan W. Grill#, Carl-Philipp Heisenberg#
Forces driving epithelial spreading in zebrafish gastrulation.
Science, 338(6104) 257-260 (2012)
DOI
Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.


S Fürthauer, M. Strempel, Stephan W. Grill#, Frank Jülicher#
Active chiral fluids.
Eur Phys J E Soft Matter, 35(9) Art. No. 89 (2012)
DOI
Active processes in biological systems often exhibit chiral asymmetries. Examples are the chirality of cytoskeletal filaments which interact with motor proteins, the chirality of the beat of cilia and flagella as well as the helical trajectories of many biological microswimmers. Here, we derive constitutive material equations for active fluids which account for the effects of active chiral processes. We identify active contributions to the antisymmetric part of the stress as well as active angular momentum fluxes. We discuss four types of elementary chiral motors and their effects on a surrounding fluid. We show that large-scale chiral flows can result from the collective behavior of such motors even in cases where isolated motors do not create a hydrodynamic far field.


Sebastian Fürthauer, Maaike Neeft, Stephan W. Grill, Karsten Kruse, Frank Jülicher
The Taylor-Couette motor: spontaneous flows of active polar fluids between two coaxial cylinders
New J Phys, 14(2) Art. No. e023001 (2012)
Open Access PDF DOI
We study the dynamics of active polar fluids in a Taylor–Couette geometry where the fluid is confined between two rotating coaxial cylinders. This system can spontaneously generate flow fields and thereby set the two cylinders into relative rotation either by spontaneous symmetry breaking or via asymmetric boundary conditions on the polarization field at the cylinder surfaces. In the presence of an externally applied torque, the system can act as a rotatory motor and perform mechanical work. The relation between the relative angular velocity of the cylinders and the externally applied torque exhibits rich behaviors such as dynamic instabilities and the coexistence of multiple stable steady states for certain ranges of parameter values and boundary conditions.


Martin Depken, Juan MR Parrondo , Stephan W. Grill
Irregular transcription dynamics for rapid production of high-fidelity transcripts
arXiv, Art. No. 1201.5344 [q-bio.BM] (2012)
Open Access

Mirjam Mayer, Guillaume Salbreux, Stephan W. Grill
Biophysics of Cell Developmental Processes: A Lasercutter's Perspective
Comprehensive Biophysics, 7 194-207 (2012)


2011
Louis Leung✳︎, Abigail Klopper✳︎, Stephan W. Grill, William A Harris, Caren Norden
Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia.
Development, 138(22) 5003-5013 (2011)
PDF DOI
Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.


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)
DOI
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.


Stephan W. Grill
Growing up is stressful: biophysical laws of morphogenesis.
Curr Opin Genet Dev, 21(5) 647-652 (2011)
DOI
Would it not be nice to understand the rules that govern how a small and round zygote reforms itself into a full blown three-dimensional and structured organism? The past decades have provided us with a wealth of knowledge about molecular mechanisms, intracellular behaviors, and tissue organization. However, we still do not know how to systematically integrate molecular mechanisms into descriptions that operate at larger scales involving higher-order structures such as the actomyosin cell cortex or an entire tissue. For development, it is the biophysical laws by which these structures deform, move, and restructure that are essential for morphogenetic rearrangements at developmental length- and time-scales. Recent years have seen the advent of systematic approaches for identifying these laws and ways to determine associated physical behaviors. Here I attempt to paint an intuitive picture of the mechanical concepts that are important for large-scale developmental rearrangements, and I briefly review the technique of laser ablation for measuring associated physical quantities and testing physical models.


Matilde Galli, Javier Muñoz, Vincent Portegijs, Mike Boxem, Stephan W. Grill, Albert J R Heck, Sander van den Heuvel
aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division.
Nat Cell Biol, 13(9) 1132-1138 (2011)
DOI
The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3-PAR-6-aPKC polarity complex. However, molecular interactions between polarity proteins and LIN-5-GPR-Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.


Eric A. Galburt, Juan Parrondo, Stephan W. Grill
RNA polymerase pushing.
Biophys Chem, 157(1-3) 43-47 (2011)
DOI
Molecular motors can exhibit Brownian ratchet or power stroke mechanisms. These mechanistic categories are related to transition state position: An early transition state suggests that chemical energy is stored and then released during the step (stroke) while a late transition state suggests that the release of chemical energy rectifies thermally activated motion that has already occurred (ratchet). Cellular RNA polymerases are thought to be ratchets that can push each other forward to reduce pausing during elongation. Here, by constructing a two-dimensional energy landscape from the individual landscapes of active and backtracked enzymes, we identify a new pushing mechanism which is the result of a saddle trajectory that arises in the two-dimensional energy landscape of interacting enzymes. We show that this mechanism is more effective with an early transition state suggesting that interacting RNAPs might translocate via a power stroke.


Jonathon Howard#, Stephan W. Grill#, Justin Bois#
Turing's next steps: the mechanochemical basis of morphogenesis.
Nat Rev Mol Cell Biol, 12(6) 400-406 (2011)
PDF DOI
Nearly 60 years ago, Alan Turing showed theoretically how two chemical species, termed morphogens, diffusing and reacting with each other can generate spatial patterns. Diffusion plays a crucial part in transporting chemical signals through space to establish the length scale of the pattern. When coupled to chemical reactions, mechanical processes - forces and flows generated by motor proteins - can also define length scales and provide a mechanochemical basis for morphogenesis.


Nathan Goehring, Carsten Hoege, Stephan W. Grill#, Anthony A. Hyman#
PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos.
J Cell Biol, 193(3) 583-594 (2011)
DOI
Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.


Marcus Jahnel, Martin Behrndt, Anita Jannasch, Erik Schäffer, Stephan W. Grill
Measuring the complete force field of an optical trap.
Opt Lett, 36(7) 1260-1262 (2011)
DOI
The use of optical traps to measure or apply forces on the molecular level requires a precise knowledge of the trapping force field. Close to the trap center, this field is typically approximated as linear in the displacement of the trapped microsphere. However, applications demanding high forces at low laser intensities can probe the light-microsphere interaction beyond the linear regime. Here, we measured the full nonlinear force and displacement response of an optical trap in two dimensions using a dual-beam optical trap setup with back-focal-plane photodetection. We observed a substantial stiffening of the trap beyond the linear regime that depends on microsphere size, in agreement with Mie theory calculations. Surprisingly, we found that the linear detection range for forces exceeds the one for displacement by far. Our approach allows for a complete calibration of an optical trap.


Nicolas T Chartier, Diana P Salazar Ospina, Laura Benkemoun, Mirjam Mayer, Stephan W. Grill, Amy Shaub Maddox, Jean-Claude Labbé
PAR-4/LKB1 Mobilizes Nonmuscle Myosin through Anillin to Regulate C. elegans Embryonic Polarization and Cytokinesis.
Curr Biol, 21(4) 259-269 (2011)
PDF DOI
BACKGROUND: The serine/threonine kinase LKB1 regulates cell growth and polarity in metazoans, and loss of LKB1 function is implicated in the development of some epithelial cancers. Despite its fundamental role, the mechanism by which LKB1 regulates polarity establishment and/or maintenance is unclear. In the present study, we use the nematode C. elegans to investigate the role of the LKB1 ortholog PAR-4 in actomyosin contractility, a cellular process essential for polarity establishment and cell division in the early embryo. RESULTS: Using high-resolution time-lapse imaging of GFP-tagged nonmuscle myosin II (NMY-2), we found that par-4 mutations reduce actomyosin contractility during polarity establishment, leading to the mispositioning of anterior PAR proteins and to defects in contractile ring ingression during cytokinesis. Fluorescence recovery after photobleaching analysis revealed that the mobility of a cortical population of NMY-2 was reduced in par-4 mutants. Interestingly, the contractility defects of par-4 mutants depend on the reciprocal activity of ANI-1 and ANI-2, two C. elegans homologs of the actin cytoskeletal scaffold protein anillin. CONCLUSION: Because loss of PAR-4 promoted inappropriate accumulation of ANI-2 at the cell cortex, we propose that PAR-4 controls C. elegans embryonic polarity by regulating the activity of anillin family scaffold proteins, thus enabling turnover of cortical myosin and efficient actomyosin contractility. This work provides the first description of a cellular mechanism by which PAR-4/LKB1 mediates cell polarization.


Justin Bois, Frank Jülicher, Stephan W. Grill
Pattern formation in active fluids
Phys Rev Lett, 106(2) Art. No. 028103 (2011)
PDF
We discuss pattern formation in active fluids in which active stress is regulated by diffusing molecular components. Nonhomogeneous active stress profiles create patterns of flow which transport stress regulators by advection. Our work is motivated by the dynamics of the actomyosin cell cortex in which biochemical pathways regulate active stress. We present a mechanism in which a single diffusing species up regulates active stress, resulting in steady flow and concentration patterns. We also discuss general pattern-formation behaviors of reaction-diffusion systems placed in active fluids.


2010
Mirjam Mayer
Mechanics of the C. elegans cell cortex
Ph.D. Thesis,Technische Universität Dresden, Dresden, Germany (2010)


Stephan W. Grill
Forced to be unequal
Science, 330(6004) 597-598 (2010)
PDF

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)
PDF DOI
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.


Abigail Klopper✳︎, Gabriel Krens✳︎, Stephan W. Grill, Carl-Philipp Heisenberg
Finite-size corrections to scaling behavior in sorted cell aggregates.
Eur Phys J E Soft Matter, 33(2) 99-103 (2010)
PDF DOI
Cell sorting is a widespread phenomenon pivotal to the early development of multicellular organisms. In vitro cell sorting studies have been instrumental in revealing the cellular properties driving this process. However, these studies have as yet been limited to two-dimensional analysis of three-dimensional cell sorting events. Here we describe a method to record the sorting of primary zebrafish ectoderm and mesoderm germ layer progenitor cells in three dimensions over time, and quantitatively analyze their sorting behavior using an order parameter related to heterotypic interface length. We investigate the cell population size dependence of sorted aggregates and find that the germ layer progenitor cells engulfed in the final configuration display a relationship between total interfacial length and system size according to a simple geometrical argument, subject to a finite-size effect.


Mirjam Mayer, Martin Depken, Justin Bois, Frank Jülicher, Stephan W. Grill
Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows.
Nature, 467(7315) 617-621 (2010)
PDF DOI
Asymmetric cell divisions are essential for the development of multicellular organisms. To proceed, they require an initially symmetric cell to polarize. In Caenorhabditis elegans zygotes, anteroposterior polarization is facilitated by a large-scale flow of the actomyosin cortex, which directs the asymmetry of the first mitotic division. Cortical flows appear in many contexts of development, but their underlying forces and physical principles remain poorly understood. How actomyosin contractility and cortical tension interact to generate large-scale flow is unclear. Here we report on the subcellular distribution of cortical tension in the polarizing C. elegans zygote, which we determined using position- and direction-sensitive laser ablation. We demonstrate that cortical flow is associated with anisotropies in cortical tension and is not driven by gradients in cortical tension, which contradicts previous proposals. These experiments, in conjunction with a theoretical description of active cortical mechanics, identify two prerequisites for large-scale cortical flow: a gradient in actomyosin contractility to drive flow and a sufficiently large viscosity of the cortex to allow flow to be long-ranged. We thus reveal the physical requirements of large-scale intracellular cortical flow that ensure the efficient polarization of the C. elegans zygote.


Abigail Klopper, Justin Bois, Stephan W. Grill
Influence of secondary structure on recovery from pauses during early stages of RNA transcription.
Phys Rev E, 81(3 Pt 1) Art. No. 030904 (2010)
PDF
The initial stages of transcription by RNA polymerase are frequently marked by pausing and stalling events. These events have been linked to an inactive backtracked state in which the polymerase diffuses along the template DNA. We investigate theoretically the influence of RNA secondary structure in confining this diffusion. The effective confinement length peaks at transcript lengths commensurate with early stalling. This finite-size effect accounts for slow progress at the beginning of transcription, which we illustrate via stochastic hopping models for backtracking polymerases.


2009
Nicholas Licata, Stephan W. Grill
The first-passage problem for diffusion through a cylindrical pore with sticky walls.
Eur Phys J E Soft Matter, 30(4) 439-447 (2009)
PDF DOI
We calculate the first-passage time distribution for diffusion through a cylindrical pore with sticky walls. A particle diffusively explores the interior of the pore through a series of binding and unbinding events with the cylinder wall. Through a diagrammatic expansion we obtain first-passage time statistics for the particle's exit from the pore. Connections between the model and nucleocytoplasmic transport in cells are discussed.


Eric A. Galburt, Stephan W. Grill, Carlos Bustamante
Single molecule transcription elongation.
Methods, 48(4) 323-332 (2009)
PDF DOI
Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.


Martin Depken, Eric A. Galburt, Stephan W. Grill
The origin of short transcriptional pauses.
Biophys J, 96(6) 2189-2193 (2009)
PDF DOI
RNA polymerases are protein molecular machines that transcribe genetic information from DNA into RNA. The elongation of the RNA molecule is frequently interrupted by pauses, the detailed nature of which remains controversial. Here we ask whether backtracking, the central mechanism behind long pauses, could also be responsible for short pauses normally attributed to the ubiquitous pause state. To this end, we model backtracking as a force-biased random walk, giving rise to a broad distribution of pause durations as observed in experiments. Importantly, we find that this single mechanism naturally generates two populations of pauses that are distinct both in duration and trajectory: long-time pauses with the expected behavior of diffusive backtracks, and a new class of short-time backtracks with characteristics similar to those of the ubiquitous pause. These characteristics include an apparent force insensitivity and immobility of the polymerase. Based on these results and a quantitative comparison to published pause trajectories measured with optical tweezers, we suggest that a significant fraction of short pauses are simply due to backtracking.


2008
Gohta Goshima, Mirjam Mayer, Nan Zhang, Nico Stuurman, Ronald D Vale
Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle.
J Cell Biol, 181(3) 421-429 (2008)
PDF DOI
Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.


2007
Eric A. Galburt✳︎, Stephan W. Grill✳︎, Anna Wiedmann, Lucyna Lubkowska, Jason Choy, Eva Nogales, Mikhail Kashlev, Carlos Bustamante
Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner.
Nature, 446(7137) 820-823 (2007)
PDF DOI
RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.


2006
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)
PDF DOI
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.


Yongli Zhang, Corey L Smith, Anjanabha Saha, Stephan W. Grill, Shirley Mihardja, Steven B Smith, Bradley R Cairns, Craig L Peterson, Carlos Bustamante
DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.
Mol Cell, 24(4) 559-568 (2006)
PDF DOI
ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.


2005
Stephan W. Grill, Karsten Kruse, Frank Jülicher
Theory of mitotic spindle oscillations.
Phys Rev Lett, 94(10) 108104-108104 (2005)
PDF
During unequal cell division the mitotic spindle is positioned away from the center of the cell before cell cleavage. In many biological systems this repositioning is accompanied by oscillatory movements of the spindle. We present a theoretical description for mitotic spindle oscillations. We show that the cooperative attachment and detachment of cortical force generators to astral microtubules leads to spontaneous oscillations beyond a critical number of force generators. This mechanism can quantitatively describe the spindle oscillations observed during unequal division of the one cell stage Caenorhabditis elegans embryo.


2003
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)
PDF DOI
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.


Kelly Colombo, Stephan W. Grill, Randall J Kimple, Francis S Willard, David P Siderovski, Pierre Gönczy
Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos.
Science, 300(5627) 1957-1961 (2003)
PDF DOI
Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. We found that the GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, were essential for generation of proper pulling forces. GPR-1/2 interacted with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2.


2001
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)
PDF DOI
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.