Biology subjects

Grill, S. W.

Publications and source records attributed to Grill, S. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Self-centering steady-state flows emerge in confined actomyosin networks

The actin cytoskeleton drives shape changes and transport in cells and supports mechanical signal transmission. However, how cells control and use active cytoskeletal flows at the mesoscale is not well understood. We reconstituted an active cytoskeleton in water-in-oil emulsion droplets of Xenopus laevis egg extract and observed, above a critical droplet size, the emergence of a 3D radially convergent steady-state flow of polymeric actin, maintained by continuous actin turnover. The flow condensed lipid-rich cellular debris into a centered inclusion. Steady-state F-actin density and flow velocity profiles roughly collapse onto scale-invariant master curves. This behavior can be explained by a physical model representing the network as an isotropic active viscous fluid with a percolation threshold. The contracting network behaves as an active swimmer with complex internal dynamics that centers itself and the central inclusion inside the droplets without physical boundary attachment. Active contraction, crosslinking and polymerization dynamics in an actin network can thus generate cell-scale flow patterns that sense the confining geometry and external signals and exert forces that are likely sufficient to move and localize organelles in cells.

biophysics↗

bioRxiv2026

Actomyosin active torques determine body plan handedness in C. elegans

Left-right (LR) asymmetries are a defining feature of bilaterian body plans, but the mechanisms that convert molecular chirality into a conserved body-axis handedness remain unclear. In C. elegans, LR axis specification proceeds via a chiral spindle skew that establishes a handed cell-cell contact pattern (CCP) at the 6-cell stage; an arrangement that, when reversed, leads to situs inversus nematodes [1]. Chiral acto-myosin flows that arise from active chiral torques have been previously identified as the driver of this LR asymmetric spindle skew [2]. Here, we show that high amounts of Lifeact::mKate2, widely used to visualize F-actin, reverse the handedness of active torques in the cortex. We find that this results in reversed chiral acto-myosin flows during LR symmetry breaking, leading to mirrored 6-cell embryos and situs inversus nematodes. Our findings demonstrate that reversing the handedness of active torques changes the LR handedness at the organismal level and establish active chiral torques as instructive for LR axis specification in worms.

biophysics↗

bioRxiv2025

An active torque dipole across tissue layers drives avian left-right symmetry breaking

Unlike in mice, frogs, and fish, left-right (L/R) body axis formation in avian embryos does not arise from the chiral beat of cilia. Instead, a counter-clockwise tissue rotation around Hensens node, the organizer of amniote development, repositions cells expressing L/R sidedness genes. Yet, the physical origin of this rotation remains elusive. Here, we provide evidence that in quail embryos, the node tissue generates an active chiral torque of [~]6{micro}N{micro}m to drive the chiral tissue flow. Microsurgery experiments reveal that this torque depends on actomyosin molecular activity, is generated within the dorsal node tissue, and requires the underlying ventral meso-/endoderm to act as a mechanical substrate sustaining the counter-torque. We conclude that a dorsoventrally oriented tissue-scale active torque dipole at the node translates cell-scale chirality to organismal L/R asymmetry, adding a mechanical dimension to the canonical function of embryonic organizers as signaling hubs.

biophysics↗

bioRxiv2025

A protein-DNA surface hydrogel mechanically protects the cell nucleus

The nuclear envelope protects the genome from mechanical stress during processes such as migration, division, and compression1-6, but how it buffers forces at the scale of DNA remains unclear. Here, we utilize optical tweezers to show that a multivalent protein-DNA co-condensate containing the nuclear envelope protein LEM27,8 and the DNA-binding protein BAF 9,10 shield DNA beyond its melting point at 65 pN11. Under load, their collective assembly induces an unconventional DNA stiffening effect that provides mechanical reinforcement, dependent on the intrinsically disordered region (IDR) of LEM2. At the nuclear surface, these components form an elastic surface hydrogel in which LEM2 IDR-IDR interactions contract the surface hydrogel relative to its relaxed state, introducing a pre-stress in the lamin network. Inside cells, this surface hydrogel model can recapitulate elastic properties of the nuclear envelope measured via AFM indentation experiments as well as nuclear morphology, using parameters obtained at the molecular scale by use of optical tweezers. Disruption of the surface hydrogel increases DNA damage and micronuclei formation during nuclear deformation. These findings reveal a load-bearing, mesoscale surface hydrogel that reinforces the nucleus and expands the functional repertoire of biomolecular condensates to include DNA protection under mechanical stress.

biophysics↗

bioRxiv2024

Repeated extrinsic and anisotropic mechanical inputs promote polarized adherens junction elongation

A key challenge in development is understanding how complex organisms physically coordinate the morphogenesis of multiple tissues. Here, using biophysical approaches, we investigate how muscles under the epidermis specifically stimulate the extension of anterior-posterior (AP-oriented) epidermal adherens junctions during late C. elegans embryonic elongation. First, light-sheet imaging shows that asynchronous patterns of muscle contractions drive embryo rotations. In turn, junctions between the lateral and dorso-ventral epidermis repeatedly oscillate between a folded, hypotensed state, and an extended, hypertensed state. Second, FRAP (Fluorescence Recovery After Photobleaching) analysis of an E-cadherin::GFP construct shows that muscle contractions stimulate E-cadherin turnover. Moreover, a mechano-chemical model backed by genetic tests suggests that E-cadherin trafficking modulates junction elongation due to lower line tension. Altogether, our results illustrate how muscle contractions fluidize epidermal adherens junctions, which, combined with anisotropic tension in the epidermis, drives their polarized extension.

developmental biology↗

bioRxiv2024

Actin polymerization counteracts prewetting of N-WASP on supported lipid bilayers

Cortical condensates, transient punctate-like structures rich in actin and the actin nucleation pathway member N-WASP, form during activation of the actin cortex in the C. elegans oocyte. Their emergence and spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, the physical process that drives the onset of cortical condensate formation near membranes remains unexplored. Here, using a reconstituted phase separation assay of cortical condensate proteins, we demonstrate that the key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via a prewetting transition. Actin partitions into the condensates, where it polymerizes and counteracts the N-WASP prewetting transition. Taken together, the dynamics of condensate-assisted cortex formation appear to be controlled by a balance between surface-assisted condensate formation and polymer-driven condensate dissolution. This opens new perspectives for understanding how the formation of complex intracellular structures is affected and controlled by phase separation.

biophysics↗

bioRxiv2023

A cytokinetic ring-driven cell rotation achieves Hertwig's rule in early development

Cells tend to divide along the direction in which they are longest, as famously stated by Oscar Hertwig in 1884 in his long axis rule1,2. The orientation of the mitotic spindle determines the cell division axis3, and Hertwigs long axis rule is usually ensured by forces stemming from microtubules4. Pulling on the spindle from the cell cortex can give rise to unstable behaviors5,6, and we here set out to understand how Hertwigs long axis rule is realized in early embryonic divisions where cortical pulling forces are prevalent. We focus on early C. elegans development, where we compressed embryos to reveal that cortical pulling forces favor an alignment of the spindle with the cells short axis. Strikingly, we find that this misalignment is corrected by an actomyosin-based mechanism that rotates the entire cell, including the mitotic spindle. We uncover that myosin-driven contractility in the cytokinetic ring generates inward forces that align it with the short axis, and thereby the spindle with the long axis. A theoretical model together with experiments using slightly compressed mouse zygotes suggest that a constricting cytokinetic ring can ensure Hertwigs long axis rule in cells that are free to rotate inside a confining structure, thereby generalizing the underlying principle.

developmental biology↗

bioRxiv2023

Assembly of tight junction belts by surface condensation and actin elongation

Formation of biomolecular condensates via phase separation enables compartmentation of many cellular processes. However, how cells can control condensation at specific locations to create complex cellular structures remains poorly understood. Here, we investigated the mechanism of tight junction formation, which involves condensation of scaffold proteins at cell-cell contacts and elongation of the condensates into a belt around the cellular perimeter. Using cell biology, reconstitution, and thermodynamic theory, we discovered that cells use surface phase transitions to control local condensation at the membrane far below bulk saturation. Surface condensation of junctional ZO-scaffold proteins is mediated by receptor binding and regulated by the receptors oligomerization state. Functionally, ZO surface condensation is directly coupled to actin polymerization and bundling, which drives elongation of receptor-ZO-actin condensates similar to tight junction belt formation in cells. We conclude that surface phase transitions provide a robust mechanism to control the position and shape of protein condensates. One-Sentence SummaryLocal surface binding of cytosolic scaffold proteins provides spatial control of protein condensation to assemble adhesion junctions.

biophysics↗

bioRxiv2023

Axis convergence in C. elegans embryos

Embryos develop in a surrounding that guides key aspects of their development. For example, the anteroposterior (AP) body axis is always aligned with the geometric long axis of the surrounding eggshell in fruit flies and worms. The mechanisms that ensure convergence of the AP axis with the long axis of the eggshell remain unresolved. We investigate axis convergence in early C. elegans development, where the nascent AP axis, when misaligned, actively re-aligns to converge with the long axis of the egg. Here, we identify two physical mechanisms that underlie axis convergence. First, bulk cytoplasmic flows, driven by actomyosin cortical flows, can directly reposition the AP axis. Second, active forces generated within the pseudocleavage furrow, a transient actomyosin structure similar to a contractile ring, can drive a mechanical re-orientation such that it becomes positioned perpendicular to the long axis of the egg. This in turn ensures AP axis convergence. Numerical simulations, together with experiments that either abolish the pseudocleavage furrow or change the shape of the egg, demonstrate that the pseudocleavage furrow-dependent mechanism is the key driver of axis convergence. We conclude that active force generation within the actomyosin cortical layer drives axis convergence in the early nematode.

biophysics↗

bioRxiv2022

GTPase-dependent cyclic flexibility transitions drive the two-component EEA1-Rab5 molecular motor

The recognition of vesicles by their correct target compartment depends on the pairing of small GTPases with effector proteins (1-3). Whereas ATPases can cyclically convert the free energy of ATP hydrolysis into mechanical work, the function of small GTPases is predominantly associated with signal transduction processes, and their role in mechano-transduction is less established (4-6). However, binding of the GTPase Rab5 to the long coiled-coil tethering protein EEA1 on an early endosome induces a rigidity transition resulting in a large conformational change in EEA1 from a rigid and extended to a flexible and collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer (7). It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperones. Here, we use fluorescence correlation spectroscopy to reveal that EEA1 in bulk solution can undergo multiple flexibility transition cycles that are associated with the binding and release of Rab5(GTP) and Rab5(GDP). Using semi-flexible polymer theory we provide evidence that the cyclic transitioning of Rab5-EEA1 between extended and collapsed conformations is driven by the energetics of Rab5 binding/unbinding and GTP hydrolysis. Cyclic flexibility transitions represent a complete mechanical work cycle that is able to perform up to 20 kBT of mechanical work per cycle, against an opposing force. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from GTP hydrolysis by Rab5. We conclude that coiled-coil tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

biophysics↗

bioRxiv2021

A condensate dynamic instability orchestrates oocyte actomyosin cortex activation

A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition. We here report on the discovery that in C. elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP, and ARP2/3 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow, and then switch to disassembly before dissolving completely. We find that in contrast to condensate growth via diffusion, the growth dynamics of cortical condensates are chemically driven. Remarkably, the associated chemical reactions obey mass action kinetics despite governing both composition and size. We suggest that the resultant condensate dynamic instability suppresses coarsening of the active micro-emulsion, ensures reaction kinetics that are independent of condensate size, and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.

biophysics↗

bioRxiv2021

Co-condensation of proteins with single- and double-stranded DNA

Biomolecular condensates provide distinct compartments that can localize and organize biochemistry inside cells. Recent evidence suggests that condensate formation is prevalent in the cell nucleus. To understand how different components of the nucleus interact during condensate formation is an important challenge. In particular, the physics of co-condensation of proteins together with nucleic acids remains elusive. Here, we use optical tweezers to study how the prototypical prion-like protein Fused-in-Sarcoma (FUS) forms liquid-like assemblies in vitro, by co-condensing together with individual DNA molecules. Through progressive DNA unpeeling, buffer exchange and force measurements, we show that FUS adsorbing in a single layer on DNA effectively generates a sticky FUS-DNA polymer that can collapse to form a liquid-like FUS-DNA co-condensate. Condensation occurs at constant DNA tension for double-stranded DNA, which is a signature of phase separation. We suggest that co-condensation mediated by protein adsorption on nucleic acids is an important mechanism for intracellular compartmentalization.

biophysics↗

bioRxiv2021

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking

1Proper left-right symmetry breaking is essential for animal development and in many species the actin cytoskeleton plays an instrumental role in this process. Active torque generation in the actomyosin layer promotes left-right symmetry breaking in C. elegans embryos by driving chiral counter-rotating cortical flows. While both Formins and Myosins have been implied in left-right symmetry breaking, and both can rotate actin filaments in vitro, it remains unclear if 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 counter-rotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counter-rotation 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. 2 SignificanceActive torque generation in the actin cytoskeleton has been implicated in driving left-right symmetry breaking of developing embryos, but which molecules generate the active torque and how active torque generation is organized subcellularly remains unclear. This study shows that cortical Formin, recruited to cortical regions where RhoA signaling is active, promotes active torque generation in the actomyosin layer. We find that active torque tends to locally rotate the cortex in a clockwise fashion, which drives the emergence of chiral counter-rotating flows with consistent handedness and facilitates left-right symmetry breaking of C. elegans embryos.

developmental biology↗

bioRxiv2020

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

In mammals HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1, HP1{beta}, and HP1{gamma}, 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 HP1-DNA condensates, HP1 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 HP1{beta}. Finally, we find that differences in each HP1 paralogs 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.

biochemistry↗

bioRxiv2020

Surface condensation of a pioneer transcription factor on DNA

In the last decade, extensive studies on the properties of non-membrane-bound compartments in the cellular cytoplasm have shown that concepts in phase separation drawn from physical chemistry can describe their formation and behaviour1-4. Current evidence also suggests that phase separation plays a role in the organization inside the cell nucleus5-8. However, the influence and role of DNA on the physical chemistry of phase separation is not well understood. Here, we are interested in the role of interactions between phase separating proteins and the DNA surface. The interaction of liquid phases with surfaces has been extensively studied in soft matter physics, in the context of macroscopic surfaces and non-biological liquids9-11. The conditions in the nucleus are different from those studied in conventional soft matter physics because DNA with a diameter of about 2 nm12 provides a microscopic surface, and liquid-like phases are complex mixtures of proteins subject to a myriad of biochemical modifications13. Transcriptional condensates, which are thought to serve as regulatory hubs in gene expression14-21, provide an accessible system to investigate the physics of condensates that emerge from DNA-protein and protein-protein interactions. These condensates are typically small22, and the mechanisms that determine their size are unknown. Whether they can be understood as phase separated compartments has been subject to debate23-26. Here, we use optical tweezers to directly observe the condensation of the pioneer transcription factor Klf427,28 on DNA in vitro. We demonstrate that Klf4 forms microphases that are enabled by interaction with the DNA surface. This sets their typical size and allows them to form below the saturation concentration for liquid-liquid phase separation. We combine experiment with theory to show that these microphases can be understood as forming by surface condensation on DNA via a switch-like transition similar to prewetting. Polymer surface mediated condensation reconciles several observations that were previously thought to be at odds with the idea of phase separation as an organizing principle in the nucleus.

biophysics↗

bioRxiv2020

A hydraulic instability drives the cell death decision in the nematode germline

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

developmental biology↗

bioRxiv2020

Thermal fluctuations assist mechanical signal propagation in coiled-coil proteins

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 novel motor-like activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. To elucidate how binding of a single signaling protein can globally change the stiffness of a 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, changing the configuration of the entire molecule and thereby affecting its stiffness. Our work sheds light onto intramolecular communication and force generation in long coiled-coil proteins.

biophysics↗

bioRxiv2020

Machine learning pattern recognition and differential network analysis of gastric microbiome in the presence of proton pump inhibitor treatment or Helicobacter pylori infection

Although long thought to be a sterile and inhospitable environment, the stomach is inhabited by diverse microbial communities, co-existing in a dynamic balance. Long-term use of orally administered 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 the early phase. They mainly focus on the most prevalent taxa and rely on linear techniques for multivariate analysis. Here we disclose the importance of complementing linear dimensionality reduction techniques such as Principal Component Analysis and Multidimensional Scaling with nonlinear approaches derived from the physics of complex systems. Then, we show the importance to complete multivariate pattern analysis with differential network analysis, to reveal mechanisms of re-organizations which emerge from combinatorial microbial variations induced by a medical treatment (PPIs) or an infectious state (H. pylori).

systems biology↗