Cortical Myosin Research Articles

MRCK ensures cortex-chromatin "social distancing" to enable egg spindle rotation.

During the second meiotic cell division, egg cells discard one set of chromatids to the polar body to produce a large haploid gamete. Meiotic spindle rotation is a critical step to ensure proper polar body extrusion. In this issue, Bourdais et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202211029) have identified MRCKβ as an essential kinase for efficient spindle rotation. MRCK activates cortical myosin II rings overlying the spindle to prevent the notoriously sticky interaction between the cell cortex and chromatin to facilitate spindle rotation. Furthermore, Bourdais et al. found that the same MRCK-myosin II pathway also operates in zygotes to promote parental genome unification.

Nuclear lamin facilitates collective border cell invasion into confined spaces in vivo.

Cells migrate collectively through confined environments during development and cancer metastasis. The nucleus, a stiff organelle, impedes single cells from squeezing into narrow channels within artificial environments. However, how nuclei affect collective migration into compact tissues is unknown. Here, we use border cells in the fly ovary to study nuclear dynamics in collective, confined in vivo migration. Border cells delaminate from the follicular epithelium and squeeze into tiny spaces between cells called nurse cells. The lead cell nucleus transiently deforms within the lead cell protrusion, which then widens. The nuclei of follower cells deform less. Depletion of the Drosophila B-type lamin, Lam, compromises nuclear integrity, hinders expansion of leading protrusions, and impedes border cell movement. In wildtype, cortical myosin II accumulates behind the nucleus and pushes it into the protrusion, whereas in Lam-depleted cells, myosin accumulates but does not move the nucleus. These data suggest that the nucleus stabilizes lead cell protrusions, helping to wedge open spaces between nurse cells.

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, we found that M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, we developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Aurora A and cortical flows promote polarization and cytokinesis by inducing asymmetric ECT-2 accumulation.

In the early Caenorhabditis elegans embryo, cell polarization and cytokinesis are interrelated yet distinct processes. Here, we sought to understand a poorly understood aspect of cleavage furrow positioning. Early C. elegans embryos deficient in the cytokinetic regulator centralspindlin form furrows, due to an inhibitory activity that depends on aster positioning relative to the polar cortices. Here, we show polar relaxation is associated with depletion of cortical ECT-2, a RhoGEF, specifically at the posterior cortex. Asymmetric ECT-2 accumulation requires intact centrosomes, Aurora A (AIR-1), and myosin-dependent cortical flows. Within a localization competent ECT-2 fragment, we identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. During both polarization and cytokinesis, our results suggest that centrosomal AIR-1 breaks symmetry via ECT-2 phosphorylation; this local inhibition of ECT-2 is amplified by myosin-driven flows that generate regional ECT-2 asymmetry. Together, these mechanisms cooperate to induce polarized assembly of cortical myosin, contributing to both embryo polarization and cytokinesis.

Axis specification: Breaking symmetry with a myosin patch in the egg

Drosophila anterior-posterior axis specification occurs in the oocyte, but the initial symmetry break has been unclear. A new study reveals that a posterior domain of cortical myosin is induced with unique post-translational modification and dynamics and that this domain recruits downstream posterior determinants.

Nuclear Deformation Lets Cells Gauge Their Physical Confinement.

How cells sense their physical microenvironment remains incompletely understood. In two recent Science articles, Lomakin etal. (2020) and Venturini etal. (2020) demonstrate that progressive nuclear deformation associated with cellular confinement triggers intracellular events that promote cell contractility and migration, revealing the nucleus to serve as a central mechanosensor.

Polar relaxation by dynein-mediated removal of cortical myosin II.

Nearly six decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction, whereas suppression of dynein activity delayed furrowing. We show that dynein-mediated removal of myosin II from the polar cortexes triggers a bidirectional cortical flow toward the cell equator, which induces the assembly of the actomyosin contractile ring. These results provide a molecular mechanism for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

Cellular Blebs and Membrane Invaginations Are Coupled through Membrane Tension Buffering

Bleb-type cellular protrusions play key roles in a range of biological processes. It was recently found that bleb growth is facilitated by a local supply of membrane from tubular invaginations, but the interplay between the expanding bleb and the membrane tubes remains poorly understood. On the one hand, the membrane area stored in tubes may serve as a reservoir for bleb expansion. On the other hand, the sequestering of excess membrane in stabilized invaginations may effectively increase the cell membrane tension, which suppresses spontaneous protrusions. Here, we investigate this duality through physical modeling and in vivo experiments. In agreement with observations, our model describes the transition into a tube-flattening mode of bleb expansion while also predicting that the blebbing rate is impaired by elevating the concentration of the curved membrane proteins that form the tubes. We show both theoretically and experimentally that the stabilizing effect of tubes could be counterbalanced by the cortical myosin contractility. Our results largely suggest that proteins able to induce membrane tubulation, such as those containing N-BAR domains, can buffer the effective membrane tension—a master regulator of all cell deformations.

Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm.Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

Epithelial vertex models with active biochemical regulation of contractility can explain organized collective cell motility.

Collective motions of groups of cells are observed in many biological settings such as embryo development, tissue formation, and cancer metastasis. To effectively model collective cell movement, it is important to incorporate cell specific features such as cell size, cell shape, and cell mechanics, as well as active behavior of cells such as protrusion and force generation, contractile forces, and active biochemical signaling mechanisms that regulate cell behavior. In this paper, we develop a comprehensive model of collective cell migration in confluent epithelia based on the vertex modeling approach. We develop a method to compute cell-cell viscous friction based on the vertex model and incorporate RhoGTPase regulation of cortical myosin contraction. Global features of collective cell migration are examined by computing the spatial velocity correlation function. As active cell force parameters are varied, we found rich dynamical behavior. Furthermore, we find that cells exhibit nonlinear phenomena such as contractile waves and vortex formation. Together our work highlights the importance of active behavior of cells in generating collective cell movement. The vertex modeling approach is an efficient and versatile approach to rigorously examine cell motion in the epithelium.

Role of membrane-tension gated Ca2+ flux in cell mechanosensation.

Eukaryotic cells are sensitive to mechanical forces they experience from the environment. The process of mechanosensation is complex, and involves elements such as the cytoskeleton and active contraction from myosin motors. Ultimately, mechanosensation is connected to changes in gene expression in the cell, known as mechanotransduction. While the involvement of the cytoskeleton in mechanosensation is known, the processes upstream of cytoskeletal changes are unclear. In this paper, by using a microfluidic device that mechanically compresses live cells, we demonstrate that Ca2+ currents and membrane tension-sensitive ion channels directly signal to the Rho GTPase and myosin contraction. In response to membrane tension changes, cells actively regulate cortical myosin contraction to balance external forces. The process is captured by a mechanochemical model where membrane tension, myosin contraction and the osmotic pressure difference between the cytoplasm and extracellular environment are connected by mechanical force balance. Finally, to complete the picture of mechanotransduction, we find that the tension-sensitive transcription factor YAP family of proteins translocate from the nucleus to the cytoplasm in response to mechanical compression.

Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells

Asymmetric cell division, creating sibling cells with distinct developmental potentials, can be manifested in sibling cell size asymmetry. This form of physical asymmetry occurs in several metazoan cells, but the underlying mechanisms and function are incompletely understood. Here we use Drosophila neural stem cells to elucidate the mechanisms involved in physical asymmetry establishment. We show that Myosin relocalizes to the cleavage furrow via two distinct cortical Myosin flows: at anaphase onset, a polarity induced, basally directed Myosin flow clears Myosin from the apical cortex. Subsequently, mitotic spindle cues establish a Myosin gradient at the lateral neuroblast cortex, necessary to trigger an apically directed flow, removing Actomyosin from the basal cortex. On the basis of the data presented here, we propose that spatiotemporally controlled Myosin flows in conjunction with spindle positioning and spindle asymmetry are key determinants for correct cleavage furrow placement and cortical expansion, thereby establishing physical asymmetry.

Genome-scale single-cell mechanical phenotyping reveals disease-related genes involved in mitotic rounding

To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular pressure, which the contractile actomyosin cortex directs into shape. Here, we introduce a genome-scale microcantilever- and RNAi-based approach to phenotype the contribution of > 1000 genes to the rounding of single mitotic cells against confinement. Our screen analyzes the rounding force, pressure and volume of mitotic cells and localizes selected proteins. We identify 49 genes relevant for mitotic rounding, a large portion of which have not previously been linked to mitosis or cell mechanics. Among these, depleting the endoplasmic reticulum-localized protein FAM134A impairs mitotic progression by affecting metaphase plate alignment and pressure generation by delocalizing cortical myosin II. Furthermore, silencing the DJ-1 gene uncovers a link between mitochondria-associated Parkinson’s disease and mitotic pressure. We conclude that mechanical phenotyping is a powerful approach to study the mechanisms governing cell shape.

Myosin efflux promotes cell elongation to coordinate chromosome segregation with cell cleavage

Chromatid segregation must be coordinated with cytokinesis to preserve genomic stability. Here we report that cells clear trailing chromatids from the cleavage site by undergoing two phases of cell elongation. The first phase relies on the assembly of a wide contractile ring. The second phase requires the activity of a pool of myosin that flows from the ring and enriches the nascent daughter cell cortices. This myosin efflux is a novel feature of cytokinesis and its duration is coupled to nuclear envelope reassembly and the nuclear sequestration of the Rho-GEF Pebble. Trailing chromatids induce a delay in nuclear envelope reassembly concomitant with prolonged cortical myosin activity, thus providing forces for the second elongation. We propose that the modulation of cortical myosin dynamics is part of the cellular response triggered by a “chromatid separation checkpoint” that delays nuclear envelope reassembly and, consequently, Pebble nuclear sequestration when trailing chromatids are present at the midzone.

A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility.

During female meiosis, haploid eggs are generated from diploid oocytes. This reduction in chromosome number occurs through two highly asymmetric cell divisions, resulting in one large egg and two small polar bodies. Unlike mitosis, where an actomyosin contractile ring forms between the sets of segregating chromosomes, the meiotic contractile ring forms on the cortex adjacent to one spindle pole, then ingresses down the length of the spindle to position itself at the exact midpoint between the two sets of segregating chromosomes. Depletion of casein kinase 1 gamma (CSNK-1) in Caenorhabditis elegans led to the formation of large polar bodies that contain all maternal DNA, because the contractile ring ingressed past the spindle midpoint. Depletion of CSNK-1 also resulted in the formation of deep membrane invaginations during meiosis, suggesting an effect on cortical myosin. Both myosin and anillin assemble into dynamic rho-dependent cortical patches that rapidly disassemble in wild-type embryos. CSNK-1 was required for disassembly of both myosin patches and anillin patches. Disassembly of anillin patches was myosin independent, suggesting that CSNK-1 prevents expulsion of the entire meiotic spindle into a polar body by negatively regulating the rho pathway rather than through direct inhibition of myosin.

Local and global remodelling of cortical myosin by the mitotic kinase Aurora-A

Local and global remodelling of cortical myosin by the mitotic kinase Aurora-A

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity.

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.

The biophysical nature of cells: potential cell behaviours revealed by analytical and computational studies of cell surface mechanics.

BackgroundThe biophysical characteristics of cells determine their shape in isolation and when packed within tissues. Cells can form regular or irregular epithelial structures, round up and form clusters, or deform and attach to substrates. The acquired shape of cells and tissues is a consequence of (i) internal cytoskeletal processes, such as actin polymerisation and cortical myosin contraction, (ii) adhesion molecules within the cell membrane that interact with substrates and neighbouring cells, and (iii) processes that regulate cell volume. Although these processes seem relatively simple, when combined they unleash a rich variety of cellular behaviour that is not readily understandable outside a theoretical framework.MethodsWe perform a mathematical analysis of a commonly used class of model formalisms that describe cell surface mechanics using an energy-based approach. Predictions are then confirmed through comparison with the computational outcomes of a Vertex model and 2D and 3D simulations of the Cellular Potts model.ResultsThe analytical study reveals the complete possible spectrum of single cell behaviour and tissue packing in both 2D and 3D, by taking the typical core elements of cell surface mechanics into account: adhesion, cortical tension and volume conservation. We show that from an energy-based description, forces and tensions can be derived, as well as the prediction of cell behaviour and tissue packing, providing an intuitive and biologically relevant mapping between modelling parameters and experiments.ConclusionsThe quantitative cellular behaviours and biological insights agree between the analytical study and the diverse computational model formalisms, including the Cellular Potts model. This illustrates the generality of energy-based approaches for cell surface mechanics and highlights how meaningful and quantitative comparisons between models can be established. Moreover, the mathematical analysis reveals direct links between known biophysical properties and specific parameter settings within the Cellular Potts model.

Myosin II controls cellular branching morphogenesis and migration in three dimensions by minimizing cell-surface curvature.

In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration.