Actomyosin Network Research Articles

Regulation of Actin Dynamics in the C. elegans Somatic Gonad.

The reproductive system of the hermaphroditic nematode C. elegans consists of a series of contractile cell types—including the gonadal sheath cells, the spermathecal cells and the spermatheca–uterine valve—that contract in a coordinated manner to regulate oocyte entry and exit of the fertilized embryo into the uterus. Contraction is driven by acto-myosin contraction and relies on the development and maintenance of specialized acto-myosin networks in each cell type. Study of this system has revealed insights into the regulation of acto-myosin network assembly and contractility in vivo.

Aurora-A Breaks Symmetry in Contractile Actomyosin Networks Independently of Its Role in Centrosome Maturation

Cell polarity is facilitated by a rearrangement of the actin cytoskeleton at the cell cortex. The program triggering the asymmetric remodeling of contractile actomyosin networks remains poorly understood. Here, we show that polarization of Caenorhabditis elegans zygotes is established through sequential downregulation of cortical actomyosin networks by the mitotic kinase, Aurora-A. Aurora-A accumulates around centrosomes to locally disrupt the actomyosin contractile activity at the proximal cortex, thereby promoting cortical flows during symmetry breaking. Aurora-A later mediates global disassembly of cortical actomyosin networks, which facilitates the initial polarization through suppression of centrosome-independent cortical flows. Translocation of Aurora-A from the cytoplasm to the cortex is sufficient to interfere with the cortical actomyosin networks independently of its roles in centrosome maturation and cell-cycle progression. We propose that Aurora-A activity serves as a centrosome-mediated cue that breaks symmetry in actomyosin contractile activity, and facilitates the initial polarization through global suppression of cortical actomyosin networks.

Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos

SUMMARYEnvironmental stress threatens the fidelity of embryonic morphogenesis. Heat, for example, is a teratogen. Yet how heat affects morphogenesis is poorly understood. Here, we identify a heat-inducible actin stress response (ASR) in Drosophila embryos that is mediated by the activation of the actin regulator Cofilin. Similar to ASR in adult mammalian cells, heat stress in fly embryos triggers the assembly of intra-nuclear actin rods. Rods measure up to a few microns in length, and their assembly depends on elevated free nuclear actin concentration and Cofilin. Outside the nucleus, heat stress causes Cofilin-dependent destabilization of filamentous actin (F-actin) in actomyosin networks required for morphogenesis. F-actin destabilization increases the chance of morphogenesis mistakes. Blocking the ASR by reducing Cofilin dosage improves the viability of heat-stressed embryos. However, improved viability correlates with restoring F-actin stability, not rescuing morphogenesis. Thus, ASR endangers embryos, perhaps by shifting actin from cytoplasmic filaments to an elevated nuclear pool.

Scaling behaviour in steady-state contracting actomyosin networks.

Contractile actomyosin network flows are crucial for many cellular processes including cell division and motility, morphogenesis and transport. How local remodeling of actin architecture tunes stress production and dissipation and regulates large-scale network flows remains poorly understood. Here, we generate contracting actomyosin networks with rapid turnover in vitro, by encapsulating cytoplasmic Xenopus egg extracts into cell-sized ‘water-in-oil’ droplets. Within minutes, the networks reach a dynamic steady-state with continuous inward flow. The networks exhibit homogeneous, density-independent contraction for a wide range of physiological conditions, implying that the myosin-generated stress driving contraction and the effective network viscosity have similar density dependence. We further find that the contraction rate is roughly proportional to the network turnover rate, but this relation breaks down in the presence of excessive crosslinking or branching. Our findings suggest that cells use diverse biochemical mechanisms to generate robust, yet tunable, actin flows by regulating two parameters: turnover rate and network geometry.

Quantifying Dissipation in Actomyosin Networks

The self-organization of actomyosin networks is known to be intricately controlled by concentrations of filaments and associated proteins, however a general physical explanation of the emergence of different dynamical states (such as vortices in gliding assays) in these active matter systems has not yet been firmly established. It has been hypothesized that the dissipation of free energy by active matter systems is optimized during self-organization, leading to the emergence of more highly dissipative dynamical states, but this idea has not yet been evidenced in actomyosin systems. Here we establish a methodology for testing this hypothesis in actomyosin networks using MEDYAN, an agent-based simulation platform for studying active networks. We extend the capabilities of MEDYAN to allow quantification of the rates of dissipation resulting from chemical reactions and relaxation of mechanical stresses during simulation trajectories, and apply these methods here to characterize the trajectory of dissipation rates accompanying the self-organization of small disordered actomyosin networks at varying concentrations of myosin and cross-linkers, as well as the distributions of these dissipation rates. In the data presented here we do not observe network reorganizations that lead to increases in the total dissipation rate as predicted by the dissipation-driven adaptation hypothesis mentioned above, however we discuss possible future experiments utilizing this new methodology that could carefully test the applicability of this principle in actomyosin networks.

Rapid Treadmilling and Myosin Motors Synergistically Induce Formation of Ring-Like Actomyosin Architectures and Cortexes

Semiflexible actin filaments crosslinked by myosins control cell motility and morphology via dynamic remodeling process - treadmilling. In less chemically dynamic reconstituted actomyosin networks, motor proteins induce global geometric contraction, creating cluster-like structures. However, filaments treadmill much faster in living cells and are often found to be abundant at the cell periphery, frequently forming ring-like structures or thin, contractile sub-plasma-membrane cortexes. To explore the interplay between treadmilling and myosin induced contractility, we used an advanced computational model MEDYAN, which couples stochastic reaction-diffusion scheme with polymer physics modeling. Our results indicate that the actomyosin network geometry is tightly regulated by filament treadmilling. In our simulation, non-treadmilling and slowly treadmilling actomyosin networks would geometrically collapse and form clusters. On the other hand, enhanced filament treadmilling generates ring-like structures that are dense and mechanically contractile with their stability regulated by myosin concentration. We found that rapid treadmilling towards boundary helps filaments escape from geometric collapse, while myosin prevents filaments from growing against the boundary by altering their orientation such that those filaments are rescued from destruction.

Probing Fluctuations and Avalanches in the Cytoskeleton with Active Micropost Arrays

The cellular actomyosin network plays an important role in a wide range of cell behavior, including motility, morphology and mechanotransduction. Although the building blocks of actomyosin networks are well understood at the molecular level, the picture connecting these microscale units to cell-scale behavior remains incomplete. Here we present results using active micropost array detectors (AMPADS) to characterize the dynamical fluctuations and local rheology of cellular actomyosin networks in detail. AMPADS are poly(dimethylsiloxane) (PDMS) micropillar arrays with embedded magnetic nanowires that enable mechanical actuation of an adherent cell. We classified the cell-associated microposts based on their traction force, and identified two distinct populations, one containing posts coupled to stress fibers and the other containg posts coupled to the actomyosin cortex. Both groups show weak power law rheology, but we find that the fluctuations of stress fiber-associated posts are more active and highly anisotropic in comparison to the cortically-associated posts. Notably, the fluctuations of both populations are highly heterogenous and resemble a Lévy process. Specifically, the mean-squared displacements of both types of posts display broadly distributed amplitudes and super-diffusive behavior. We find that the amplitude distribution is fat-tailed, and is dominated by a corresponding distribution of intermittent, large step-like displacements, resembling avalanches and earthquakes in physical systems. Our regular array of detectors also allows us to determine the spatial extent and average symmetry of the largest rearrangements in the cortex, which are both spatially and temporally complex, resembling those in plastic solids. These results suggest that the dynamics of the cellular actomyosin network resembles what is seen in soft matter systems such as sandpiles and foams, where constituent components self-organize into marginally stable, plastic networks, with significant implications for future models of the cortex.

The Physical Bases of Forming a Smooth Boundary between an Expanding Arp 2/3 Actin Network and a Contractile Actomyosin Network

The Physical Bases of Forming a Smooth Boundary between an Expanding Arp 2/3 Actin Network and a Contractile Actomyosin Network

The N-cadherin interactome in primary cardiomyocytes as defined using quantitative proximity proteomics.

ABSTRACTThe junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.

Differential Actin Binding Affinity Leads to Protein Sorting in a Reconstituted Active Composite Layer

Various functional cell surface proteins bind to cortical actin and undergo transient clustering driven by actomyosin flows. Examples include the GPI-anchored proteins, Ras-signalling proteins, T-cell receptors and many glycoproteins such as CD44. Given the density and diversity of protein and lipid species in the plasma membrane, the question arises how the local accumulation of specific molecules is achieved in a timely manner. Here we explore the possibility that the combination of differential binding of surface molecules to actin and the non-equilibrium acto-myosin dynamics leads to molecular patterning and cluster formation of membrane proteins. Using reconstituted acto-myosin networks tethered to supported lipid bilayers, as well as theoretical simulations, we provide evidence for this proposed active sorting mechanism. We show that differential affinity for actin templates a variety of patterns of membrane tethered proteins, including complete segregation, bull's eye and mixed pattern. Our results suggest that this mechanism could help in understanding the spatio-temporal organization and regulation of various processes at the cell surface.

Anillin regulates epithelial cell mechanics by structuring the medial-apical actomyosin network.

Cellular forces sculpt organisms during development, while misregulation of cellular mechanics can promote disease. Here, we investigate how the actomyosin scaffold protein anillin contributes to epithelial mechanics in Xenopus laevis embryos. Increased mechanosensitive recruitment of vinculin to cell-cell junctions when anillin is overexpressed suggested that anillin promotes junctional tension. However, junctional laser ablation unexpectedly showed that junctions recoil faster when anillin is depleted and slower when anillin is overexpressed. Unifying these findings, we demonstrate that anillin regulates medial-apical actomyosin. Medial-apical laser ablation supports the conclusion that that tensile forces are stored across the apical surface of epithelial cells, and anillin promotes the tensile forces stored in this network. Finally, we show that anillin's effects on cellular mechanics impact tissue-wide mechanics. These results reveal anillin as a key regulator of epithelial mechanics and lay the groundwork for future studies on how anillin may contribute to mechanical events in development and disease.

Mimicking Active Biopolymer Networks with a Synthetic Hydrogel.

Stiffening due to internal stress generation is of paramount importance in living systems and is the foundation for many biomechanical processes. For example, cells stiffen their surrounding matrix by pulling on collagen and fibrin fibers. At the subcellular level, molecular motors prompt fluidization and actively stiffen the cytoskeleton by sliding polar actin filaments in opposite directions. Here, we demonstrate that chemical cross-linking of a fibrous matrix of synthetic semiflexible polymers with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) produces internal stress by induction of a coil-to-globule transition upon crossing the lower critical solution temperature of PNIPAM, resulting in a macroscopic stiffening response that spans more than 3 orders of magnitude in modulus. The forces generated through collapsing PNIPAM are sufficient to drive a fluid material into a stiff gel within a few seconds. Moreover, rigidified networks dramatically stiffen in response to applied shear stress featuring power law rheology with exponents that match those of reconstituted collagen and actomyosin networks prestressed by molecular motors. This concept holds potential for the rational design of synthetic materials that are fluid at room temperature and rapidly rigidify at body temperature to form hydrogels mechanically and structurally akin to cells and tissues.

Centrosome Aurora A regulates RhoGEF ECT-2 localisation and ensures a single PAR-2 polarity axis in C. elegans embryos.

Proper establishment of cell polarity is essential for development. In the one-cell C. elegans embryo, a centrosome-localised signal provides spatial information for polarity establishment. It is hypothesised that this signal causes local inhibition of the cortical actomyosin network, and breaks symmetry to direct partitioning of the PAR proteins. However, the molecular nature of the centrosomal signal that triggers cortical anisotropy in the actomyosin network to promote polarity establishment remains elusive. Here, we discover that depletion of Aurora A kinase (AIR-1 in C. elegans) causes pronounced cortical contractions on the embryo surface, and this creates more than one PAR-2 polarity axis. This function of AIR-1 appears to be independent of its role in microtubule nucleation. Importantly, upon AIR-1 depletion, centrosome positioning becomes dispensable in dictating the PAR-2 axis. Moreover, we uncovered that a Rho GEF, ECT-2, acts downstream of AIR-1 in regulating contractility and PAR-2 localisation, and, notably, AIR-1 depletion influences ECT-2 cortical localisation. Overall, this study provides a novel insight into how an evolutionarily conserved centrosome Aurora A kinase inhibits promiscuous PAR-2 domain formation to ensure singularity in the polarity establishment axis.

Distinct Actin-Dependent Nanoscale Assemblies Underlie the Dynamic and Hierarchical Organization of E-Cadherin

Intercellular adhesion mediated by E-cadherin is pivotal in maintaining epithelial tissue integrity and for tissue morphogenesis. Adhesion requires homophilic interactions between extracellular domains of E-cadherin molecules from neighboring cells. The interaction of its cytoplasmic domains with the cortical acto-myosin network, appears to strengthen adhesion, although, it is unclear how cortical actin affects the organization and function of E-cadherin dynamically. Here we use the ectopic expression of Drosophila E-cadherin (E-cad) in larval hemocytes, which lack endogenous E-cad, to recapitulate functional cell-cell junctions in a convenient model system. We used fluorescence emission anisotropy-based microscopy and Fluorescence Correlation Spectroscopy (FCS) to probe the nanoscale organization of E-cad. We find that E-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization, similar to that reported for the adherens junction in the developing embryonic epithelial tissue. Further, we find that extra-junctional E-cad is also organized as relatively immobile nanoclusters as well as diffusive and more loosely packed oligomers and monomers. These oligomers are promoted by cis­-interactions of the ectodomain and, strikingly, their growth is constantly counteracted by cortical actomyosin. Oligomers in turn assist in generating nanoclusters that are stabilized by cortical acto-myosin. Thus, actin remodels oligomers and stabilizes nanoclusters, revealing a requirement for actin in the dynamic organization of E-cad at the nanoscale. This dynamic organization is also present at cell-cell contacts (junction), and its disruption affects junctional integrity in the hemocyte system, as well as in the embryo. Our observations uncover a hierarchical mechanism for the nanoscale organization of E-cad, which is necessary for dynamic adhesion and maintaining junctional integrity in the face of extensive remodeling.

Assessing the Contribution of Active and Passive Stresses in C. elegans Elongation.

The role of the actomyosin network is investigated in the elongation of C. elegans during embryonic morphogenesis. We present a model of active elongating matter that combines prestress and passive stress in nonlinear elasticity. Using this model we revisit recently published data from laser ablation experiments to account for why cells under contraction can lead to an opening fracture. By taking into account the specific embryo geometry, we obtain quantitative predictions for the contractile forces exerted by the molecular motors myosin II for an elongation up to 70% of the initial length. This study demonstrates the importance of active processes in embryonic morphogenesis and the interplay between geometry and nonlinear mechanics during morphological events. In particular, it outlines the role of each connected layer of the epidermis compressed by an apical extracellular matrix that distributes the stresses during elongation.

Origin, Organization, Dynamics, and Function of Actin and Actomyosin Networks at the T Cell Immunological Synapse.

The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.

Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex.

Dynamic reorganization of the actomyosin cytoskeleton allows fast modulation of the cell surface, which is vital for many cellular functions. Myosin-II motors generate the forces required for this remodeling by imparting contractility to actin networks. However, myosin-II activity might also have a more indirect contribution to cytoskeletal dynamics; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by enhancing disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but the reassembly of actin in these assays was limited by factors such as diffusional constraints and the use of stabilized actin filaments. Here, we present the reconstitution of a minimal dynamic actin cortex, where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which the actin network undergoes constant turnover. Our findings suggest a multifaceted role of myosin-II in the dynamics of the eukaryotic actin cortex. This article has an associated First Person interview with the first author of the paper.

Vimentin on the move:new developments in cell migration.

The vimentin gene ( VIM) encodes one of the 71 human intermediate filament (IF) proteins, which are the building blocks of highly ordered, dynamic, and cell type-specific fiber networks. Vimentin is a multi-functional 466 amino acid protein with a high degree of evolutionary conservation among vertebrates. Vim −/− mice, though viable, exhibit systemic defects related to development and wound repair, which may have implications for understanding human disease pathogenesis. Vimentin IFs are required for the plasticity of mesenchymal cells under normal physiological conditions and for the migration of cancer cells that have undergone epithelial–mesenchymal transition. Although it was observed years ago that vimentin promotes cell migration, the molecular mechanisms were not completely understood. Recent advances in microscopic techniques, combined with computational image analysis, have helped illuminate vimentin dynamics and function in migrating cells on a precise scale. This review includes a brief historical account of early studies that unveiled vimentin as a unique component of the cell cytoskeleton followed by an overview of the physiological vimentin functions documented in studies on Vim −/− mice. The primary focus of the discussion is on novel mechanisms related to how vimentin coordinates cell migration. The current hypothesis is that vimentin promotes cell migration by integrating mechanical input from the environment and modulating the dynamics of microtubules and the actomyosin network. These new findings undoubtedly will open up multiple avenues to study the broader function of vimentin and other IF proteins in cell biology and will lead to critical insights into the relevance of different vimentin levels for the invasive behaviors of metastatic cancer cells.

Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms

Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.

Principles of Actomyosin Regulation In Vivo

The actomyosin cytoskeleton is responsible for most force-driven processes in cells and tissues. How it assembles into the necessary structures at the right time and place is an important question. Here, we focus on molecular mechanisms of actomyosin regulation recently elucidated in animal models, and highlight several common principles that emerge. The architecture of the actomyosin network - an important determinant of its function - results from actin polymerization, crosslinking and turnover, localized myosin activation, and contractility-driven self-organization. Spatiotemporal regulation is achieved by tissue-specific expression and subcellular localization of Rho GTPase regulators. Subcellular anchor points of actomyosin structures control the outcome of their contraction, and molecular feedback mechanisms dictate whether they are transient, cyclic, or persistent.