Actomyosin Network Research Articles

Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling

Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.

CRX haploinsufficiency compromises photoreceptor precursor translocation and differentiation in human retinal organoids

BackgroundThe CRX-associated autosomal dominant retinopathies suggest a possible pathogenic mechanism of gene haploinsufficiency. However, based on reported human patient cases and studies with mouse models, it is hard to confirm the specific weight of haploinsufficiency in pathogenesis due to the interspecies gaps between gene expression and function.MethodsWe created monoallelic CRX by replacing one allele with tdTomato in human embryonic stem cells (hESCs) and subsequently dissect pathogenesis in hESCs-derived retinal organoids. We used transcriptome and immunofluorescence analyses to dissect phenotypic differences between CRX-monoallelic knockout and control wildtype organoids. For location analysis of CRX+ cells, a CRX-expression-tracing system was constructed in control hESCs. We implemented long-term live-cell imaging to describe the translocation of CRX+ cells between two groups in early organoid differentiation. The expression pattern of these dynamic differences was validated using RNA-seq and immunofluorescence assays.ResultsWe identified delayed differentiation of outer nuclear layer (ONL) stratification along with thinner ONL, serious loss of photoreceptor outer segments, as well as downregulated expression of gene for phototransduction and inner/outer segment formation. By live-cell imaging and immunostaining, we observed the overtension of actomyosin network and the arrested translocation of monoallelic CRX+ cells in the early stage of retinal differentiation.ConclusionsWe confirmed that gene haploinsufficiency is the mechanism for the dominant pathogenicity of CRX and discovered that CRX regulated postmitotic photoreceptor precursor translocation in addition to its specification of photoreceptor cell fates during human retinal development. These findings revealed a new underlying mechanism of CRX dominant pathogenesis and provided a new clue for the treatment of CRX-associated human retinopathies.

Dynamic high-order buckling and spontaneous recovery of active epithelial tissues

Active epithelial tissues can adapt to quasi-static compressive forces through buckling instability, but their responses to dynamic forces at shorter timescales remain elusive. We firstly establish a cytoarchitectural model that can accurately capture the experimentally observed high-order buckling and postbuckling (e.g., spontaneous flattening and stress recovery) behaviors of epithelia under fast compression. It is found that the stress evolution of epithelia can be divided into three stages: loading, phase transition, and stress recovery. In the loading stage, we observe the high-order instability with a buckling mode highly correlated with the strain rate, and derive its analytical relation, showing that the rate-dependent buckling mode is quantitatively determined by the viscoelastic and geometrical characteristics of epithelia. In the phase transition and stress recovery stages, we demonstrate that the postbuckling process is governed by the active tension generated by the actomyosin network. Furthermore, by proposing a minimal model, we obtain the explicit solutions of the flattening time and stress recovery extent as functions of the applied strain or strain rate, which are in quantitative agreement with our simulations and relevant experiments. In addition, depending on the stress evolution route, we construct a universal phase diagram for the morphology evolution of the epithelia in a wide range of strain and strain rate. This study elucidates the dominative roles of the activity and rheological characteristics of active soft materials in their dynamic mechanical behaviors, offering an approach for studying the complex morphology evolution.

Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division.

Friction patterns guide actin network contraction

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation.

Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. Here, we demonstrate that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. We show that while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.

Molecular-scale substrate anisotropy, crowding and division drive collective behaviours in cell monolayers.

The ability of cells to reorganize in response to external stimuli is important in areas ranging from morphogenesis to tissue engineering. While nematic order is common in biological tissues, it typically only extends to small regions of cells interacting via steric repulsion. On isotropic substrates, elongated cells can co-align due to steric effects, forming ordered but randomly oriented finite-size domains. However, we have discovered that flat substrates with nematic order can induce global nematic alignment of dense, spindle-like cells, thereby influencing cell organization and collective motion and driving alignment on the scale of the entire tissue. Remarkably, single cells are not sensitive to the substrate's anisotropy. Rather, the emergence of global nematic order is a collective phenomenon that requires both steric effects and molecular-scale anisotropy of the substrate. To quantify the rich set of behaviours afforded by this system, we analyse velocity, positional and orientational correlations for several thousand cells over days. The establishment of global order is facilitated by enhanced cell division along the substrate's nematic axis, and associated extensile stresses that restructure the cells' actomyosin networks. Our work provides a new understanding of the dynamics of cellular remodelling and organization among weakly interacting cells.

Architecture of the cortical actomyosin network driving apical constriction in C. elegans.

Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Our results demonstrate that C. elegans endodermal precursor cells apically constrict using a mixed-polarity actin filament network and with myosin and a myosin activator distributed throughout the network. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction.

A cell membrane model that reproduces cortical flow-driven cell migration and collective movement.

Many fundamental biological processes are dependent on cellular migration. Although the mechanical mechanisms of single-cell migration are relatively well understood, those underlying migration of multiple cells adhered to each other in a cluster, referred to as cluster migration, are poorly understood. A key reason for this knowledge gap is that many forces-including contraction forces from actomyosin networks, hydrostatic pressure from the cytosol, frictional forces from the substrate, and forces from adjacent cells-contribute to cell cluster movement, making it challenging to model, and ultimately elucidate, the final result of these forces. This paper describes a two-dimensional cell membrane model that represents cells on a substrate with polygons and expresses various mechanical forces on the cell surface, keeping these forces balanced at all times by neglecting cell inertia. The model is discrete but equivalent to a continuous model if appropriate replacement rules for cell surface segments are chosen. When cells are given a polarity, expressed by a direction-dependent surface tension reflecting the location dependence of contraction and adhesion on a cell boundary, the cell surface begins to flow from front to rear as a result of force balance. This flow produces unidirectional cell movement, not only for a single cell but also for multiple cells in a cluster, with migration speeds that coincide with analytical results from a continuous model. Further, if the direction of cell polarity is tilted with respect to the cluster center, surface flow induces cell cluster rotation. The reason why this model moves while keeping force balance on cell surface (i.e., under no net forces from outside) is because of the implicit inflow and outflow of cell surface components through the inside of the cell. An analytical formula connecting cell migration speed and turnover rate of cell surface components is presented.

A deep learning framework for quantitative analysis of actin microridges

Microridges are evolutionarily conserved actin-rich protrusions present on the apical surface of squamous epithelial cells. In zebrafish epidermal cells, microridges form self-evolving patterns due to the underlying actomyosin network dynamics. However, their morphological and dynamic characteristics have remained poorly understood owing to a lack of computational methods. We achieved ~95% pixel-level accuracy with a deep learning microridge segmentation strategy enabling quantitative insights into their bio-physical-mechanical characteristics. From the segmented images, we estimated an effective microridge persistence length of ~6.1 μm. We discovered the presence of mechanical fluctuations and found relatively greater stresses stored within patterns of yolk than flank, indicating distinct regulation of their actomyosin networks. Furthermore, spontaneous formations and positional fluctuations of actin clusters within microridges were associated with pattern rearrangements over short length/time-scales. Our framework allows large-scale spatiotemporal analysis of microridges during epithelial development and probing of their responses to chemical and genetic perturbations to unravel the underlying patterning mechanisms.

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT.

Epithelial-to-mesenchymal transition (EMT) is a fundamental process whereby epithelial cells acquire mesenchymal phenotypes and the ability to migrate. EMT is the hallmark of gastrulation, an evolutionarily conserved developmental process. In mammals, epiblast cells ingress at the primitive streak to form mesoderm. Cells ingress and exit the epiblast epithelial layer and the associated EMT is dynamically regulated and involves a stereotypical sequence of cell behaviors. 3D time-lapse imaging of gastrulating mouse embryos combined with cell and tissue scale data analyses revealed the asynchronous ingression of epiblast cells at the primitive streak. Ingressing cells constrict their apical surfaces in a pulsed ratchet-like fashion through asynchronous shrinkage of apical junctions. A quantitative analysis of the distribution of apical proteins revealed the anisotropic and reciprocal enrichment of members of the actomyosin network and Crumbs2 complexes, potential regulators of asynchronous shrinkage of cell junctions. Loss of function analyses demonstrated a requirement for Crumbs2 in myosin II localization and activity at apical junctions, and as a candidate regulator of actomyosin anisotropy.

Inferring scale-dependent non-equilibrium activity using carbon nanotubes.

In living systems, irreversible, yet stochastic, molecular interactions form multiscale structures (such as cytoskeletal networks), which mediate processes (such as cytokinesis and cellular motility) in a close relationship between the structure and function. However, owing to a lack of methods to quantify non-equilibrium activity, their dynamics remain poorly characterized. Here, by measuring the time-reversal asymmetry encoded in the conformational dynamics of filamentous single-walled carbon nanotubes embedded in the actomyosin network of Xenopus egg extract, we characterize the multiscale dynamics of non-equilibrium activity encoded in bending-mode amplitudes. Our method is sensitive to distinct perturbations to the actomyosin network and the concentration ratio of adenosine triphosphate to adenosine diphosphate. Thus, our method can dissect the functional coupling of microscopic dynamics to the emergence of larger scale non-equilibrium activity. We relate the spatiotemporal scales of non-equilibrium activity to the key physical parameters of a semiflexible filament embedded in a non-equilibrium viscoelastic environment. Our analysis provides a general tool to characterize steady-state non-equilibrium activity in high-dimensional spaces.

A Typical Workflow to Simulate Cytoskeletal Systems.

Many cytoskeletal systems are now sufficiently well known to permit their precise quantitative modeling. Microtubule and actin filaments are well characterized, and the associated proteins are often known, as well as their abundance and the interactions between these elements. Thus, computer simulations can be used to investigate the collective behavior of the system precisely, in a way that is complementary to experiments. Cytosim is an Open Source cytoskeleton simulation suite designed to handle large systems of flexible filaments with associated proteins such as molecular motors. It also offers the possibility to simulate passive crosslinkers, diffusible crosslinkers, nucleators, cutters, and discrete versions of the motors that only step on unoccupied lattice sites on a filament. Other objects complement the filaments by offering spherical or more complicated geometry that can be used to represent chromosomes, the nucleus, or vesicles in the cell. Cytosim offers simple command-line tools for running a simulation and displaying its results, which are versatile and do not require programming skills. In this workflow, step-by-step instructions are given to i) install the necessary environment on a new computer, ii) configure Cytosim to simulate the contraction of a 2D actomyosin network, and iii) produce a visual representation of the system. Next, the system is probed by systematically varying a key parameter: the number of crosslinkers. Finally, the visual representation of the system is complemented by the numerical quantification of contractility to view, in a graph, how contractility depends on the composition of the system. Overall, these different steps constitute a typical workflow that can be applied with few modifications to tackle many other problems in the cytoskeletal field.

Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks.

During morphogenesis, large-scale changes of tissue primordia are coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. We show that the single Drosophila Alp/Enigma-family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. We reveal that Zasp52 contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin-binding activity. Using endogenously-tagged lines, we identify that Zasp52 interacts with junctional components, including APC2, Polychaetoid and Sidekick, and actomyosin regulators. Analysis of zasp52 mutant embryos reveals that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur where actomyosin cables are found during embryogenesis, and in vivo and in silico analyses suggest a model whereby supracellular Zasp52-containing cables aid to insulate morphogenetic changes from one another.

Zyxin contributes to coupling between cell junctions and contractile actomyosin networks during apical constriction

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and β-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved.

State transitions of a confined actomyosin system controlled through contractility and polymerization rate

Pattern formation induced by symmetry breaking is a fundamental concept underlying biological phenomena across different scales, from single cells to tissues. However, the mechanics behind the pattern formation of the actomyosin system remains elusive due to complex biochemical regulations in living cells. In this study, we report the transition between distinct patterns of cytoplasmic actomyosin networks: steady actin flow and periodic actin waves, which are confined to a quasi-two-dimensional cell-like compartment. By combining molecular perturbations and numerical simulations of the active fluid model, we show that contractility and actin polymerization rate are the critical factors for the state transition from the steady actin flow to periodic actin waves. These patterns emerge either when active stress outweighs the diffusive relaxation of actin filaments or when the actin polymerization rate is sufficiently slow to accumulate actin filaments close to the surface of the circular confinement. Furthermore, our active fluid model predicts that the spatial heterogeneity at the onset of contraction leads to a rotational actin wave, which is stable only at the phase boundary between the steady actin flow and periodic actin waves. This study provides an integrative understanding of the distinct pattern formation of active gels confined in cell-sized spaces.

Two-point optical manipulation reveals mechanosensitive remodeling of cell–cell contacts in vivo

Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding our understanding of how mechanical stresses remodel cell-cell contacts. Here, we develop a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows us to produce junction extension and shrinkage through different push and pull manipulations at the edges of junctions. We use these observations to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, we show that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether our work provides insight into how stress produces efficient deformation of cell-cell contacts invivo and identifies unanticipated mechanosensitive features of their remodeling.

Bending actin filaments: twists of fate.

In many cellular contexts, intracellular actomyosin networks must generate directional forces to carry out cellular tasks such as migration and endocytosis, which play important roles during normal developmental processes. A number of different actin binding proteins have been identified that form linear or branched actin, and that regulate these filaments through activities such as bundling, crosslinking, and depolymerization to create a wide variety of functional actin assemblies. The helical nature of actin filaments allows them to better accommodate tensile stresses by untwisting, as well as to bend to great curvatures without breaking. Interestingly, this latter property, the bending of actin filaments, is emerging as an exciting new feature for determining dynamic actin configurations and functions. Indeed, recent studies using in vitro assays have found that proteins including IQGAP, Cofilin, Septins, Anillin, α-Actinin, Fascin, and Myosins-alone or in combination-can influence the bending or curvature of actin filaments. This bending increases the number and types of dynamic assemblies that can be generated, as well as the spectrum of their functions. Intriguingly, in some cases, actin bending creates directionality within a cell, resulting in a chiral cell shape. This actin-dependent cell chirality is highly conserved in vertebrates and invertebrates and is essential for cell migration and breaking L-R symmetry of tissues/organs. Here, we review how different types of actin binding protein can bend actin filaments, induce curved filament geometries, and how they impact on cellular functions.

Cell cycle-dependent palmitoylation of protocadherin 7 by ZDHHC5 promotes successful cytokinesis.

Cell division requires dramatic reorganization of the cell cortex, which is primarily driven by the actomyosin network. We previously reported that protocadherin 7 (PCDH7) gets enriched at the cell surface during mitosis, which is required to build up the full mitotic rounding pressure. Here, we report that PCDH7 interacts with and is palmitoylated by the palmitoyltransferase, ZDHHC5. PCDH7 and ZDHHC5 colocalize at the mitotic cell surface and translocate to the cleavage furrow during cytokinesis. The localization of PCDH7 depends on the palmitoylation activity of ZDHHC5. Silencing PCDH7 increases the percentage of multinucleated cells and the duration of mitosis. Loss of PCDH7 expression correlates with reduced levels of active RhoA and phospho-myosin at the cleavage furrow. This work uncovers a palmitoylation-dependent translocation mechanism for PCDH7, which contributes to the reorganization of the cortical cytoskeleton during cell division.