Actomyosin Contractility Research Articles

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, their largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

Podoplanin Drives Dedifferentiation and Amoeboid Invasion of Melanoma

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilise amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by the RhoA signalling pathway. Migrastatic drugs targeting actin polymerization and contractility to inhibit invasion and metastasis are therefore a promising treatment option. To predict amoeboid invasion and metastatic potential, there is a need for biomarkers functionally linked to contractility pathways. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts, and is overexpressed in several cancer types. Here, we show that podoplanin enhances amoeboid invasion in melanoma. Expression of podoplanin in murine melanoma models drives rounded cell morphology, increasing motility and invasion in vivo. Podoplanin expression is upregulated in a subset of dedifferentiated human melanoma, and in vitro is sufficient to suppress melanogenesis and upregulate melanoma-associated markers Mitf and Pou3f2 . Together, our data indicates that podoplanin is both a potential biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

Who Needs a Contractile Actomyosin Ring? The Plethora of Alternative Ways to Divide a Protozoan Parasite.

Cytokinesis, or the division of the cytoplasm, following the end of mitosis or meiosis, is accomplished in animal cells, fungi, and amoebae, by the constriction of an actomyosin contractile ring, comprising filamentous actin, myosin II, and associated proteins. However, despite this being the best-studied mode of cytokinesis, it is restricted to the Opisthokonta and Amoebozoa, since members of other evolutionary supergroups lack myosin II and must, therefore, employ different mechanisms. In particular, parasitic protozoa, many of which cause significant morbidity and mortality in humans and animals as well as considerable economic losses, employ a wide diversity of mechanisms to divide, few, if any, of which involve myosin II. In some cases, cell division is not only myosin II-independent, but actin-independent too. Mechanisms employed range from primitive mechanical cell rupture (cytofission), to motility- and/or microtubule remodeling-dependent mechanisms, to budding involving the constriction of divergent contractile rings, to hijacking host cell division machinery, with some species able to utilize multiple mechanisms. Here, I review current knowledge of cytokinesis mechanisms and their molecular control in mammalian-infective parasitic protozoa from the Excavata, Alveolata, and Amoebozoa supergroups, highlighting their often-underappreciated diversity and complexity. Billions of people and animals across the world are at risk from these pathogens, for which vaccines and/or optimal treatments are often not available. Exploiting the divergent cell division machinery in these parasites may provide new avenues for the treatment of protozoal disease.

Protein kinase A activity is regulated by actomyosin contractility during cell migration and is required for durotaxis.

Dynamic subcellular regulation of protein kinase A (PKA) activity is important for the motile behavior of many cell types, yet the mechanisms governing PKA activity during cell migration remain largely unknown. The motility of SKOV-3 epithelial ovarian cancer (EOC) cells has been shown to be dependent both on localized PKA activity and, more recently, on mechanical reciprocity between cellular tension and extracellular matrix rigidity. Here, we investigated the possibility that PKA is regulated by mechanical signaling during migration. We find that localized PKA activity in migrating cells rapidly decreases upon inhibition of actomyosin contractility (specifically, of myosin ATPase, Rho kinase, or myosin light-chain kinase activity). Moreover, PKA activity is spatially and temporally correlated with cellular traction forces in migrating cells. Additionally, PKA is rapidly and locally activated by mechanical stretch in an actomyosin contractility-dependent manner. Finally, inhibition of PKA activity inhibits mechanically guided migration, also known as durotaxis. These observations establish PKA as a locally regulated effector of cellular mechanotransduction and as a regulator of mechanically guided cell migration.

Loss of Vimentin Enhances Cell Motility through Small Confining Spaces.

The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F-actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin-driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild-type and vimentin-null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto-myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.

Volume Adaptation Controls Stem Cell Mechanotransduction.

Recent studies have found discordant mechanosensitive outcomes when comparing 2D and 3D, highlighting the need for tools to study mechanotransduction in 3D across a wide spectrum of stiffness. A gelatin methacryloyl (GelMA) hydrogel with a continuous stiffness gradient ranging from 5 to 38 kPa was developed to recapitulate physiological stiffness conditions. Adipose-derived stem cells (ASCs) were encapsulated in this hydrogel, and their morphological characteristics and expression of both mechanosensitive proteins (Lamin A, YAP, and MRTFa) and differentiation markers (PPARγ and RUNX2) were analyzed. Low-stiffness regions (∼8 kPa) permitted increased cellular and nuclear volume and enhanced mechanosensitive protein localization in the nucleus. This trend was reversed in high stiffness regions (∼30 kPa), where decreased cellular and nuclear volumes and reduced mechanosensitive protein nuclear localization were observed. Interestingly, cells in soft regions exhibited enhanced osteogenic RUNX2 expression, while those in stiff regions upregulated the adipogenic regulator PPARγ, suggesting that volume, not substrate stiffness, is sufficient to drive 3D stem cell differentiation. Inhibition of myosin II (Blebbistatin) and ROCK (Y-27632), both key drivers of actomyosin contractility, resulted in reduced cell volume, especially in low-stiffness regions, causing a decorrelation between volume expansion and mechanosensitive protein localization. Constitutively active and inactive forms of the canonical downstream mechanotransduction effector TAZ were stably transfected into ASCs. Activated TAZ resulted in higher cellular volume despite increasing stiffness and a consistent, stiffness-independent translocation of YAP and MRTFa into the nucleus. Thus, volume adaptation as a function of 3D matrix stiffness can control stem cell mechanotransduction and differentiation.

Confinement hinders motility by inducing RhoA-mediated nuclear influx, volume expansion, and blebbing.

Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.

Phosphorylation Reduces the Mechanical Stability of the α-Catenin/ β-Catenin Complex.

The α‐catenin/β‐catenin complex serves as a critical molecular interface involved in cadherin–catenin‐based mechanosensing at the cell–cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro‐environmental perturbation. However, the mechanical stability of this complex has yet to be quantified. Here, we directly quantified the mechanical stability of the α‐catenin/β‐catenin complex and showed that it has enough mechanical stability to survive for tens to hundreds of seconds within physiological level of forces up to 10 pN. Phosphorylation or phosphotyrosine‐mimetic mutations (Y142E or/and T120E) on β‐catenin shorten the mechanical lifetime of the complex by tens of fold over the same force range. These results provide insights into the regulation of the α‐catenin/β‐catenin complex by phosphorylation.

Phosphorylation Reduces the Mechanical Stability of the α‐Catenin/ β‐Catenin Complex

AbstractThe α‐catenin/β‐catenin complex serves as a critical molecular interface involved in cadherin–catenin‐based mechanosensing at the cell–cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro‐environmental perturbation. However, the mechanical stability of this complex has yet to be quantified. Here, we directly quantified the mechanical stability of the α‐catenin/β‐catenin complex and showed that it has enough mechanical stability to survive for tens to hundreds of seconds within physiological level of forces up to 10 pN. Phosphorylation or phosphotyrosine‐mimetic mutations (Y142E or/and T120E) on β‐catenin shorten the mechanical lifetime of the complex by tens of fold over the same force range. These results provide insights into the regulation of the α‐catenin/β‐catenin complex by phosphorylation.

Actomyosin contractility scales with myoblast elongation and enhances differentiation through YAP nuclear export

Skeletal muscle fibers are formed by the fusion of mononucleated myoblasts into long linear myotubes, which differentiate and reorganize into multinucleated myofibers that assemble in bundles to form skeletal muscles. This fundamental process requires the elongation of myoblasts into a bipolar shape, although a complete understanding of the mechanisms governing skeletal muscle fusion is lacking. To address this question, we consider cell aspect ratio, actomyosin contractility and the Hippo pathway member YAP as potential regulators of the fusion of myoblasts into myotubes. Using fibronectin micropatterns of different geometries and traction force microscopy, we investigated how myoblast elongation affects actomyosin contractility. Our findings indicate that cell elongation enhances actomyosin contractility in myoblasts, which regulate their actin network to their spreading area. Interestingly, we found that the contractility of cell pairs increased after their fusion and raise on elongated morphologies. Furthermore, our findings indicate that myoblast elongation modulates nuclear orientation and triggers cytoplasmic localization of YAP, increasing evidence that YAP is a key regulator of mechanotransduction in myoblasts. Taken together, our findings support a mechanical model where actomyosin contractility scales with myoblast elongation and enhances the differentiation of myoblasts into myotubes through YAP nuclear export.

HAX1 impact on collective cell migration, cell adhesion, and cell shape is linked to the regulation of actomyosin contractility.

HAX1 protein is involved in the regulation of apoptosis, cell motility and calcium homeostasis. Its overexpression was reported in several tumors, including breast cancer. This study demonstrates that HAX1 has an impact on collective, but not single-cell migration, thus indicating the importance of cell–cell contacts for the HAX1-mediated effect. Accordingly, it was shown that HAX1 knockdown affects cell–cell junctions, substrate adhesion, and epithelial cell layer integrity. As demonstrated here, these effects can be attributed to the modulation of actomyosin contractility through changes in RhoA and septin signaling. Additionally, it was shown that HAX1 does not influence invasive potential in the breast cancer cell line, suggesting that its role in breast cancer progression may be linked instead to collective invasion of the epithelial cells but not single-cell dissemination.

Keratin intermediate filaments: intermediaries of epithelial cell migration.

Migration of epithelial cells is fundamental to multiple developmental processes, epithelial tissue morphogenesis and maintenance, wound healing and metastasis. While migrating epithelial cells utilize the basic acto-myosin based machinery as do other non-epithelial cells, they are distinguished by their copious keratin intermediate filament (KF) cytoskeleton, which comprises differentially expressed members of two large multigene families and presents highly complex patterns of post-translational modification. We will discuss how the unique mechanophysical and biochemical properties conferred by the different keratin isotypes and their modifications serve as finely tunable modulators of epithelial cell migration. We will furthermore argue that KFs together with their associated desmosomal cell-cell junctions and hemidesmosomal cell-extracellular matrix (ECM) adhesions serve as important counterbalances to the contractile acto-myosin apparatus either allowing and optimizing directed cell migration or preventing it. The differential keratin expression in leaders and followers of collectively migrating epithelial cell sheets provides a compelling example of isotype-specific keratin functions. Taken together, we conclude that the expression levels and specific combination of keratins impinge on cell migration by conferring biomechanical properties on any given epithelial cell affecting cytoplasmic viscoelasticity and adhesion to neighboring cells and the ECM.

Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction.

The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane protein nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton.

Two S. pombe septation phases differ in ingression rate, septum structure, and response to F-actin loss.

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

Differential Expression of a Classic Cadherin Directs Tissue-Level Contractile Asymmetry during Neural Tube Closure.

Embryos control force generation at tissue boundaries, but how they do so remains poorly understood. Here we show how tissue-specific expression of the type II cadherin, Cadherin2, patterns actomyosin contractility along tissue boundaries to control zippering and neural tube closure in the basal chordate, Ciona robusta. Cadherin2 is differentially expressed and homotypically enriched in neural cells along the neural/epidermal (Ne/Epi) boundary, where RhoA and myosin are activated during zipper progression. Homotypically enriched Cadherin2 sequesters the Rho GTPase-activating protein, Gap21/23, to homotypic junctions. Gap21/23 in turn redirects RhoA/myosin activity to heterotypic Ne/Epi junctions. By activating myosin II along Ne/Epi junctions ahead ofthe zipper and inhibiting myosin II along newlyformed Ne/Ne junctions behind the zipper, Cadherin2 promotes tissue-level contractile asymmetry to drive zipper progression. We propose that dynamic coupling of junction exchange to local changes in contractility may control fusion and separation of epithelia in many other contexts.

Mechanosensitive Junction Remodeling Promotes Robust Epithelial Morphogenesis

Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodeling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behavior under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviors, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodeling and continuous strain relaxation. First, junctions must overcome a critical strain threshold to trigger tension remodeling, resulting in irreversible junction length changes. Second, there is a continuous relaxation of junctional strain that removes mechanical memory from the system. This enables pulsatile contractions to further remodel cell shape via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodeling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.

The phosphatase inhibitor Sds23 regulates cell division symmetry in fission yeast.

Animal and fungal cells divide through the assembly, anchoring, and constriction of a contractile actomyosin ring (CAR) during cytokinesis. The timing and position of the CAR must be tightly controlled to prevent defects in cell division, but many of the underlying signaling events remain unknown. The conserved heterotrimeric protein phosphatase PP2A controls the timing of events in mitosis, and upstream pathways including Greatwall–Ensa regulate PP2A activity. A role for PP2A in CAR regulation has been less clear, although loss of PP2A in yeast causes defects in cytokinesis. Here, we report that Sds23, an inhibitor of PP2A family protein phosphatases, promotes the symmetric division of fission yeast cells through spatial control of cytokinesis. We found that sds23∆ cells divide asymmetrically due to misplaced CAR assembly, followed by sliding of the CAR away from its assembly site. These mutant cells exhibit delayed recruitment of putative CAR anchoring proteins including the glucan synthase Bgs1. Our observations likely reflect a broader role for regulation of PP2A in cell polarity and cytokinesis because sds23∆ phenotypes were exacerbated when combined with mutations in the fission yeast Ensa homologue, Igo1. These results identify the PP2A regulatory network as a critical component in the signaling pathways coordinating cytokinesis.

Diverse roles of non-muscle myosin II contractility in 3D cell migration.

All is flux, nothing stays still. Heraclitus of Ephesus' characterization of the universe holds true for cells within animals and for proteins within cells. In this review, we examine the dynamics of actin and non-muscle myosin II within cells, and how their dynamics power the movement of cells within tissues. The 3D environment that migrating cells encounter along their path also changes over time, and cells can adopt various mechanisms of motility, depending on the topography, mechanics and chemical composition of their surroundings. We describe the differential spatio-temporal regulation of actin and myosin II-mediated contractility in mesenchymal, lobopodial, amoeboid, and swimming modes of cell migration. After briefly reviewing the biochemistry of myosin II, we discuss the role actomyosin contractility plays in the switch between modes of 3D migration that cells use to adapt to changing environments.

The cellular and molecular mechanisms that establish the mechanics of Drosophila gastrulation.

In this review, we cover advances in the field that have contributed to our mechanistic understanding of how tissues internalize during Drosophila melanogaster gastrulation. The changes in tissue shape and architecture that are associated with mesoderm and endoderm invagination in the early Drosophila embryo are accompanied by cell shape changes which are driven by actomyosin contractility. The activation of signal transduction pathways is patterned by embryonic transcription factors, which define distinct geometries of gene expression and the tissue contractile domains. At the subcellular level, outputs from signaling pathways that activate actomyosin contractility are highly polarized and their behavior is fine-tuned by a balance of both positive and negative regulation. Cells are mechanically linked through adherens junctions, allowing forces that are generated by cells to be integrated across the tissue, ensuring coordinated cell behavior during tissue invagination. The transmission of force between cells also enables mechanical feedback whereby force generation influences both cell and cytoskeletal behavior. Finally, after tissue invagination, mesoderm cells undergo an epithelial-to-mesenchymal transition and cell spreading. We highlight studies that have utilized this model system to uncover fundamental principles at molecular-, cell-, and tissue-levels, which have contributed to our understanding of similar tissue morphogenetic processes across different organisms.