Actomyosin Contraction Research Articles

Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling.

Barrier function is critical for epithelial tissues. Epithelial cells maintain barrier function via tight junctions, which must be remodeled to allow for cell- and tissue-scale shape changes. How barrier function is maintained and remodeled as epithelial cells change shape remains unclear.The scaffolding protein Anillin is required for generating effective actomyosin contraction to reinforce damaged tight junctions; lack of reinforcement leads to repeated barrier leaks.These findings highlight a novel role for Anillin in tight junction remodeling and suggest that Anillin's ability to tune the level and duration of local Rho activation affects the contractile output.

Tubulin-Targeted Therapy in Melanoma Increases the Cell Migration Potential by Activation of the Actomyosin Cytoskeleton─An In Vitro Study.

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines─WM35 and WM266-4─representing the less and more invasive melanomas. We investigated the mechanisms of cellular processes regulating the activation of actomyosin as an effect of colchicine treatment. Additionally, we investigated the biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632) and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy (AFM) and microconstriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers, compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased the level of cancer cell invasiveness. We emphasized that a cellular pathway of Rho-ROCK-dependent actomyosin contraction is responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

Inverse blebs operate as hydraulic pumps during mouse blastocyst formation.

During preimplantation development, mouse embryos form a fluid-filled lumen. Pressurized fluid fractures cell-cell contacts and accumulates into pockets, which coarsen into a single lumen. How the embryo controls intercellular fluid movement during coarsening is unknown. Here we report inverse blebs growing into cells at adhesive contacts. Throughout the embryo we observed hundreds of inverse blebs, each filling with intercellular fluid and retracting within a minute. Inverse blebs grow due to pressure build-up resulting from fluid accumulation and cell-cell adhesion, which locally confines fluid. Inverse blebs retract due to actomyosin contraction, practically pushing fluid within the intercellular space. Importantly, inverse blebs occur infrequently at contacts formed by multiple cells, which effectively serve as fluid sinks. Manipulation of the embryo topology reveals that without sinks inverse blebs pump fluid into one another in futile cycles. We propose that inverse blebs operate as hydraulic pumps to promote luminal coarsening, thereby constituting an instrument used by cells to control fluid movement.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension

Plakophilin 4 (PKP4) is a component of cell–cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Myosin light chain phosphatase is a downstream target of Rho-kinase in endothelin-1-induced transactivation of the TGF-β receptor.

Rho-kinase (ROCK) regulates actomyosin contraction, coronary vasospasm, and cytoskeleton dynamics. ROCK and of NADPH oxidase (NOX) play an essential role in cardiovascular disease and proteoglycan synthesis, which promotes atherosclerosis by trapping low density lipoprotein. ROCK is activated by endothelin-1 (ET1) and transactivates the transforming growth factor beta receptor (TGFβR1), intensifying Smad signaling and proteoglycan production. This study aimed to identify the role of myosin light chain phosphatase (MLCP) as a downstream target of ROCK in TβR1 transactivation. Vascular smooth muscle cells were treated with ET1 and inhibitors of ROCK and MLCP were added. The phosphorylation levels of Smad2C, myosin light chain (MLC), and MLCP were monitored by western blot, and the mRNA expression of chondroitin 4-O-sulfotransferase 1 (C4ST1) was assessed by quantitative real-time PCR. We examined ROCK's role in ET1-induced TGFβR1 activation. ROCK phosphorylated MLCP at the MYPT1 T853 residue, blocked by the ROCK inhibitor Y27632. ROCK also increased MLC phosphorylation and actomyosin contraction in response to ET1, enhanced by the phosphatase inhibitor Calyculin A. Calyculin A also increased C4ST1 expression, GAG-chain synthesizing enzymes. This work suggests that ROCK is involved in ET1-mediated TβR1 activation through increased MLCP phosphorylation, which leads to Smad2C phosphorylation and stimulates C4ST1 expression.

Two Septin complexes mediate actin dynamics during cell wound repair

Cells have robust wound repair systems to prevent further damage or infection and to quickly restore cell cortex integrity when exposed to mechanical and chemical stress. Actomyosin ring formation and contraction at the wound edge are major events during closure of the plasma membrane and underlying cytoskeleton during cell wound repair. Here, we show that all five Drosophila Septins are required for efficient cell wound repair. Based on their different recruitment patterns and knockdown/mutant phenotypes, two distinct Septin complexes, Sep1/Sep2/Pnut and Sep4/Sep5/Pnut, are assembled to regulate actin ring assembly, contraction, and remodeling during the repair process. Intriguingly, we find that these two Septin complexes have different F-actin bending activities. In addition, we find that Anillin regulates the recruitment of only one of two Septin complexes upon wounding. Our results demonstrate that two functionally distinct Septin complexes work side by side to discretely regulate actomyosin ring dynamics during cell wound repair.

Confinement induces internal flows in adherent cell aggregates.

During mesenchymal migration, F-actin protrusion at the leading edge and actomyosin contraction determine the retrograde flow of F-actin within the lamella. The coupling of this flow to integrin-based adhesions determines the force transmitted to the extracellular matrix and the net motion of the cell. In tissues, motion may also arise from convection, driven by gradients in tissue-scale surface tensions and pressures. However, how migration coordinates with convection to determine the net motion of cellular ensembles is unclear. To explore this, we study the spreading of cell aggregates on adhesive micropatterns on compliant substrates. During spreading, a cell monolayer expands from the aggregate towards the adhesive boundary. However, cells are unable to stabilize the protrusion beyond the adhesive boundary, resulting in retraction of the protrusion and detachment of cells from the matrix. Subsequently, the cells move upwards and rearwards, yielding a bulk convective flow towards the centre of the aggregate. The process is cyclic, yielding a steady-state balance between outward (protrusive) migration along the surface, and 'retrograde' (contractile) flows above the surface. Modelling the cell aggregates as confined active droplets, we demonstrate that the interplay between surface tension-driven flows within the aggregate, radially outward monolayer flow and conservation of mass leads to an internal circulation.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

Paxillin phase separation promotes focal adhesion assembly and integrin signaling.

Focal adhesions (FAs) are transmembrane protein assemblies mediating cell-matrix connection. Although protein liquid-liquid phase separation (LLPS) has been tied to the organization and dynamics of FAs, the underlying mechanisms remain unclear. Here, we experimentally tune the LLPS of PXN/Paxillin, an essential scaffold protein of FAs, by utilizing a light-inducible Cry2 system in different cell types. In addition to nucleating FA components, light-triggered PXN LLPS potently activates integrin signaling and subsequently accelerates cell spreading. In contrast to the homotypic interaction-driven LLPS of PXN in vitro, PXN condensates in cells are associated with the plasma membrane and modulated by actomyosin contraction and client proteins of FAs. Interestingly, non-specific weak intermolecular interactions synergize with specific molecular interactions to mediate the multicomponent condensation of PXN and are efficient in promoting FA assembly and integrin signaling. Thus, our data establish an active role of the PXN phase transition into a condensed membrane-associated compartment in promoting the assembly/maturation of FAs.

A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow during Drosophila gastrulation

Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension

Convergent extension (CE) requires the coordinated action of the planar cell polarity (PCP) proteins1,2 and the actin cytoskeleton,3,4,5,6 but this relationship remains incompletely understood. For example, PCP signaling orients actomyosin contractions, yet actomyosin is also required for the polarized localization of PCP proteins.7,8 Moreover, the actin-regulating Septins play key roles in actin organization9 and are implicated in PCP and CE in frogs, mice, and fish5,6,10,11,12 but execute only a subset of PCP-dependent cell behaviors. Septin loss recapitulates the severe tissue-level CE defects seen after core PCP disruption yet leaves overt cell polarity intact.5 Together, these results highlight the general fact that cell movement requires coordinated action by distinct but integrated actin populations, such as lamella and lamellipodia in migrating cells13 or medial and junctional actin populations in cells engaged in apical constriction.14,15 In the context of Xenopus mesoderm CE, three such actin populations are important, a superficial meshwork known as the "node-and-cable" system,4,16,17,18 a contractile network at deep cell-cell junctions,6,19 and mediolaterally oriented actin-rich protrusions, which are present both superficially and deeply.4,19,20,21 Here, we exploited the amenability of the uniquely "two-dimensional" node and cable system to probe the relationship between PCP proteins, Septins, and the polarization of this actin network. We find that the PCP proteins Vangl2 and Prickle2 and Septins co-localize at nodes, and that the node and cable system displays a cryptic, PCP- and Septin-dependent anteroposterior (AP) polarity in its organization and dynamics.

Medioapical contractile pulses coordinated between cells regulate Drosophila eye morphogenesis.

Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.

Direct Reprogramming of Mouse Fibroblasts to Osteoblast-like Cells Using Runx2/Dlx5 Factors on Engineered Stiff Hydrogels.

Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.

Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability.

We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca2+) influx as a mechanism of trauma-induced endothelial permeability. Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca2+ signaling may play a role. Ex vivo plasma from injured patients with "Low Injury/Low Shock" (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and "High Injury/High Shock" (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca2+ channel. Live cell fluorescence microscopy and ECIS were used to assess cytosolic Ca2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain (MLC) phosphorylation and actomyosin contraction. Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca2+ increase. Cytosolic Ca2+ increase, MLC phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca2+ present. High Injury/High Shock plasma did not induce endothelial permeability without extracellular Ca2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca2+ influx and permeability. This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca2+ influx via the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca2+-targeted therapies and interventions in the care of severely injured patients.

CSIG-27. FLUID SHEAR STRESS ACTIVATES A MECHANO-METASTATIC CASCADE TO PROMOTE MEDULLOBLASTOMA METASTASIS

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Dissemination of MB cells into cerebrospinal fluid (CSF) and blood initiates metastasis in the central nervous system, which significantly worsens patient prognosis. Disseminated MB cells survive the nutrient-deprived CSF environment and are subjected to fluid shear stress (FSS) by CSF flow. Whether and how FSS contributes to MB metastasis is completely unknown. We computationally simulated FSS dynamics in MB patients using MRI-informed CSF flow models. Based on CSF flow rates and force magnitudes in patients, we engineered two FSS application systems: (1) an orbital shaker system that applies FSS to large populations of cells, and (2) a microfluidic system compatible with live-cell and high-resolution imaging. By applying physiologically relevant FSS to MB cells, we discovered that FSS-treated MB cells metastasize more frequently and form larger metastatic tumors along the spinal cords of mice. Mechanistically, FSS induces actomyosin contraction, which promotes cell clustering, glucose transporter 1 (GLUT1) localization at the cell surface, and GLUT1-dependent glucose uptake. FSS elevates intracellular calcium through mechanosensitive ion channel PIEZO2, which is required for FSS-induced actomyosin contraction, cell clustering, and GLUT1 activity. Genetically perturbing PIEZO2 or pharmacologically inhibiting GLUT1 robustly suppresses MB metastasis in mice. Collectively, we discover that MB cells perceive FSS, define an FSS-activated mechano-metastatic cascade, and demonstrate that this cascade is clinically targetable to mitigate MB metastasis.

Local glycolysis fuels actomyosin contraction during axonal retraction.

In response to repulsive cues, axonal growth cones can quickly retract. This requires the prompt activity of contractile actomyosin, which is formed by the non-muscle myosin II (NMII) bound to actin filaments. NMII is a molecular motor that provides the necessary mechanical force at the expense of ATP. Here, we report that this process is energetically coupled to glycolysis and is independent of cellular ATP levels. Induction of axonal retraction requires simultaneous generation of ATP by glycolysis, as shown by chemical inhibition and genetic knock-down of GAPDH. Co-immunoprecipitation and proximal-ligation assay showed that actomyosin associates with ATP-generating glycolytic enzymes and that this association is strongly enhanced during retraction. Using microfluidics, we confirmed that the energetic coupling between glycolysis and actomyosin necessary for axonal retraction is localized to the growth cone and near axonal shaft. These results indicate a tight coupling between on-demand energy production by glycolysis and energy consumption by actomyosin contraction suggesting a function of glycolysis in axonal guidance.

Vinculin is required for interkinetic nuclear migration (INM) and cell cycle progression.

Vinculin is an actin-binding protein (ABP) that strengthens the connection between the actin cytoskeleton and adhesion complexes. It binds to β-catenin/N-cadherin complexes in apical adherens junctions (AJs), which maintain cell-to-cell adhesions, and to talin/integrins in the focal adhesions (FAs) that attach cells to the basal membrane. Here, we demonstrate that β-catenin targets vinculin to the apical AJs and the centrosome in the embryonic neural tube (NT). Suppression of vinculin slows down the basal-to-apical part of interkinetic nuclear migration (BAINM), arrests neural stem cells (NSCs) in the G2 phase of the cell cycle, and ultimately dismantles the apical actin cytoskeleton. In the NSCs, mitosis initiates when an internalized centrosome gathers with the nucleus during BAINM. Notably, our results show that the first centrosome to be internalized is the daughter centrosome, where β-catenin and vinculin accumulate, and that vinculin suppression prevents centrosome internalization. Thus, we propose that vinculin links AJs, the centrosome, and the actin cytoskeleton where actomyosin contraction forces are required.

Syndapin and GTPase RAP-1 control endocytic recycling via RHO-1 and non-muscle myosin II

After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required invivo for basolateral endocytic recycling in the C.elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling invivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C.elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.

Autoregulatory mission impossible: when afferent arterioles lose contractility

The myogenic response of afferent arterioles is a key autoregulatory mechanism that protects the glomeruli from barotrauma. Afferent arteriolar smooth muscle cells contract to increased intraluminal pressure through mechanosensitive cation channels and interactions between integrin and extracellular matrix that trigger calcium-dependent actomyosin contraction. The study by Feng etal. provides evidence supporting the concept that increased matrix metalloproteinase 9 in kidney microvessels of Dahl salt-sensitive rats interferes with integrin-matrix binding and promotes phenotypic transformation of afferent arterioles, causing loss of myogenic constriction and hypertensive nephropathy.