Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion

Epithelia can eliminate apoptotic cells by apical extrusion. This is a complex morphogenetic event where expulsion of the apoptotic cell is accompanied by rearrangement of its immediate neighbors to form a rosette. A key mechanism for extrusion is constriction of an actomyosin network that neighbor cells form at their interface with the apoptotic cell. Here we report a complementary process of cytoskeletal relaxation that occurs when cortical contractility is down-regulated at the junctions between those neighbor cells themselves. This reflects a mechanosensitive Src family kinase (SFK) signaling pathway that is activated in neighbor cells when the apoptotic cell relaxes shortly after injury. Inhibiting SFK signaling blocks both the expulsion of apoptotic cells and the rosette formation among their neighbor cells. This reveals the complex pattern of spatially distinct contraction and relaxation that must be established in the neighboring epithelium for apoptotic cells to be extruded.     

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6–atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical–basal polarity in forming epithelia, the Cdc42–Par6–aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.     

Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT.     

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.     

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.     

Cell Shape Dynamics Reveal Balance of Elasticity and Contractility in Peripheral Arcs

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension.     

Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.     

Epithelial cell cluster size affects force distribution in response to EGF-induced collective contractility

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.     

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.     

Integrins uncouple Src-induced morphological and oncogenic transformation

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.     

Stimulation of integrin-mediated cell contractility by fibronectin polymerization

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.     

Dynamic modes of the cortical actomyosin gel during cell locomotion and division

Tight regulation of the contractility of the actomyosin cortex is essential for proper cell locomotion and division. Enhanced contractility leads, for example, to aberrations in the positioning of the mitotic spindle or to anomalous migration modes that allow tumor cells to escape anti-dissemination treatments. Spherical membrane protrusions called blebs occasionally appear during cell migration, cell division or apoptosis. We have shown that the cortex ruptures at sites where actomyosin cortical contractility is increased, leading to the formation of blebs. Here, we propose that bleb formation, which releases cortical tension, can be used as a reporter of cortical contractility. We go on to analyze the implications of spontaneous cortical contractile behaviors on cell locomotion and division and we particularly emphasize that variations in actomyosin contractility can account for a variety of migration modes.     

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.     

Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila melanogaster, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. We show that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase–binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation.     

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.     

The tumor suppressor adenomatous polyposis coli controls the direction in which a cell extrudes from an epithelium

Despite high rates of cell death, epithelia maintain intact barriers by squeezing dying cells out using a process termed cell extrusion. Cells can extrude apically into the lumen or basally into the tissue the epithelium encases, depending on whether actin and myosin contract at the cell base or apex, respectively. We previously found that microtubules in cells surrounding a dying cell target p115 RhoGEF to the actin cortex to control where contraction occurs. However, what controls microtubule targeting to the cortex and whether the dying cell also controls the extrusion direction were unclear. Here we find that the tumor suppressor adenomatous polyposis coli (APC) controls microtubule targeting to the cell base to drive apical extrusion. Whereas wild-type cells preferentially extrude apically, cells lacking APC or expressing an oncogenic APC mutation extrude predominantly basally in cultured monolayers and zebrafish epidermis. Thus APC is essential for driving extrusion apically. Surprisingly, although APC controls microtubule reorientation and attachment to the actin cortex in cells surrounding the dying cell, it does so by controlling actin and microtubules within the dying cell. APC disruptions that are common in colon and breast cancer may promote basal extrusion of tumor cells, which could enable their exit and subsequent migration.     

Mechanical tension induces lateral movement of intramembrane components of the tight junction: studies on mouse mammary cells in culture

Occluding junctions of mammary epithelial cells in nonproliferating primary culture occasionally display an atypical pattern of intramembrane strands oriented predominantly perpendicular, instead of roughly parallel, to the apical border of the junction. To test whether the orienting influence was a centripetal cytoskeletal tension often observed in epithelial sheets on fixed substrates, we seeded cells at low density; this allows them to spread maximally while forming a barely confluent pavement. The result was a fourfold increase in the percentage of junctions with the strongly aligned, atypical pattern. Closely similar configurations were observed as the earliest detectable effect of chelation of extracellular Ca++, which induced pronounced centripetal contraction of the cell body. Externally imposed tension, applied so as to stretch cells in one direction only, affected the positions of strands in stretched junctions as might be predicted, by flattening their undulations, increasing their alignment parallel to the apical border. Thus mechanical tension alone, whether inherent in the cytoskeleton or imposed on the cell surface by exogenous force, can cause coordinate lateral displacement of macromolecular assemblies within the membranes of both joined cells.     

Cellular contractility and substrate elasticity: a numerical investigation of the actin cytoskeleton and cell adhesion

Numerous experimental studies have established that cells can sense the stiffness of underlying substrates and have quantified the effect of substrate stiffness on stress fibre formation, focal adhesion area, cell traction, and cell shape. In order to capture such behaviour, the current study couples a mixed mode thermodynamic and mechanical framework that predicts focal adhesion formation and growth with a material model that predicts stress fibre formation, contractility, and dissociation in a fully 3D implementation. Simulations reveal that SF contractility plays a critical role in the substrate-dependent response of cells. Compliant substrates do not provide sufficient tension for stress fibre persistence, causing dissociation of stress fibres and lower focal adhesion formation. In contrast, cells on stiffer substrates are predicted to contain large amounts of dominant stress fibres. Different levels of cellular contractility representative of different cell phenotypes are found to alter the range of substrate stiffness that cause the most significant changes in stress fibre and focal adhesion formation. Furthermore, stress fibre and focal adhesion formation evolve as a cell spreads on a substrate and leading to the formation of bands of fibres leading from the cell periphery over the nucleus. Inhibiting the formation of FAs during cell spreading is found to limit stress fibre formation. The predictions of this mutually dependent material-interface framework are strongly supported by experimental observations of cells adhered to elastic substrates and offer insight into the inter-dependent biomechanical processes regulating stress fibre and focal adhesion formation.