Local VE-cadherin mechanotransduction triggers long-ranged remodeling of endothelial monolayers.
ABSTRACT: In this study, we present results demonstrating that mechanotransduction by vascular endothelial cadherin (VE-cadherin, also known as CDH5) complexes in endothelial cells triggers local cytoskeletal remodeling, and also activates global signals that alter peripheral intercellular junctions and disrupt cell-cell contacts far from the site of force application. Prior studies have documented the impact of actomyosin contractile forces on adherens junction remodeling, but the role of VE-cadherin in force sensation and its ability to influence endothelial cell and tissue mechanics globally have not been demonstrated. Using mechanical manipulation of VE-cadherin bonds and confocal imaging, we demonstrate VE-cadherin-based mechanotransduction. We then demonstrate that it requires homophilic VE-cadherin ligation, an intact actomyosin cytoskeleton, Rho-associated protein kinase 1 (ROCK1) and phosphoinositide 3-kinase. VE-cadherin-mediated mechanotransduction triggered local actin and vinculin recruitment, as well as global signals that altered focal adhesions and disrupted peripheral intercellular junctions. Confocal imaging revealed that VE-cadherin-specific changes appear to propagate across cell junctions to disrupt distant inter-endothelial junctions. These results demonstrate the central role of VE-cadherin adhesions and the actomyosin cytoskeleton within an integrated, mechanosensitive network that both induces local cytoskeletal remodeling at the site of force application and regulates the global integrity of endothelial tissues.
Project description:The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.
Project description:Mechanical forces drive the remodeling of tissues during morphogenesis. This relies on the transmission of forces between cells by cadherin-based adherens junctions, which couple the force-generating actomyosin cytoskeletons of neighboring cells. Moreover, components of cadherin adhesions adopt force-dependent conformations that induce changes in the composition of adherens junctions, enabling transduction of mechanical forces into an intracellular response. Cadherin mechanotransduction can mediate reinforcement of cell-cell adhesions to withstand forces but also induce biochemical signaling to regulate cell behavior or direct remodeling of cell-cell adhesions to enable cell rearrangements. By transmission and transduction of mechanical forces, cadherin adhesions coordinate cellular behaviors underlying morphogenetic processes of collective cell migration, cell division, and cell intercalation. Here, we review recent advances in our understanding of this central role of cadherin adhesions in force-dependent regulation of morphogenesis.
Project description:Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the ?-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.
Project description:Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes.
Project description:Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell–cell junctions. Both Arp2/3 complex–dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex–positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin–NMII bundles are located more distally from the VE-cadherin–rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push–pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes.
Project description:The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.
Project description:Fluid shear stress due to blood flow on the vascular endothelium regulates blood vessel development, remodeling, physiology, and pathology [1, 2]. A complex consisting of PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs) that resides at endothelial cell-cell junctions transduces signals important for flow-dependent vasodilation, blood vessel remodeling, and atherosclerosis. PECAM-1 transduces forces to activate src family kinases (SFKs), which phosphorylate and transactivate VEGFRs [3-5]. By contrast, VE-cadherin functions as an adaptor that interacts with VEGFRs through their respective cytoplasmic domains and promotes VEGFR activation in flow . Indeed, shear stress triggers rapid increases in force across PECAM-1 but decreases the force across VE-cadherin, in close association with downstream signaling . Interestingly, VE-cadherin cytoplasmic tyrosine Y658 can be phosphorylated by SFKs , which is maximally induced by low shear stress in vitro and in vivo . These considerations prompted us to address the involvement of VE-cadherin cytoplasmic tyrosines in flow sensing. We found that phosphorylation of a small pool of VE-cadherin on Y658 is essential for flow sensing through the junctional complex. Y658 phosphorylation induces dissociation of p120ctn, which allows binding of the polarity protein LGN. LGN is then required for multiple flow responses in vitro and in vivo, including activation of inflammatory signaling at regions of disturbed flow, and flow-dependent vascular remodeling. Thus, endothelial flow mechanotransduction through the junctional complex is mediated by a specific pool of VE-cadherin that is phosphorylated on Y658 and bound to LGN.
Project description:Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with ?-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly.
1000-01-01 | S-EPMC4150062 | BioStudies
Project description:Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+-signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ?-catenin/plakoglobin mask VE-cadherin-Y731, and leukocyte-docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin mediated mechanical force exerted on the VE-cadherin-catenin complex.
Project description:Maintenance and remodeling of endothelial cell junctions critically depend on the VE-cadherin/catenin complex and its interaction with the actin filament cytoskeleton. Here we demonstrate that local lack of vascular endothelial (VE)-cadherin at established cell junctions causes actin-driven and actin-related protein 2/3 complex (ARP2/3)-controlled lamellipodia to appear intermittently at those sites. Lamellipodia overlap the VE-cadherin-free adjacent plasma membranes and facilitate formation of new VE-cadherin adhesion sites, which quickly move into the junctions, driving VE-cadherin dynamics and remodeling. Inhibition of the ARP2/3 complex by expression of the N-WASP (V)CA domain or application of two ARP2/3 inhibitors, CK-548 and CK-666, blocks VE-cadherin dynamics and causes intercellular gaps. Furthermore, expression of carboxy-terminal-truncated VE-cadherin increases the number of ARP2/3-controlled lamellipodia, whereas overexpression of wild-type VE-cadherin largely blocks it and decreases cell motility. The data demonstrate a functional interrelationship between VE-cadherin-mediated cell adhesion and actin-driven, ARP2/3-controlled formation of new VE-cadherin adhesion sites via intermittently appearing lamellipodia at established cell junctions. This coordinated mechanism controls VE-cadherin dynamics and cell motility and maintains monolayer integrity, thus potentially being relevant in disease and angiogenesis.