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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. Refereed/Peer-reviewed

Refereed/Peer-reviewed

Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation.1 Several different mechanisms exist for apoptotic clearance, including efferocytosis2,3 and apical extrusion.4,5 We found that extrusion was the first-line response to apoptosis in cultured monolayers and in zebrafish epidermis. During extrusion, the apoptotic cell elicited active lamellipodial protrusions and assembly of a contractile extrusion ring in its neighbors. Depleting E-cadherin compromised both the contractile ring and extrusion, implying that a cadherin-dependent pathway allows apoptotic cells to engage their neighbors for extrusion. We identify RhoA as the cadherin-dependent signal in the neighbor cells and show that it is activated in response to contractile tension from the apoptotic cell. This mechanical stimulus is conveyed by a myosin-VI-dependent mechanotransduction pathway that is necessary both for extrusion and to preserve the epithelial barrier when apoptosis was stimulated. Earlier studies suggested that release of sphingosine-1-phosphate (S1P) from apoptotic cells might define where RhoA was activated. However, we found that, although S1P is necessary for extrusion, its contribution does not require a localized source of S1P in the epithelium. We therefore propose a unified view of how RhoA is stimulated to engage neighbor cells for apoptotic extrusion. Here, tension-sensitive mechanotransduction is the proximate mechanism that activates RhoA specifically in the immediate neighbors of apoptotic cells, but this also must be primed by S1P in the tissue environment. Together, these elements provide a coincidence detection system that confers robustness on the extrusion response. Refereed/Peer-reviewed

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P₂) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P₂, or disabling the interaction between FMNL2 and PtdIns(4,5)P₂. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton. Refereed/Peer-reviewed

Forces play diverse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development., Mechanical forces play a crucial role during morphogenesis, but how these are sensed and transduced in vivo is not fully understood. Here the authors apply a FRET tension sensor to live zebrafish and study changes in VE-cadherin tension at endothelial cell-cell junctions during arterial maturation.

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across alpha E-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia. Refereed/Peer-reviewed

ROCK1 is the prominent isoform responsible for molecular organization of epithelial zonula adherens (ZA) and its contractile properties. ROCK1 selectively localizes NMIIA to ZA and supports cortical tension and GTP-Rho at the ZA. NMIIA, in a feedback loop, promotes cortical localization of ROCK1., Rho kinases (ROCK1 and ROCK2) function downstream of the small GTPase RhoA to drive actomyosin cytoskeletal remodeling. It has often been believed that ROCK1 and ROCK2 may be functionally redundant, as they share a highly conserved kinase domain. However, in this study, we report differential functional effects for these ROCKs at the epithelial zonula adherens (ZA). Using specific siRNA, we found that ROCK1 depletion disrupted cadherin organization at the ZA, accompanied by loss of F-actin and NMIIA, whereas ROCK2 knockdown had no significant effect. Further, ROCK1, but not ROCK2, was necessary to stabilize GTP-RhoA at the ZA, thereby sustaining junctional tension and inhibiting intraepithelial cell movement. We also found that nonmuscle myosin IIA is a major determinant of ROCK1 cortical stability. Thus, despite sharing the catalytic domain with ROCK2, ROCK1 appears to be the dominant kinase essential for junctional integrity and contractile tension at epithelial ZA.

The ability of mesenchymal stem cells to sense nanoscale variations in extracellular matrix (ECM) compositions in their local microenvironment is crucial to their survival and their fate; however, the underlying molecular mechanisms defining how such fates are temporally modulated remain poorly understood. In this work, we have utilized self-assembled block copolymer surfaces to present nanodomains of an adhesive peptide found in many ECM proteins at different lateral spacings (from 30 to 60 nm) and studied the temporal response (2 h to 14 days) of human mesenchymal stem cells (hMSCs) using a panel of real-time localization and activity biosensors. Our findings revealed that within the first 4 to 24 h postadhesion and spreading, hMSCs on smaller nanodomain spacings recruit more activated FAK and Src proteins to produce larger, longer-lived, and increased numbers of focal adhesions (FAs). The adhesions formed on smaller nanospacings rapidly recruit higher amounts of nonmuscle myosin IIA and vinculin and experience tension forces (by >5 pN/FA) significantly higher than those observed on larger nanodomain spacings. The transmission of higher levels of tension into the cytoskeleton at short times was accompanied by higher Rac1, cytosolic β-catenin, and nuclear localization of YAP/TAZ and RUNX2, which together biased the commitment of hMSCs to an osteogenic fate. This investigation provides mechanistic insights to confirm that smaller lateral spacings of adhesive nanodomains alter hMSC mechanosensing and biases mechanotransduction at short times via differential coupling of FAK/Src/Rac1/myosin IIA/YAP/TAZ signaling pathways to support longer-term changes in stem cell differentiation and state Refereed/Peer-reviewed

RhoA stimulates cell contractility by recruiting downstream effectors to the cortical plasma membrane.We now show that direct binding by anillin is required for effective signaling: this antagonizes the otherwise labile membrane association of GTP-RhoA to promote effect or recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membranephosphoinositide-4,5-P2 (PIP2) to promote signaling.We propose and test a sequential pathway where GTP-RhoA first binds to anillin and then is retained at the membrane by PIP2 after it disengages from anillin. Importantly, re-binding of membrane GTP-RhoA to anillin, regulated by the cortical density of anillin, creates cycles through this pathway. These cycles repeatedly reset the dissociation kinetics of GTP-RhoA, substantially increasing its dwell time to recruit effectors. Thus, anillin regulates RhoA signaling by a paradigm of kinetic scaffolding that may apply to other signals whose efficacy dependson their cortical dwell times. Refereed/Peer-reviewed

The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the beta subunit harbors the active site, it requires the a subunit for its activity. Here we explore the other functions of the FTase alpha subunit in addition to its established role in protein prenylation. We found that in the absence of the beta subunit, the alpha subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase alpha using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase alpha subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase alpha controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase alpha subunit that includes control of intracellular membrane trafficking. Refereed/Peer-reviewed

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric G alpha 12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly. Refereed/Peer-reviewed

Mechanotransduction is a strong driver of mesenchymal stem cell (MSC) fate. In vitro, variations in matrix mechanics invoke changes in MSC proliferation, migration and differentiation. However, when incorporating MSCs within injectable, inherently soft hydrogels, this dominance over MSC response substantially limits our ability to couple the ease of application of hydrogels with efficiently directed MSC differentiation, especially in the case of bone generation. Here, we identify differential miRNA expression in response to varying hydrogel stiffness and RhoA activity. We show that modulation of miR-100-5p and miR-143-3p can be used to bias MSC fate and provide mechanistic insight by demonstrating convergence on mTOR signalling. By modulating these mechanosensitive miRNAs, we can enhance osteogenesis in a soft 3D hydrogel. The outcomes of this study provide new understanding of the mechanisms regulating MSC mechanotransduction and differentiation, but also a novel strategy with which to drive MSC fate and significantly impact MSC-based tissue-engineering applications., Mesenchymal stem cell (MSC) fate can be mechanically regulated by substrate stiffness but this is difficult to control in a 3D hydrogel. Here the authors identify miRNAs that change expression in response to substrate stiffness and RhoA signalling and show that they can bias MSC fate in a 3D soft hydrogel.

The inflammasome generates caspase-1 p20/p10, presumed to be the active protease. Boucher et al. demonstrate that the inflammasome contains an active caspase-1 species, p33/p10, and functions as a holoenzyme. Further caspase-1 self-processing generates and releases p20/p10 to terminate protease activity., Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome–caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses., Graphical Abstract

Adherens junctions in epithelia are contractile structures, where coupling of adhesion to the actomyosin cytoskeleton generates mechanical tension for morphogenesis and homeostasis. In established monolayers, junctional contractility is supported by the interplay between cell signals and scaffolding proteins. However, less is known about how contractile junctions develop, especially during the establishment of epithelial monolayers. Here, we show that junctional tension increases concomitant with accumulation of actomyosin networks as Caco-2 epithelia become confluent. This is associated with development of a zone of RhoA signaling at junctions. Further, we find that the low levels of RhoA signaling and contractility found in subconfluent cultures reflect a mechanism for their active suppression. Specifically, the RhoA antagonist, SRGAP1, is present at subconfluent junctions to a greater extent than in confluent cultures and SRGAP1 RNAi restores RhoA signaling and contractility in subconfluent cultures to levels seen in confluent cells. Overall, these observations suggest that regulated changes in junctional contractility mediated by modulation of RhoA signaling occur as epithelial monolayers mature. Refereed/Peer-reviewed

We use a two-dimensional cellular Potts model to represent the behavior of an epithelial cell layer and describe its dynamics in response to a microscopic wound. Using an energy function to describe properties of the cells, we found that the interaction between contractile tension along cell-cell junctions and cell-cell adhesion plays an important role not only in determining the dynamics and morphology of cells in the monolayer, but also in influencing whether or not a wound in the monolayer will close. Our results suggest that, depending on the balance between cell-cell adhesion and junctional tension, mechanics of the monolayer can either correspond to a hard or a soft regime that determines cell morphology and polygonal organization in the monolayer. Moreover, the presence of a wound in a hard regime, where junctional tension is significant, can lead to two results: (1) wound closure or (2) an initial increase and expansion of the wound area towards an equilibrium value. Theoretical approximations and simulations allowed us to determine the thresholds in the values of cell-cell adhesion and initial wound size that allow the system to lead to wound closure. Overall, our results suggest that around the site of injury, changes in the balance between contraction and adhesion determine whether or not non-monotonous wound closure occurs. Refereed/Peer-reviewed

Apoptosis is a ubiquitous mode of programmed cell death that is found in healthy organs and can be stimulated by many toxic stresses. When it occurs in epithelia, apoptosis presents major challenges to tissue integrity. Apoptotic corpses can promote inflammatory and autoimmune responses if they are retained, and the cellular fragmentation that accompanies apoptosis can potentially compromise the epithelial barrier. Here we discuss 2 homeostatic mechanisms that allow epithelia to circumvent these potential risks: clearance of apoptotic corpses by professional and non-professional phagocytes and physical expulsion of apoptotic cells by apical extrusion. Extrusion and phagocytosis may represent complementary responses that preserve epithelial integrity despite the inevitable challenge of apoptosis. Refereed/Peer-reviewed

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model., Author summary Mathematical models play a key role in uncovering mechanisms responsible for the formation of patterns in cells and tissues. The well known Turing mechanism based on nonlinear reaction kinetics and differential diffusion explains the formation of static patterns, while positive feedback interactions can generate dynamical structures such as propagating fronts and excitable pulses. Recent studies have demonstrated the importance of mechanical forces that can lead to novel mechanisms of pattern formation such as clustering and oscillations in contractile systems. Here we investigate how contractile forces in mechanically active media can affect bistable front propagation. We found that contraction regulates the front speed or can fully suppress its propagation in space to create a static localized zone. The proposed model provides a new mechanism for cross-talk between mechanical activity of cells and biochemical signaling.

The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic “skeleton” is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b −/− zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b −/− mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechanoprotection. Refereed/Peer-reviewed

Contractile adherens junctions support cell−cell adhesion, epithelial integrity, and morphogenesis. Much effort has been devoted to understanding how contractility is established; however, less is known about whether contractility can be actively downregulated at junctions nor what function this might serve. We now identify such an inhibitory pathway that is mediated by the cytoskeletal scaffold, cortactin. Mutations of cortactin that prevent its tyrosine phosphorylation downregulate RhoA signaling and compromise the ability of epithelial cells to generate a contractile zonula adherens. This is mediated by the RhoA antagonist, SRGAP1. We further demonstrate that this mechanism is co-opted by hepatocyte growth factor to promote junctional relaxation and motility in epithelial collectives. Together, our findings identify a novel function of cortactin as a regulator of RhoA signaling that can be utilized by morphogenetic regulators for the active downregulation of junctional contractility., Epithelial cell-cell adhesions are contractile junctions, but whether contractility can be down-regulated is not known. Here the authors report how tyrosine dephosphorylation of the cytoskeletal scaffold, cortactin, recruits the RhoA antagonist SRGAP1 to relax adherens junctions in response to HGF.

The physical properties of cells reflect how the structure and dynamics of the actomyosin cortex are coupled to the plasma membrane. In epithelia, adhesive E-cadherin clusters associate with the cell cortex to assemble the junctional actomyosin that participates in epithelial morphogenesis. E-cadherin is present not only at the apical zonula adherens (ZA), but also distributed throughout the lateral adherens junction (LAJ) below the ZA. However, the organizational dynamics of the actomyosin network at the LAJs remains elusive. To address this, we used quantitative real-time imaging to characterize the dynamics of actomyosin contractility at lateral cadherin contacts. Here, we report that contractility is coordinated into smaller actomyosin rings that link cadherin clusters together within the larger cortical network at the lateral junctions. We conclude that Myosin II activity determines the contractility of actomyosin cables between cadherin clusters to propagate pulsatility across lateral cell-cell contacts. Refereed/Peer-reviewed

Background. Actomyosin-based contractility acts on cadherin junctions to support tissue integrity and morphogenesis. The actomyosin apparatus of the epithelial zonula adherens (ZA) is built by coordinating junctional actin assembly with Myosin II activation. However, the physical interaction between Myosin and actin filaments that is necessary for contractility can induce actin filament turnover, potentially compromising the contractile apparatus itself. Results. We now identify tension-sensitive actin assembly as one cellular solution to this design paradox. We show that junctional actin assembly is maintained by contractility in established junctions and increases when contractility is stimulated. The underlying mechanism entails the tension-sensitive recruitment of vinculin to the ZA. Vinculin, in turn, directly recruits Mena/VASP proteins to support junctional actin assembly. By combining strategies that uncouple Mena/VASP from vinculin or ectopically target Mena/VASP to junctions, we show that tension-sensitive actin assembly is necessary for junctional integrity and effective contractility at the ZA. Conclusions. We conclude that tension-sensitive regulation of actin assembly represents a mechanism for epithelial cells to resolve potential design contradictions that are inherent in the way that the junctional actomyosin system is assembled. This emphasizes that maintenance and regulation of the actin scaffolds themselves influence how cells generate contractile tension. Refereed/Peer-reviewed

We generated a computational approach to analyze the biomechanics of epithelial cell aggregates, either island or stripes or entire monolayers, that combines both vertex and contact-inhibition-of-locomotion models to include both cell-cell and cell-substrate adhesion. Examination of the distribution of cell protrusions (adhesion to the substrate) in the model predicted high order profiles of cell organization that agree with those previously seen experimentally. Cells acquired an asymmetric distribution of basal protrusions, traction forces and apical aspect ratios that decreased when moving from the edge to the island center. Our in silico analysis also showed that tension on cell-cell junctions and apical stress is not homogeneous across the island. Instead, these parameters are higher at the island center and scales up with island size, which we confirmed experimentally using laser ablation assays and immunofluorescence. Without formally being a 3-dimensional model, our approach has the minimal elements necessary to reproduce the distribution of cellular forces and mechanical crosstalk as well as distribution of principal stress in cells within epithelial cell aggregates. By making experimental testable predictions, our approach would benefit the mechanical analysis of epithelial tissues, especially when local changes in cell-cell and/or cell-substrate adhesion drive collective cell behavior., Comment: 39 pages, 8 Figures. Supplementary Information is included

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur. Refereed/Peer-reviewed

Cell-cell contacts are major determinants of tissue organization in both health and disease. Adhesive interactions, especially those mediated by classical cadherin receptors, influence cell-cell recognition and tissue patterning during development. Conversely, cadherin dysfunction promotes tumor progression to invasion and metastasis. Over the past three decades, we have learnt a great deal about the molecular mechanisms responsible for cadherin-based cell-cell interactions. Yet our knowledge remains incomplete. The intersection between cell biology and mechanical forces has long been suspected to be an important missing factor in understanding cadherin biology. However, tangible evidence remained elusive until recently, when several reports began to elucidate the role of cadherins and the cytoskeleton in mechanotransduction. In this review, we examine these advances and discuss their implications. Refereed/Peer-reviewed

Epithelial tissues form physically integrated barriers against the external environment protecting organs from infection and invasion. Within each tissue, epithelial cells respond to different challenges that can potentially compromise tissue integrity. In particular, cells collectively respond to injuries by reorganizing their cell-cell junctions and migrating directionally towards the sites of damage. Notwithstanding, the mechanisms that drive collective responses in epithelial aggregates remain poorly understood. In this work, we develop a minimal mechanistic model that is able to capture the essential features of epithelial collective responses to injuries. We show that a model that integrates the mechanics of cells at the cell-cell and cell-substrate interfaces as well as contact inhibition of locomotion (CIL) correctly predicts two key properties of epithelial response to injury as: (1) local relaxation of the tissue and (2) collective reorganization involving the extension of cryptic lamellipodia that extend, on average, up to 3 cell diameters from the site of injury and morphometric changes in the basal regions. Our model also suggests that active responses (like the actomyosin purse string and softening of cell-cell junctions) are needed to drive morphometric changes in the apical region. Therefore, our results highlight the importance of the crosstalk between junctional biomechanics, cell substrate adhesion, and CIL, as well as active responses, in guiding the collective rearrangements that are required to preserve the epithelial barrier in response to injury. European Commission - European Regional Development Fund Higher Education Authority Irish Research Council National Health and Medical Research Council of Australia Australian Research Council Australian Cancer Research Foundation

The role of myosin IIB in junctional contractility and its mode of regulation are not well understood. It is demonstrated that junctional recruitment of myosin IIB requires the activation of a receptor-type protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway. This reinforces the concept that E-cadherin–based signaling recruits distinct myosin II paralogues to generate contractile tension., 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.

Actomyosin at the epithelial zonula adherens (ZA) generates junctional tension for tissue integrity and morphogenesis. This requires the RhoA GTPase, which establishes a strikingly stable active zone at the ZA. Mechanisms must then exist to confer robustness on junctional RhoA signalling at the population level. We now identify a feedback network that generates a stable mesoscopic RhoA zone out of dynamic elements. The key is scaffolding of ROCK1 to the ZA by myosin II. ROCK1 protects junctional RhoA by phosphorylating Rnd3 to prevent the cortical recruitment of the Rho suppressor, p190B RhoGAP. Combining predictive modelling and experimentation, we show that this network constitutes a bistable dynamical system that is realized at the population level of the ZA. Thus, stability of the RhoA zone is an emergent consequence of the network of interactions that allow myosin II to feedback to RhoA. Refereed/Peer-reviewed

Cells are linked together dynamically by adhesion molecules, such as the classical cadherins. E-cadherin, which mediates epithelial cell–cell interactions, plays fundamental roles in tissue organization and is often perturbed in diseases such as cancer. It has long been recognized that the biology of E-cadherin arises from cooperation between adhesion and the actin cytoskeleton. A major feature is the generation of contractile forces at junctions, yielding patterns of tension that contribute to tissue integrity and patterning. Here we discuss recent developments in understanding how cadherin junctions integrate signaling and cytoskeletal dynamics to sense and generate force. Refereed/Peer-reviewed

This Perspective considers how classical cadherin cell-cell adhesion receptors are organized at the nanoscale to generate lateral clusters. Recent advances in optical microscopy reveal that clustering constitutes a general feature of cadherin organization, but one that takes diverse forms. Here we consider the molecular mechanisms responsible for cadherin clustering and their functional implications. We frame our discussion in light of what is known about how nanoscale organization is conferred upon the plasma membrane, through protein-protein interactions, regulation of the cortical actin cytoskeleton, and the lipid environment of the membrane. Refereed/Peer-reviewed

In neurosecretory cells, secretory vesicles (SVs) undergo Ca2(+)-dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking. Refereed/Peer-reviewed

The findings presented here demonstrate the role of α-catenin in cadherin-based adhesion and mechanotransduction in different mechanical contexts. Bead-twisting measurements in conjunction with imaging, and the use of different cell lines and α-catenin mutants reveal that the acute local mechanical manipulation of cadherin bonds triggers vinculin and actin recruitment to cadherin adhesions in an actin- and α-catenin-dependent manner. The modest effect of α-catenin on the two-dimensional binding affinities of cell surface cadherins further suggests that force-activated adhesion strengthening is due to enhanced cadherin–cytoskeletal interactions rather than to α-catenin-dependent affinity modulation. Complementary investigations of cadherin-based rigidity sensing also suggest that, although α-catenin alters traction force generation, it is not the sole regulator of cell contractility on compliant cadherin-coated substrata.

Epithelial cells generate contractile forces at their cell-cell contacts. These are concentrated at the specialized apical junction of the zonula adherens (ZA), where a ring of stabilized E-cadherin lies adjacent to prominent actomyosin bundles. Coupling of adhesion and actomyosin contractility yields tension in the junction. The biogenesis of junctional contractility requires actin assembly at the ZA as well as the recruitment of nonmuscle myosin II, but the molecular regulators of these processes are not yet fully understood. We now report a role for tropomyosins 5NM1 (Tm5NM1) and 5NM2 (Tm5NM2) in their generation. Both these tropomyosin isoforms were found at the ZA and their depletion by RNAi or pharmacological inhibition reduced both F-actin and myosin II content at the junction. Photoactivation analysis revealed that the loss of F-actin was attributable to a decrease in filament stability. These changes were accompanied by a decrease in E-cadherin content at junctions. Ultimately, both long-term depletion of Tm5NM1/2 and acute inhibition with drugs caused junctional tension to be reduced. Thus these tropomyosin isoforms are novel contributors to junctional contractility and integrity. Refereed/Peer-reviewed

The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells. Refereed/Peer-reviewed

E-cadherin cell-cell junctions couple the contractile cortices of epithelial cells together, generating tension within junctions that influences tissue organization. Although junctional tension is commonly studied at the apical zonula adherens, we now report that E-cadherin adhesions induce the contractile actomyosin cortex throughout the apical lateral axis of junctions. However, cells establish distinct regions of contractile activity even within individual contacts, producing high tension at the zonula adherens but substantially lower tension elsewhere. We demonstrate that N-WASP (also known as WASL) enhances apical junctional tension by stabilizing local F-actin networks, which otherwise undergo stress-induced turnover. Further, we find that cells are extruded from monolayers when this pattern of intra-junctional contractility is disturbed, either when N-WASP redistributes into lateral junctions in H-Ras(V12)-expressing cells or on mosaic redistribution of active N-WASP itself. We propose that local control of actin filament stability regulates the landscape of intra-junctional contractility to determine whether or not cells integrate into epithelial populations. Refereed/Peer-reviewed

Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin assembly at the ZA. Refereed/Peer-reviewed

Epithelial junctions are fundamental determinants of tissue organization, subject to regulation by tyrosine phosphorylation. Homophilic binding of E-cadherin activates tyrosine kinases, such as Src, that control junctional integrity. Protein tyrosine phosphatases (PTPs) also contribute to cadherin-based adhesion and signaling, but little is known about their specific identity or functions at epithelial junctions. Here, we report that the receptor PTP RPTPɑ (human gene name PTPRA) is recruited to epithelial adherens junctions at the time of cell-cell contact, where it is in molecular proximity to E-cadherin. RPTPɑ is required for appropriate cadherin-dependent adhesion and for cyst architecture in three-dimensional culture. Loss of RPTPɑ impairs adherens junction integrity, as manifested by defective E-cadherin accumulation and peri-junctional F-actin density. These effects correlate with a role for RPTPɑ in cellular (c)-Src activation at sites of E-cadherin engagement. Mechanistically, RPTPɑ is required for appropriate tyrosine phosphorylation of cortactin, a major Src substrate and a cytoskeletal actin organizer. Expression of a phosphomimetic cortactin mutant in RPTPɑ-depleted cells partially rescues F-actin and E-cadherin accumulation at intercellular contacts. These findings indicate that RPTPɑ controls cadherinmediated signaling by linking homophilic E-cadherin engagement to cortactin tyrosine phosphorylation through c-Src. Refereed/Peer-reviewed

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell-cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function. (c) 2013 International Society of Differentiation. Refereed/Peer-reviewed

WAVE2–Arp2/3 is a major nucleator of actin assembly at the zonula adherens and likely acts in response to junctional Rac signaling. It supports myosin II recruitment to, and tension generation at, the junction., 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.

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Forster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes. Refereed/Peer-reviewed

The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. This can potentially be determined by the balance between molecules that promote nucleotide exchange or GTP hydrolysis. However, how these activities may be coordinated is poorly understood. We now report a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. We identify an extramitotic activity of the centralspindlin complex, better understood as a cytokinetic regulator, which localizes to the interphase zonula adherens by interacting with the cadherin-associated protein, alpha-catenin. Centralspindlin recruits the RhoGEF, ECT2, to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localization of p190 B RhoGAP, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is used in interphase cells to control the Rho GTPase cycle at the zonula adherens. Refereed/Peer-reviewed

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyl-transferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution. Refereed/Peer-reviewed

GAP-43 (growth-associated protein-43) is it dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association., subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys(3) and Cys(4) of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid (foes affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together. these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via in Arf6-associated endocytic vesicular pathway. Refereed/Peer-reviewed

Caveolar proteins and caveolae negatively regulate a second clathrin-independent endocytic CLIC/GEEC pathway; caveolin-1 affects membrane diffusion properties of raft-associated CLIC cargo, and the scaffolding domain of caveolin-1 is required and sufficient for endocytic inhibition., Several studies have suggested crosstalk between different clathrin-independent endocytic pathways. However, the molecular mechanisms and functional relevance of these interactions are unclear. Caveolins and cavins are crucial components of caveolae, specialized microdomains that also constitute an endocytic route. Here we show that specific caveolar proteins are independently acting negative regulators of clathrin-independent endocytosis. Cavin-1 and Cavin-3, but not Cavin-2 or Cavin-4, are potent inhibitors of the clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, in a process independent of caveola formation. Caveolin-1 (CAV1) and CAV3 also inhibit the CLIC/GEEC pathway upon over-expression. Expression of caveolar protein leads to reduction in formation of early CLIC/GEEC carriers, as detected by quantitative electron microscopy analysis. Furthermore, the CLIC/GEEC pathway is upregulated in cells lacking CAV1/Cavin-1 or with reduced expression of Cavin-1 and Cavin-3. Inhibition by caveolins can be mimicked by the isolated caveolin scaffolding domain and is associated with perturbed diffusion of lipid microdomain components, as revealed by fluorescence recovery after photobleaching (FRAP) studies. In the absence of cavins (and caveolae) CAV1 is itself endocytosed preferentially through the CLIC/GEEC pathway, but the pathway loses polarization and sorting attributes with consequences for membrane dynamics and endocytic polarization in migrating cells and adult muscle tissue. We also found that noncaveolar Cavin-1 can act as a modulator for the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. This work provides new insights into the regulation of noncaveolar clathrin-independent endocytosis by specific caveolar proteins, illustrating multiple levels of crosstalk between these pathways. We show for the first time a role for specific cavins in regulating the CLIC/GEEC pathway, provide a new tool to study this pathway, identify caveola-independent functions of the cavins and propose a novel mechanism for inhibition of the CLIC/GEEC pathway by caveolin., Author Summary Endocytosis is the process that allows cells to take up molecules from the environment. Several endocytic pathways exist in mammalian cells. While the best understood endocytic pathway uses clathrin, recent years have seen a great increase in our understanding of clathrin-independent endocytic pathways. Here we characterize the crosstalk between caveolae, flask-shaped specialized microdomains present at the plasma membrane, and a second clathrin-independent pathway, the CLIC/GEEC Cdc42-regulated endocytic pathway. These pathways are segregated in migrating cells with caveolae at the rear and CLIC/GEEC endocytosis at the leading edge. Here we find that specific caveolar proteins, caveolins and cavins, can also negatively regulate the CLIC/GEEC pathway. With the help of several techniques, including quantitative electron microscopy analysis and real-time live-cell imaging, we demonstrate that expression of caveolar proteins affects early carrier formation, causes cellular lipid changes, and changes the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. The functional consequences of loss of caveolar proteins on the CLIC/GEEC pathway included inhibition of polarized cell migration and increased endocytosis in tissue explants.

The zonula adherens (ZA) of epithelial cells is a site of cell-cell adhesion where cellular forces are exerted and resisted. Increasing evidence indicates that E-cadherin adhesion molecules at the ZA serve to sense force applied on the junctions and coordinate cytoskeletal responses to those forces. Efforts to understand the role that cadherins play in mechanotransduction have been limited by the lack of assays to measure the impact of forces on the ZA. In this study we used 4D imaging of GFP-tagged E-cadherin to analyse the movement of the ZA. Junctions in confluent epithelial monolayers displayed prominent movements oriented orthogonal (perpendicular) to the ZA itself. Two components were identified in these movements: a relatively slow unidirectional (translational) component that could be readily fitted by least-squares regression analysis, upon which were superimposed more rapid oscillatory movements. Myosin IIB was a dominant factor responsible for driving the unilateral translational movements. In contrast, frequency spectrum analysis revealed that depletion of Myosin IIA increased the power of the oscillatory movements. This implies that Myosin IIA may serve to dampen oscillatory movements of the ZA. This extends our recent analysis of Myosin II at the ZA to demonstrate that Myosin IIA and Myosin IIB make distinct contributions to junctional movement at the ZA. Refereed/Peer-reviewed