Preexisting tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia.

Apical extrusion is a tissue-intrinsic process that allows epithelia to eliminate unfit or surplus cells. This is exemplified by the early extrusion of apoptotic cells, which is critical to maintain the epithelial barrier and prevent inflammation. Apoptotic extrusion is an active mechanical process, which involves mechanotransduction between apoptotic cells and their neighbors, as well as local changes in tissue mechanics. Here we report that the preexisting mechanical tension at adherens junctions (AJs) conditions the efficacy of apoptotic extrusion. Specifically, increasing baseline mechanical tension by overexpression of a phosphomimetic Myosin II regulatory light chain (MRLC) compromises apoptotic extrusion. This occurs when tension is increased in either the apoptotic cell or its surrounding epithelium. Further, we find that the proinflammatory cytokine, TNFα, stimulates Myosin II and increases baseline AJ tension to disrupt apical extrusion, causing apoptotic cells to be retained in monolayers. Importantly, reversal of mechanical tension with an inhibitory MRLC mutant or tropomyosin inhibitors is sufficient to restore apoptotic extrusion in TNFα-treated monolayers. Together, these findings demonstrate that baseline levels of tissue tension are important determinants of apoptotic extrusion, which can potentially be coopted by pathogenetic factors to disrupt the homeostatic response of epithelia to apoptosis.

Caveola mechanotransduction reinforces the cortical cytoskeleton to promote epithelial resilience.

As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signaling, recruitment of FMNL2 formin, and enhanced-cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.

Apical extrusion prevents apoptosis from activating an acute inflammatory program in epithelia.

Apoptosis is traditionally considered to be an immunologically silent form of cell death. Multiple mechanisms exist to ensure that apoptosis does not stimulate the immune system to cause inflammation or autoimmunity. Against this expectation, we now report that epithelia are programmed to provoke, rather than suppress, inflammation in response to apoptosis. We found that an acute inflammatory response led by neutrophils occurs in zebrafish and cell culture when apoptotic epithelial cells cannot be expelled from the monolayer by apical extrusion. This reflects an intrinsic circuit where ATP released from apoptotic cells stimulates epithelial cells in the immediate vicinity to produce interleukin-8 (IL-8). Apical extrusion therefore prevents inappropriate epithelial inflammation by physically eliminating apoptotic cells before they can activate this pro-inflammatory circuit. This carries the implication that epithelia may be predisposed to inflammation, elicited by sporadic or induced apoptosis, if apical extrusion is compromised.

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis.

Epithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP)1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ)3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening4-10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself11,12, or proximate to the junctions13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

Enhanced RhoA signalling stabilizes E-cadherin in migrating epithelial monolayers.

Epithelia migrate as physically coherent populations of cells. Previous studies have revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also increases due to an increase in RhoA-mediated junctional contractility. We found that active RhoA levels were stimulated by p114 RhoGEF (also known as ARHGEF18) at the junctions between migrating MCF-7 monolayers, and this was accompanied by increased levels of actomyosin and mechanical tension. Applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin (CDH1) during epithelial migration and promoted orderly collective movement. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion to protect against the mechanical stresses of migration. This article has an associated First Person interview with the first author of the paper.

Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion.

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.

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.

Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor Cells.

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.

Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell-matrix adhesion.

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and elicited by stimuli ranging from apoptosis to oncogenic transformation. To explore whether the morphogenetic transcription factor Snail (SNAI1) induces extrusion, we inducibly expressed a stabilized Snail6SA transgene in confluent MCF-7 monolayers. When expressed in small clusters (less than three cells) within otherwise wild-type confluent monolayers, Snail6SA expression induced apical cell extrusion. In contrast, larger clusters or homogenous cultures of Snail6SA cells did not show enhanced apical extrusion, but eventually displayed sporadic basal delamination. Transcriptomic profiling revealed that Snail6SA did not substantively alter the balance of epithelial and mesenchymal genes. However, we identified a transcriptional network that led to upregulated RhoA signalling and cortical contractility in cells expressing Snail6SA Enhanced contractility was necessary, but not sufficient, to drive extrusion, suggesting that Snail collaborates with other factors. Indeed, we found that the transcriptional downregulation of cell-matrix adhesion cooperates with contractility to mediate basal delamination. This provides a pathway for Snail to influence epithelial morphogenesis independently of classic epithelial-to-mesenchymal transition.

Anillin Promotes Cell Contractility by Cyclic Resetting of RhoA Residence Kinetics.

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 effector recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membrane phosphoinositide-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 depends on their cortical dwell times.

Author Correction: Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1.

A correction to this article has been published and is linked from the HTML version of this article.

Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1.

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.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions.

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.

Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin 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.

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.

Tension-sensitive actin assembly supports contractility at the epithelial zonula adherens.

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.

Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.

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.

Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia.

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.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells.

The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution.

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.