ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

Cell Adhesion: Sorting out Cell Mixing with Echinoid?

Cadherins control intercellular adhesion in epithelial cells. This property relies on the ability to recruit actin filaments at adherens junctions via beta-catenin and alpha-catenin. A recent study shows that Echinoid, a member of the immunoglobulin domain containing protein family, is a modulator of intercellular adhesion in Drosophila that controls cell sorting.     

The cadherin-11 cytoplasmic juxtamembrane domain promotes alpha-catenin turnover at adherens junctions and intercellular motility

Cadherins mediate homophilic cell adhesion and contribute to tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of alpha-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 deltaJMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these data implicate the cadherin-11 cytoplasmic JMD as a regulator of alpha-catenin turnover at adherens junctions and actin-cytoskeletal organization that is critical for intercellular motility and rearrangement in multicellular clusters.     

Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium

To enable stratification and barrier function, the epidermis must permit self-renewal while maintaining adhesive connections. By generating K14-GFP-actin mice to monitor actin dynamics in cultured primary keratinocytes, we uncovered a role for the actin cytoskeleton in establishing cellular organization. During epidermal sheet formation, a polarized network of nascent intercellular junctions and radial actin cables assemble in the apical plane of the monolayer. These actin fibers anchor to a central actin-myosin network, creating a tension-based plane of cytoskeleton across the apical surface of the sheet. Movement of the sheet surface relative to its base expands the zone of intercellular overlap, catalyzing new sites for nascent intercellular junctions. This polarized cytoskeleton is dependent upon alpha-catenin, Rho, and Rock, and its regulation may be important for wound healing and/or stratification, where coordinated tissue movements are involved.     

Magic touch: how does cell-cell adhesion trigger actin assembly?

The formation of cadherin-mediated cell-cell junctions is accompanied by a profound remodeling of the actin cytoskeleton. The Arp2/3 complex and its activator cortactin drive the assembly of branching actin-filament arrays, and formin-1 promotes the nucleation of non-branching actin filaments. Recruitment of these actin nucleators to nascent adhesions, formation of the links between them and the cytoplasmic cadherin domain, and triggering of their actin-polymerizing functions are vital steps in the development of cell-cell junctions. In this article, I discuss recent results showing that proteins involved in the regulation of actin polymerization have crucial roles in the development and maintenance of the cadherin junctions.     

Regulation of adherens junction protein p120(ctn) by 10 nM CCK precedes actin breakdown in rat pancreatic acini

The initial pathophysiological events that characterize CCK-hyperstimulation pancreatitis include the breakdown of the actin filament system and disruption of cadherin-catenin protein complexes. Cadherins and catenins are part of adherens junctions, which may act as anchor for the cellular actin filament system. We examined the composition and regulation of adherens junctions during CCK-induced acinar cell damage. Freshly isolated CCK-stimulated rat pancreatic acini were examined for actin filaments and functional adherens junctions by immunocytology and laser confocal scanning microscopy or by coprecipitation and immunoblotting for E-cadherin, beta- and alpha-catenin, p120(ctn), and phosphotyrosine. In addition to E-cadherin and beta-catenin, acinar cells express the cadherin-regulatory protein p120(ctn) and the attachment protein alpha-catenin. Both colocalize and coimmunoprecipitate with E-cadherin in one complex, and all colocalize with the terminal actin web. Supramaximal secretory CCK concentrations (10 nM) initiated tyrosine phosphorylation of p120(ctn) but not of beta-catenin within 2 min, preceding the breakdown of the terminal actin web by several minutes. Under these conditions, the cadherin-catenin association within the adherens junction complex remained intact. We describe for the first time supramaximal CCK-dependent tyrosine phosphorylation of the adherens junction protein p120(ctn) and demonstrate the presence of an intact adherens junction protein complex in acinar cells. p120(ctn) may participate in the actin filament breakdown during experimental conditions mimicking pancreatitis.     

α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.     

Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Actin dynamics and cell-cell adhesion in epithelia

Recent advances in the field of intercellular adhesion highlight the importance of adherens junction association with the underlying actin cytoskeleton. In skin epithelial cells a dynamic feature of adherens junction formation involves filopodia, which physically project into the membrane of adjacent cells, catalyzing the clustering of adherens junction protein complexes at their tips. In turn, actin polymerization is stimulated at the cytoplasmic interface of these complexes. Although the mechanism remains unclear, the VASP/Mena family of proteins seems to be involved in organizing actin polymerization at these sites. In vivo, adherens junction formation appears to rely upon filopodia in processes where epithelial sheets must be physically moved closer to form stable intercellular connections, for example, in ventral closure in embryonic development or wound healing in the postnatal animal.     

Direct interaction of the C-terminal domain of alpha-catenin and F-actin is necessary for stabilized cell-cell adhesion

Alpha-catenin functions to anchor adherens junctions to the filamentous actin (F-actin) cytoskeleton, through direct and indirect binding mechanisms. When truncated at amino acid 865, alpha-catenin exhibited a markedly reduced F-actin binding affinity compared to wild-type. Expression of the truncated mutant in the alpha-catenin deficient colon carcinoma cell line, Clone A, could not restore an adhesive phenotype when compared. Furthermore, the truncated alpha-catenin fusion protein failed to concentrate at sites of cell-cell contact, to promote morphological changes associated with epithelial monolayers, and to stimulate resistance to shearing forces in a hanging drop aggregation assay. Subsequent attempts to isolate single residues governing the direct F-actin interaction, using neutralizing charge or reverse charge mutations of basic residues within a homology modeled alpha-catenin C-terminal 5-helix bundle, had no effect on F-actin cosedimentation. We conclude that direct attachment of alpha-catenin to F-actin is required to promote cadherin-mediated contact formation and strong cell-cell adhesive states.     

Biochemical and structural definition of the l-afadin- and actin-binding sites of alpha-catenin

alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.     

DLG-1 Is a MAGUK Similar to SAP97 and Is Required for Adherens Junction Formation

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.     

ABI2-deficient mice exhibit defective cell migration, aberrant dendritic spine morphogenesis, and deficits in learning and memory

The Abl-interactor (Abi) family of adaptor proteins has been linked to signaling pathways involving the Abl tyrosine kinases and the Rac GTPase. Abi proteins localize to sites of actin polymerization in protrusive membrane structures and regulate actin dynamics in vitro. Here we demonstrate that Abi2 modulates cell morphogenesis and migration in vivo. Homozygous deletion of murine abi2 produced abnormal phenotypes in the eye and brain, the tissues with the highest Abi2 expression. In the absence of Abi2, secondary lens fiber orientation and migration were defective in the eye, without detectable defects in proliferation, differentiation, or apoptosis. These phenotypes were consistent with the localization of Abi2 at adherens junctions in the developing lens and at nascent epithelial cell adherens junctions in vitro. Downregulation of Abi expression by RNA interference impaired adherens junction formation and correlated with downregulation of the Wave actin-nucleation promoting factor. Loss of Abi2 also resulted in cell migration defects in the neocortex and hippocampus, abnormal dendritic spine morphology and density, and severe deficits in short- and long-term memory. These findings support a role for Abi2 in the regulation of cytoskeletal dynamics at adherens junctions and dendritic spines, which is critical for intercellular connectivity, cell morphogenesis, and cognitive functions.     

Structure of the dimerization and beta-catenin-binding region of alpha-catenin

In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin 57-264 behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly.     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis

During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.     

Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.     

Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming.

BACKGROUND: During embryonic development, epithelia with free edges must join together to create continuous tissues that seal the interior of the organism from the outside environment; failure of epithelial sealing underlies several common human birth defects. Sealing of epithelial sheets in embryos can be extremely rapid, dramatically exceeding the rate of adherens junction formation by epithelial cells in culture or during healing of epithelial wounds. Little is known about the dynamic redistribution of cellular junctional components during such events in living embryos. RESULTS: We have used time-lapse, multiphoton laser-scanning microscopy and green fluorescent protein fusion proteins to analyze the sealing of the Caenorhabditis elegans epidermis in living embryos. Rapid recruitment of alpha-catenin to sites of filopodial contact between contralateral migrating epithelial cells, concomitant with clearing of cytoplasmic alpha-catenin, resulted in formation of nascent junctions; this preceded the formation of mature junctions. Surprisingly, upon inactivation of the entire cadherin-catenin complex, only adhesive strengthening between filopodia was reproducibly affected. Other ventral epidermal cells, which did not extend filopodia and appeared to seal along the ventral midline by coordinated changes in cell shape, successfully adhered in the absence of these proteins. CONCLUSIONS: We propose that 'filopodial priming' - prealignment of bundled actin in filopodia combined with the rapid recruitment of alpha-catenin from cytoplasmic reserves at sites of filopodial contact - accounts for the rapid rate of sealing of the embryonic epidermis of C. elegans. Filopodial priming may provide a general mechanism for rapid creation of adherens junctions during epithelial-sheet sealing in embryos.     

Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.