Directed actin polymerization is the driving force for epithelial cell-cell adhesion

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.     

Mammalian formin-1 participates in adherens junctions and polymerization of linear actin cables

During epithelial sheet formation, linear actin cables assemble at nascent adherens junctions. This process requires alpha-catenin and actin polymerization, although the underlying mechanism is poorly understood. Here, we show that formin-1 interacts with alpha-catenin, localizes to adherens junctions and nucleates unbranched actin filaments. Furthermore, disruption of the alpha-catenin-formin-1 interaction blocks assembly of radial actin cables and perturbs intercellular adhesion. A fusion protein of the beta-catenin-binding domain of alpha-catenin with the actin polymerization domains of formin-1 rescues formation of adherens junctions and associated actin cables in alpha-catenin-null keratinocytes. These findings provide new insight into how alpha-catenin orchestrates actin dynamics during intercellular junction formation.     

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.     

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.     

Identification of an epidermal cell-adhesion glycoprotein.

Glycoproteins which mediate intercellular adhesion were studied by comparing the effects of trypsin and the neutral proteinase, Dispase, on human keratinocytes metabolically labelled with D-[1-14C]glucosamine or L-[1-3H]fucose. Whereas digestion of keratinocytes with trypsin/EDTA resulted in loss of both cell-substratum and intercellular adhesion, only cell-substratum adhesion was disrupted by incubation with Dispase. Analysis of the radiolabelled glycoproteins by polyacrylamide-gel electrophoresis revealed that a glycoprotein of Mr 126 000 was cleaved by trypsin/EDTA, but not by Dispase. Surface labelling of keratinocytes with galactose oxidase/NaB3H4 confirmed that this glycoprotein was exposed on the cell surface. Addition of lmM-Ca2+ prevented dispersion of keratinocytes by trypsin and concomitantly protected the glycoprotein of Mr 126 000 from digestion. These results indicate that this glycoprotein has an important role in mediating intercellular adhesion of keratinocytes.     

The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin

Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with alpha-catenin, and alpha-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion.     

Alpha-catenin: at the junction of intercellular adhesion and actin dynamics

Alpha-catenin has often been considered to be a non-regulatory intercellular adhesion protein, in contrast to beta-catenin, which has well-documented dual roles in cell-cell adhesion and signal transduction. Recently, however, alpha-catenin has been found to be important not only in connecting the E-cadherin-beta-catenin complex to the actin cytoskeleton, but also in coordinating actin dynamics and inversely correlating cell adhesion with proliferation. As the number of alpha-catenin-interacting partners increases, intriguing new connections imply even more complex regulatory functions for this protein.     

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.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

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.     

Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.     

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.     

Nuclear–cytoskeletal linkages facilitate cross talk between the nucleus and intercellular adhesions

The linker of nucleoskeleton and cytoskeleton (LINC) complex allows cells to actively control nuclear position by coupling the nucleus to the cytoplasmic cytoskeleton. Nuclear position responds to the formation of intercellular adhesions through coordination with the cytoskeleton, but it is not known whether this response impacts adhesion function. In this paper, we demonstrate that the LINC complex component SUN2 contributes to the mechanical integrity of intercellular adhesions between mammalian epidermal keratinocytes. Mice deficient for Sun2 exhibited irregular hair follicle intercellular adhesions, defective follicle structure, and alopecia. Primary mouse keratinocytes lacking Sun2 displayed aberrant nuclear position in response to adhesion formation, altered desmosome distribution, and mechanically defective adhesions. This dysfunction appeared rooted in a failure of Sun2-null cells to reorganize their microtubule network to support coordinated intercellular adhesion. Together, these results suggest that cross talk between the nucleus, cytoskeleton, and intercellular adhesions is important for epidermal tissue integrity.     

Defining the roles of beta-catenin and plakoglobin in cell-cell adhesion: isolation of beta-catenin/plakoglobin-deficient F9 cells

F9 teratocarcinoma cells in which beta-catenin and/or plakoglobin genes are knocked-out were generated and investigated in an effort to define the role of beta-catenin and plakoglobin in cell adhesion. Loss of beta-catenin expression only did not affect cadherin-mediated cell adhesion activity. Loss of both beta-catenin and plakoglobin expression, however, severely affected the strong cell adhesion activity of cadherin. In beta-catenin-deficient cells, the amount of plakoglobin associated with E-cadherin dramatically increased. In beta-catenin/plakoglobin-deficient cells, the level of E-cadherin and alpha-catenin markedly decreased. In these cells, E-cadherin formed large aggregates in cytoplasm and membrane localization of alpha-catenin was barely detected. These data confirmed that beta-catenin or plakoglobin is required for alpha-catenin to form complex with E-cadherin. It was also demonstrated that plakoglobin can compensate for the absence of beta-catenin. Moreover it was suggested that beta-catenin or plakoglobin is required not only for the cell adhesion activity but also for the stable expression and cell surface localization of E-cadherin.     

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.     

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.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

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.     

Role for E-cadherin as an inhibitory receptor on epidermal gammadelta T cells

E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), but they express another receptor for E-cadherin, α(E)(CD103)β(7) integrin, as well. In this study, we analyzed functional differences between E-cadherin-mediated homophilic binding and heterophilic binding of α(E)β(7) integrin to E-cadherin in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not α(E)β(7) integrin, was downregulated on activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated by α(E)β(7) integrin, and blocking of the binding of α(E)β(7) integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented by coligation of α(E)β(7) integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti-E-cadherin Ab, plate-bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an inhibitory receptor on DETCs, whereas α(E)β(7) integrin acts as a costimulatory receptor. Differential expression of E-cadherin and α(E)β(7) integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin and α(E)β(7) integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes.     

Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion

The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. We reported previously that E-cadherin-alpha-catenin fusion molecules showed cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Here, we constructed deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay revealed that the fusion molecules containing the ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifted the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not completely support the strong state of cell adhesion activity. Our studies showed that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity.