Sorting of cadherin-catenin-associated proteins into individual clusters.

The cytoplasmic tails of classical cadherins form a multiprotein cadherin-catenin complex (CCC) that constitutes the major structural unit of adherens junctions (AJs). The CCC in AJs forms junctional clusters, "E clusters," driven by cis and trans interactions in the cadherin ectodomain and stabilized by α-catenin-actin interactions. Additional proteins are known to bind to the cytoplasmic region of the CCC. Here, we analyze how these CCC-associated proteins (CAPs) integrate into cadherin clusters and how they affect the clustering process. Using a cross-linking approach coupled with mass spectrometry, we found that the majority of CAPs, including the force-sensing protein vinculin, interact with CCCs outside of AJs. Accordingly, structural modeling shows that there is not enough space for CAPs the size of vinculin to integrate into E clusters. Using two CAPs, scribble and erbin, as examples, we provide evidence that these proteins form separate clusters, which we term "C clusters." As proof of principle, we show, by using cadherin ectodomain monoclonal antibodies (mAbs), that mAb-bound E-cadherin forms separate clusters that undergo trans interactions. Taken together, our data suggest that, in addition to its role in cell-cell adhesion, CAP-driven CCC clustering serves to organize cytoplasmic proteins into distinct domains that may synchronize signaling networks of neighboring cells within tissues.

Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin.

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.

Basolateral protein Scribble binds phosphatase PP1 to establish a signaling network maintaining apicobasal polarity.

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions-(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Spatial and temporal organization of cadherin in punctate adherens junctions.

Adherens junctions (AJs) play a fundamental role in tissue integrity; however, the organization and dynamics of the key AJ transmembrane protein, E-cadherin, both inside and outside of AJs, remain controversial. Here we have studied the distribution and motility of E-cadherin in punctate AJs (pAJs) of A431 cells. Using single-molecule localization microscopy, we show that pAJs in these cells reach more than 1 µm in length and consist of several cadherin clusters with crystal-like density interspersed within sparser cadherin regions. Notably, extrajunctional cadherin appears to be monomeric, and its density is almost four orders of magnitude less than observed in the pAJ regions. Two alternative strategies of tracking cadherin motion within individual junctions show that pAJs undergo actin-dependent rapid-on the order of seconds-internal reorganizations, during which dense clusters disassemble and their cadherins are immediately reused for new clusters. Our results thus modify the classical view of AJs by depicting them as mosaics of cadherin clusters, the short lifetimes of which enable stable overall morphology combined with rapid internal rearrangements.

Cadherin controls nectin recruitment into adherens junctions by remodeling the actin cytoskeleton.

The mechanism that coordinates activities of different adhesion receptors is poorly understood. We investigated this mechanism by focusing on the nectin-2 and E-cadherin adherens junction receptors. We found that, cadherin was not required for the basic process of nectin junction formation because nectin-2 formed junctions in cadherin-deficient A431D cells. Formation of nectin-2 junctions in these cells, however, became regulated by cadherin as soon as E-cadherin was re-expressed. E-cadherin recruited nectin-2 into adherens junctions, where both proteins formed distinct but tightly associated clusters. Live-cell imaging showed that the appearance of E-cadherin clusters often preceded that of nectin-2 clusters at sites of junction assembly. Inactivation of E-cadherin clustering by different strategies concomitantly suppressed the formation of nectin clusters. Furthermore, cadherin significantly increased the stability of nectin clusters, thereby making them resistant to the BC-12 antibody, which targets the nectin-2 adhesion interface. By testing different E-cadherin-α-catenin chimeras, we showed that the recruitment of nectin into chimera junctions is mediated by the actin-binding domain of α-catenin. Our data suggests that E-cadherin regulates assembly of nectin junctions through α-catenin-induced remodeling of the actin cytoskeleton around the cadherin clusters.

Characterization of early and late events of adherens junction assembly.

Cadherins are transmembrane adhesion receptors. Cadherin ectodomains form adhesive 2D clusters through cooperative trans and cis interactions, whereas its intracellular region interacts with specific cytosolic proteins, termed catenins, to anchor the cadherin-catenin complex (CCC) to the actin cytoskeleton. How these two types of interactions are coordinated in the formation of specialized cell-cell adhesions, adherens junctions (AJ), remains unclear. We focus here on the role of the actin-binding domain of α-catenin (αABD) by showing that the interaction of αABD with actin generates actin-bound CCC oligomers (CCC/actin strands) incorporating up to six CCCs. The strands are primarily formed on the actin-rich cell protrusions. Once in cell-cell interface, the strands become involved in cadherin ectodomain clustering. Such combination of the extracellular and intracellular oligomerizations gives rise to the composite oligomers, trans CCC/actin clusters. To mature, these clusters then rearrange their actin filaments using several redundant pathways, two of which are characterized here: one depends on the α-catenin-associated protein, vinculin and the second one depends on the unstructured C-terminus of αABD. Thus, AJ assembly proceeds through spontaneous formation of trans CCC/actin clusters and their successive reorganization.

Spontaneous assembly and active disassembly balance adherens junction homeostasis.

The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its truncated mutant were recruited into the junctions with identical kinetics. The full-size cadherin was actively removed from the junctions by a process that was unaffected by the inactivation of its endocytic elements. The cadherin's apparent half-residence time in the junction was about 2 min. Cadherin clusters made of the truncated mutant exhibited much slower but ATP-independent junctional turnover. Taken together, our experiments showed that adherens junction homeostasis consists of three distinctive steps: cadherin spontaneous recruitment, its lateral catenin-dependent association, and its active release from the resulting clusters. The latter process, whose mechanism is not clear, may play an important role in various kinds of normal and abnormal morphogenesis.

Plakophilin 3 and Par3 facilitate desmosomes' association with the apical junctional complex.

Desmosomes (DSMs), together with adherens junctions (AJs) and tight junctions (TJs), constitute the apical cell junctional complex (AJC). While the importance of the apical and basolateral polarity machinery in the organization of AJs and TJs is well established, how DSMs are positioned within the AJC is not understood. Here we use highly polarized DLD1 cells as a model to address how DSMs integrate into the AJC. We found that knockout (KO) of the desmosomal ARM protein Pkp3, but not other major DSM proteins, uncouples DSMs from the AJC without blocking DSM assembly. DLD1 cells also exhibit a prominent extraDSM pool of Pkp3, concentrated in tricellular (tC) contacts. Probing distinct apicobasal polarity pathways revealed that neither the DSM's association with AJC nor the extraDSM pool of Pkp3 are abolished in cells with defects in Scrib module proteins responsible for basolateral membrane development. However, a loss of the apical polarity protein, Par3, completely eliminates the extraDSM pool of Pkp3 and disrupts AJC localization of desmosomes, dispersing these junctions along the entire length of cell-cell contacts. Our data are consistent with a model whereby Par3 facilitates DSM assembly within the AJC, controlling the availability of an assembly competent pool of Pkp3 stored in tC contacts.

Stable and unstable cadherin dimers: mechanisms of formation and roles in cell adhesion.

Numerous attempts to elucidate the strength of cadherin dimerization that mediates intercellular adhesion have produced controversial and inconclusive results. To clarify this issue, we compared E-cadherin dimerization on the surface of living cells with how the same process unfolds on agarose beads. In both cases, dimerization was monitored by the same site-specific cross-linking assay, greatly simplifying data interpretation. We showed that on the agarose surface under physiological conditions, E-cadherin produced a weak dimer that immediately dissociated after the depletion of calcium ions. However, either at pH 5 or in the presence of cadmium ions, E-cadherin produced a strong dimer that was unable to dissociate upon calcium depletion. Both types of dimers were W156-dependent. Remarkably, only the strong dimer was found on the surface of living cells. We also showed that the intracellular cadherin region, the clustering of which through catenins had been proposed as stabilizer of weak intercadherin interactions, was not needed, in fact, for cadherin junction assembly. Taken together, our data present convincing evidence that cadherin adhesion is based on high-affinity cadherin-cadherin interactions.

Sensing Actin Dynamics through Adherens Junctions.

We study punctate adherens junctions (pAJs) to determine how short-lived cadherin clusters and relatively stable actin bundles interact despite differences in dynamics. We show that pAJ-linked bundles consist of two distinct regions-the bundle stalk (AJ-BS) and a tip (AJ-BT) positioned between cadherin clusters and the stalk. The tip differs from the stalk in a number of ways: it is devoid of the actin-bundling protein calponin, and exhibits a much faster F-actin turnover rate. While F-actin in the stalk displays centripetal movement, the F-actin in the tip is immobile. The F-actin turnover in both the tip and stalk is dependent on cadherin cluster stability, which in turn is regulated by F-actin. The close bidirectional coupling between the stability of cadherin and associated F-actin shows how pAJs, and perhaps other AJs, allow cells to sense and coordinate the dynamics of the actin cytoskeleton in neighboring cells-a mechanism we term "dynasensing."

Endocytosis of cadherin from intracellular junctions is the driving force for cadherin adhesive dimer disassembly.

The adhesion receptor E-cadherin maintains cell-cell junctions by continuously forming short-lived adhesive dimers. Here mixed culture cross-linking and coimmunoprecipitation assays were used to determine the dynamics of adhesive dimer assembly. We showed that the amount of these dimers increased dramatically minutes after the inhibition of endocytosis by ATP depletion or by hypertonic sucrose. This increase was accompanied by the efficient recruitment of E-cadherin into adherens junctions. After 10 min, when the adhesive dimer amount had reached a plateau, the assembly of new dimers stalled completely. These cells, in a striking difference from the control, became unable to disintegrate both their intercellular contacts and adhesive dimers in response to calcium depletion. The same effects, but after a slightly longer time course, were obtained using acidic media, another potent approach inhibiting endocytosis. These data suggest that endocytosis is the main pathway for the dissociation of E-cadherin adhesive dimers. Its inhibition blocks the replenishment of the monomeric cadherin pool, thereby inhibiting new dimer formation. This suggestion has been corroborated by immunoelectron microscopy, which revealed cadherin-enriched coated pit-like structures in close association with adherens junctions.

Scribble, Erbin, and Lano redundantly regulate epithelial polarity and apical adhesion complex.

The basolateral protein Scribble (Scrib), a member of the LAP protein family, is essential for epithelial apicobasal polarity (ABP) in Drosophila However, a conserved function for this protein in mammals is unclear. Here we show that the crucial role for Scrib in ABP has remained obscure due to the compensatory function of two other LAP proteins, Erbin and Lano. A combined Scrib/Erbin/Lano knockout disorganizes the cell-cell junctions and the cytoskeleton. It also results in mislocalization of several apical (Par6, aPKC, and Pals1) and basolateral (Llgl1 and Llgl2) identity proteins. These defects can be rescued by the conserved "LU" region of these LAP proteins. Structure-function analysis of this region determined that the so-called LAPSDb domain is essential for basolateral targeting of these proteins, while the LAPSDa domain is essential for supporting the membrane basolateral identity and binding to Llgl. In contrast to the key role in Drosophila, mislocalization of Llgl proteins does not appear to be critical in the scrib ABP phenotype.

Homophilic and Heterophilic Interactions of Type II Cadherins Identify Specificity Groups Underlying Cell-Adhesive Behavior.

Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity.

Adhesive and lateral E-cadherin dimers are mediated by the same interface.

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.

Dynamic interplay between adhesive and lateral E-cadherin dimers.

E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin-permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states.

Exchange of catenins in cadherin-catenin complex.

beta-Catenin is an intracellular multifunctional protein. In complex with the transmembrane adhesive receptor E-cadherin, it becomes plasma membrane-associated and mediates intercellular adhesion. A cytosolic pool of beta-catenin interacts with DNA-binding proteins and participates in signal transduction. To reveal the possible cross-talk between these two pools, we studied whether beta-catenin is exchanged between its free and cadherin-bound states. We found that pulse-labeled beta-catenin replaces the beta-catenin bound to the cell surface prebiotinylated E-cadherin immediately after synthesis. Approximately 25% of all pulse-labeled beta-catenin destined for E-cadherin associates with this protein via this mechanism. The rest of the newly synthesized beta-catenin arrives at the plasma membrane in a complex with the E-cadherin precursor. Immediately after arrival, this beta-catenin pool is transferred to the prebiotinylated E-cadherin. beta-Catenin released from E-cadherin may participate in new exchange cycles. This beta-catenin exchange is strongly affected in cells that contain mutations in the tumor suppressor gene APC. This process may contribute significantly to both cell-cell adhesion and beta-catenin-dependent signaling.

α-Catenin-mediated cadherin clustering couples cadherin and actin dynamics.

The function of the actin-binding domain of α-catenin, αABD, including its possible role in the direct anchorage of the cadherin-catenin complex to the actin cytoskeleton, has remained uncertain. We identified two point mutations on the αABD surface that interfere with αABD binding to actin and used them to probe the role of α-catenin-actin interactions in adherens junctions. We found that the junctions directly bound to actin via αABD were more dynamic than the junctions bound to actin indirectly through vinculin and that recombinant αABD interacted with cortical actin but not with actin bundles. This interaction resulted in the formation of numerous short-lived cortex-bound αABD clusters. Our data suggest that αABD clustering drives the continuous assembly of transient, actin-associated cadherin-catenin clusters whose disassembly is maintained by actin depolymerization. It appears then that such actin-dependent αABD clustering is a unique molecular mechanism mediating both integrity and reassembly of the cell-cell adhesive interface formed through weak cis- and trans-intercadherin interactions.

Binding to F-actin guides cadherin cluster assembly, stability, and movement.

The cadherin extracellular region produces intercellular adhesion clusters through trans- and cis-intercadherin bonds, and the intracellular region connects these clusters to the cytoskeleton. To elucidate the interdependence of these binding events, cadherin adhesion was reconstructed from the minimal number of structural elements. F-actin-uncoupled adhesive clusters displayed high instability and random motion. Their assembly required a cadherin cis-binding interface. Coupling these clusters with F-actin through an α-catenin actin-binding domain (αABD) dramatically extended cluster lifetime and conferred direction to cluster motility. In addition, αABD partially lifted the requirement for the cis-interface for cluster assembly. Even more dramatic enhancement of cadherin clustering was observed if αABD was joined with cadherin through a flexible linker or if it was replaced with an actin-binding domain of utrophin. These data present direct evidence that binding to F-actin stabilizes cadherin clusters and cooperates with the cis-interface in cadherin clustering. Such cooperation apparently synchronizes extracellular and intracellular binding events in the process of adherens junction assembly.

Nectin ectodomain structures reveal a canonical adhesive interface.

Nectins are immunoglobulin superfamily glycoproteins that mediate intercellular adhesion in many vertebrate tissues. Homophilic and heterophilic interactions between nectin family members help mediate tissue patterning. We determined the homophilic binding affinities and heterophilic specificities of all four nectins and the related protein nectin-like 5 (Necl-5) from human and mouse, revealing a range of homophilic interaction strengths and a defined heterophilic specificity pattern. To understand the molecular basis of their adhesion and specificity, we determined the crystal structures of natively glycosylated full ectodomains or adhesive fragments of all four nectins and Necl-5. All of the crystal structures revealed dimeric nectins bound through a stereotyped interface that was previously proposed to represent a cis dimer. However, conservation of this interface and the results of targeted cross-linking experiments showed that this dimer probably represents the adhesive trans interaction. The structure of the dimer provides a simple molecular explanation for the adhesive binding specificity of nectins.