MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins.

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

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.

MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins.

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

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.

Adherens junction: the ensemble of specialized cadherin clusters.

The cell-cell connections in adherens junctions (AJs) are mediated by transmembrane receptors, type I cadherins (referred to here as cadherins). These cadherin-based connections (or trans bonds) are weak. To upregulate their strength, cadherins exploit avidity, the increased affinity of binding between cadherin clusters compared with isolated monomers. Formation of such clusters is a unique molecular process that is driven by a synergy of direct and indirect cis interactions between cadherins located at the same cell. In addition to their role in adhesion, cadherin clusters provide structural scaffolds for cytosolic proteins, which implicate cadherin into different cellular activities and signaling pathways. The cluster lifetime, which depends on the actin cytoskeleton, and on the mechanical forces it generates, determines the strength of AJs and their plasticity. The key aspects of cadherin adhesion, therefore, cannot be understood at the level of isolated cadherin molecules, but should be discussed in the context of cadherin clusters.

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.

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.

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.

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."

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.

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.

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.

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.

E-cadherin junction formation involves an active kinetic nucleation process.

Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity.

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell-cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell-cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell-cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell-cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell-cell contact fluidity.

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.

Afadin controls cadherin cluster stability using clathrin-independent mechanism.

Afadin is an actin-binding protein that interacts with the intracellular region of the transmembrane proteins, nectins. In collaboration with other transmembrane proteins, cadherins, nectins form adherens junctions, a major type of cell-cell adhesive structures in the multicellular organisms. To elucidate the afadin function, we studied adherens junction defects induced by afadin depletion in epithelial A431 cells. We have found that the cells lacking afadin exhibit no abnormalities in morphology or in general dynamics of adherens junctions in the confluent cell cultures. The only observed difference is a slight increase in the rate of cadherin turnover in these junctions. However, afadin depletion strongly affects the assembly of new adherens junctions immediately after two cells touch one another: initiation of new junctions is significantly delayed, the growth of the nascent junctions stagnates, and their lifetime shortens. As a result, the afadin-depleted cells need much more time to establish the mature junctional structures. This defect is not caused by the clathrin-dependent endocytosis of cadherin clusters that was monitored using live-cell imaging of A431 cells co-expressing GFP-tagged E-cadherin and mCherry-tagged clathrin light chain. Taken together our data show that afadin reinforces adherens junctions and that this process is crucial for the fast formation of adherens junctions at the sites of new cell-cell contacts.

The adherens junction: a mosaic of cadherin and nectin clusters bundled by actin filaments.

Cadherin and nectin are distinct transmembrane proteins of adherens junctions. Their ectodomains mediate adhesion, whereas their cytosolic regions couple the adhesive contact to the cytoskeleton. Both these proteins are essential for adherens junction formation and maintenance. However, some basic aspects of these proteins, such as their organization in adherence junctions, have remained open. Therefore, using super-resolution microscopy and live imaging, we focused on the subjunctional distribution of these proteins. We showed that cadherin and nectin in the junctions of A431 cells and human keratinocytes are located in separate clusters. The size of each cluster is independent of that of the adjacent clusters and can significantly fluctuate over time. Several nectin and cadherin clusters that constitute an individual adherens junction are united by the same actin-filament bundle. Surprisingly, interactions between each cluster and F-actin are not uniform, as neither vinculin nor LIM-domain actin-binding proteins match the boundaries of cadherin or nectin clusters. Thus, the adherens junction is not a uniform structure but a mosaic of different adhesive units with very diverse modes of interaction with the cytoskeleton. We propose that such a mosaic architecture of adherence junctions is important for the fast regulation of their dynamics.