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.     

Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.     

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.     

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

Cadherin exits the junction by switching its adhesive bond

The plasticity of cell-cell adhesive structures is crucial to all normal and pathological morphogenetic processes. The molecular principles of this plasticity remain unknown. Here we study the roles of two dimerization interfaces, the so-called strand-swap and X dimer interfaces of E-cadherin, in the dynamic remodeling of adherens junctions using photoactivation, calcium switch, and coimmunoprecipitation assays. We show that the targeted inactivation of the X dimer interface blocks the turnover of catenin-uncoupled cadherin mutants in the junctions of A-431 cells. In contrast, the junctions formed by strand-swap dimer interface mutants exhibit high instability. Collectively, our data demonstrate that the strand-swap interaction is a principal cadherin adhesive bond that keeps cells in firm contact. However, to leave the adherens junction, cadherin reconfigures its adhesive bond from the strand swap to the X dimer type. Such a structural transition, controlled by intercellular traction forces or by lateral cadherin alignment, may be the key event regulating adherens junction dynamics.     

E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.     

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.     

Caveolae Mediate Growth Factor-induced Disassembly of Adherens Junctions to Support Tumor Cell Dissociation

Remodeling of cell–cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell–cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell–cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.     

Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

The dynamic control of E-cadherin is critical for establishing and maintaining cell-cell junctions in epithelial cells. The concentration of E-cadherin molecules at adherens junctions (AJs) is regulated by lateral movement of E-cadherin within the plasma membrane and endocytosis. Here we set out to study the interplay between these processes and their contribution to E-cadherin dynamics. Using photoactivation (PA) and fluorescence recovery after photobleaching (FRAP), we were able to monitor the fate of E-cadherin molecules within the plasma membrane. Our results suggest that the motility of E-cadherin within and away from the cell surface are not exclusive or independent mechanisms and there is a fine balance between the two which, when perturbed, can have dramatic effects on the regulation of AJs.Key words: E-cadherin, adherens junctions, plasma membrane, protein dynamics, photoactivation, photobleaching     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Rac1, but not RhoA,signaling protects epithelial adherens junction assembly during ATP depletion

Rhofamily GTPase signaling regulates actin cytoskeleton and junctionalcomplex assembly. Our previous work showed that RhoA signaling protectstight junctions from damage during ATP depletion. Here, we examinedwhether RhoA GTPase signaling protects adherens junction assemblyduring ATP depletion. Despite specific RhoA signaling- and ATPdepletion-induced effects on adherens junction assembly, RhoA signalingdid not alter adherens junction disassembly rates during ATP depletion.This shows that RhoA signaling specifically protects tight junctionsfrom damage during ATP depletion. Rac1 GTPase signaling also regulatesadherens junction assembly and therefore may regulate adherens junctionassembly during ATP depletion. Indeed, we found that Rac1 signalingprotects adherens junctions from damage during ATP depletion. Adherensjunctions are regulated by various GTPases, including RhoA and Rac1,but adherens junctions are specifically protected by Rac1 signaling.

     

Making and breaking contacts: the cellular biology of cadherin regulation

Cadherin-mediated cell-cell interactions are dynamic processes, and cadherin function is tightly regulated in response to cellular context and signaling. Ultimately, cadherin regulation is likely to reflect the interplay between a range of fundamental cellular processes, including surface organization of receptors, cytoskeletal organization and cell trafficking, that are coordinated by signaling events. In this review we focus on recent advances in understanding how interplay with membrane trafficking and other cell-cell junctions can control cadherin function. The endocytosis of cadherins, and their post-internalization fate, influences surface expression and metabolic stability of these adhesion receptors. Similarly, at the surface, components of tight junctions provide a mode of cross-talk that regulates assembly of adherens junctions.     

Cyclic AMP-dependent protein kinase A negatively modulates adherens junction integrity and differentiation of intestinal epithelial cells

Intestinal epithelial cell differentiation is a complex process in which many different signaling pathways are likely involved. An increase in the intracellular levels of cyclic AMP (cAMP) has been shown to inhibit enterocyte differentiation; however, the mechanisms through which cAMP/PKA signaling modulates differentiation of human intestinal epithelial cells are still not well understood. Herein, we report that: (1) treatment of Caco-2/15 cells with 8Br-cAMP repressed sucrase-isomaltase and villin protein expression and strongly attenuated morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush border formation; (2) treatment of confluent Caco-2/15 cells with 8Br-cAMP led to a strong decrease in F-actin localized at cell-cell contact sites along with a reduced amount of E-cadherin and catenins, but not of ZO-1, at cell-cell interfaces concomitant with a decreased association of these proteins with the actin cytoskeleton; (3) inhibition of PKA by H89 prevented disruption of adherens junctions by extracellular calcium depletion; (4) treatment of Caco-2/15 cells with 8Br-cAMP prevented the recruitment and activation of p85/PI-3K to E-cadherin-mediated cell-cell contacts, an important event in the assembly of adherens junctions and differentiation of these cells; (5) E-cadherin appears to be phosphorylated on serine in vivo in a PKA-dependent mechanism. Conclusion: Our studies show that cAMP/PKA signaling negatively regulates adherens junction integrity as well as morphological and functional differentiation of intestinal epithelial cells.     

Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry

Nectin-1, a cell adhesion molecule belonging to the immunoglobulin superfamily, can bind to virion glycoprotein D (gD) to mediate entry of herpes simplex viruses (HSV) and pseudorabies virus (PRV). Nectin-1 colocalizes with E-cadherin at adherens junctions in epithelial cells. The disruption of cell junctions can result in the redistribution of nectin-1. To determine whether disruption of junctions by calcium depletion influenced the susceptibility of epithelial cells to viral entry, Madin-Darby canine kidney cells expressing endogenous nectin-1 or transfected human nectin-1 were tested for the ability to bind soluble forms of viral gD and to be infected by HSV and PRV, before and after calcium depletion. Confocal microscopy revealed that binding of HSV and PRV gD was localized to adherens junctions in cells maintained in normal medium but was distributed, along with nectin-1, over the entire cell surface after calcium depletion. Both the binding of gD and the fraction of cells that could be infected by HSV-1 and PRV were enhanced by calcium depletion. Taken together, these results provide evidence that nectin-1 confined to adherens junctions in epithelial cells is not very accessible to virus, whereas dissociation of cell junctions releases nectin-1 to serve more efficiently as an entry receptor.     

Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

The dynamic control of E-cadherin is critical for establishing and maintaining cell-cell junctions in epithelial cells. The concentration of E-cadherin molecules at adherens junctions (AJs) is regulated by lateral movement of E-cadherin within the plasma membrane and endocytosis. Here we set out to study the interplay between these processes and their contribution to E-cadherin dynamics. Using photoactivation (PA) and fluorescence recovery after photobleaching (FRAP) we were able to monitor the fate of E-cadherin molecules within the plasma membrane. Our results suggest that the motility of E-cadherin within, and away from, the cell surface are not exclusive or independent mechanisms and there is a fine balance between the two which when perturbed can have dramatic effects on the regulation of AJs.     

Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to Src-mediated pathway

E-cadherins play an essential role in maintaining epithelial polarity by forming Ca2+-dependent adherens junctions between epithelial cells. Here, we report that Ca2+ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca2+ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca2+ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.     

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Cadherin tales: Regulation of cadherin function by endocytic membrane trafficking

Cadherins are the primary adhesion molecules in adherens junctions and desmosomes and play essential roles in embryonic development. Although significant progress has been made in understanding cadherin structure and function, we lack a clear vision of how cells confer plasticity upon adhesive junctions to allow for cellular rearrangements during development, wound healing and metastasis. Endocytic membrane trafficking has emerged as a fundamental mechanism by which cells confer a dynamic state to adhesive junctions. Recent studies indicate that the juxtamembrane domain of classical cadherins contains multiple endocytic motifs, or “switches,” that can be used by cellular membrane trafficking machinery to regulate adhesion. The cadherin‐binding protein p120‐catenin (p120) appears to be the master regulator of access to these switches, thereby controlling cadherin endocytosis and turnover. This review focuses on p120 and other cadherin‐binding proteins, ubiquitin ligases, and growth factors as key modulators of cadherin membrane trafficking.