αE-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly

αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.     

Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

As part of the E-cadherin–β-catenin–αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell–cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin–dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell–cell adhesion.     

α-Catenin and Vinculin Cooperate to Promote High E-cadherin-based Adhesion Strength

Maintaining cell cohesiveness within tissues requires that intercellular adhesions develop sufficient strength to support traction forces applied by myosin motors and by neighboring cells. Cadherins are transmembrane receptors that mediate intercellular adhesion. The cadherin cytoplasmic domain recruits several partners, including catenins and vinculin, at sites of cell-cell adhesion. Our study used force measurements to address the role of αE-catenin and vinculin in the regulation of the strength of E-cadherin-based adhesion. αE-catenin-deficient cells display only weak aggregation and fail to strengthen intercellular adhesion over time, a process rescued by the expression of αE-catenin or chimeric E-cadherin·αE-catenins, including a chimera lacking the αE-catenin dimerization domain. Interestingly, an αE-catenin mutant lacking the modulation and actin-binding domains restores cadherin-dependent cell-cell contacts but cannot strengthen intercellular adhesion. The expression of αE-catenin mutated in its vinculin-binding site is defective in its ability to rescue cadherin-based adhesion strength in cells lacking αE-catenin. Vinculin depletion or the overexpression of the αE-catenin modulation domain strongly decreases E-cadherin-mediated adhesion strength. This supports the notion that both molecules are required for intercellular contact maturation. Furthermore, stretching of cell doublets increases vinculin recruitment and α18 anti-αE-catenin conformational epitope immunostaining at cell-cell contacts. Taken together, our results indicate that αE-catenin and vinculin cooperatively support intercellular adhesion strengthening, probably via a mechanoresponsive link between the E-cadherin·β-catenin complexes and the underlying actin cytoskeleton.     

Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.     

The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules

The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called alpha and beta catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and alpha catenin, nE alpha, nE alpha N, and nE alpha C, where the intact, amino- terminal and carboxy-terminal half of alpha catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE alpha and nE alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-catenin complex, the mechanical association of alpha catenin, especially its carboxy- terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE alpha molecules during cytokinesis was quite different from that of intact E- cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE alpha was not associated with endogenous beta catenin in transfectants, the difference in the nature of cell adhesion between nE alpha and intact E-cadherin transfectants may be explained by the function of beta catenin. The possible functions of beta catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.     

E-cadherin expression in a particular subset of sensory neurons.

We found that the dorsal root ganglia (DRG) and trigeminal ganglia of mouse embryos express the E-cadherin cell-cell adhesion molecule and analyzed its expression profile. E-cadherin expression began around Embryonic Day 12 (E12) in these ganglia, thereafter increased, and persisted to the adult stage. This cadherin was expressed by 10 and 30% of DRG neurons in E17 and postnatal animals, respectively, as well as by satellite cells and some Schwann cells. E-cadherin-positive primary sensory fibers terminated only in a narrow region of the dorsal horn of the spinal cord, which was identified as part of lamina II by double-staining for E-cadherin and substance P or somatostatin. This E-cadherin expressing area of the spinal cord extended to part of the trigeminal nucleus in the medulla. These results showed that E-cadherin is expressed in a particular subset of primary sensory neurons which may have specific functional properties. We suggest that this adhesion molecule may play a role in the selective adhesion of sensory neuronal fibers.     

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.     

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressorgene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.     

IKK&alpha; enhances human keratinocyte differentiation and determines the histological variant of epidermal squamous cell carcinomas

Squamous cell carcinomas (SCCs) of the skin display different clinical features according to their epithelial differentiation grade and histological variant. Understanding the causes of these divergences might increase the curability of SCCs. Therefore, it is important to study the mechanisms of differentiation in keratinocytes. IKK (I&kappa;B kinase) &alpha; is an important protein for epidermal morphogenesis, although the pathways through which it exerts its function are unknown and controversy exists about its role in cancer development. We show that enhanced IKK&alpha; expression increases both early and terminal differentiation of human keratinocytes through an E-cadherin-dependent mechanism. Increased expression of IKK&alpha; in mouse tumorigenic epidermal cells leads to changes in the differentiation pattern of the resulting SCCs, originating a distinct histological variant that resembles the human acantholytic SCC (ASCC) variant. Although human ASCCs have an aggressive clinical course and high risk of metastasis, nothing is known about their etiology. We show that human ASCCs, as observed in the counterpart IKK&alpha; murine tumors, express high levels of both IKK&alpha; and E-cadherin, with absence of keratins K1 and K10, usually co-expressed with IKK&alpha; and E-cadherin. The tight correlation between the properties of both murine and human ASCC variants strongly suggests that IKK&alpha; is responsible for the development of this human SCC variant.     

TGF-β1-promoted epithelial-to-mesenchymal transfor mation and cell adhesion contribute to TGF-β1-enhanced cell migration in SMMC-7721 cells

Transforming growth factor-bl (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 losesits growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cellmigration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of ct5131 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin(Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln).Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment.Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β1-catenin. These results indicated that TGF-β1-promoted cell adhesion and TGF-β1-induced epithelial-to-mesenchymal transfor-mation might be both responsible for TGF-β1-enhanced cell migration.     

E-Cadherin–dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell–cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell– cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell–cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27^kip1 and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin–neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin–dependent growth inhibition, we engineered E-cadherin–positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin–neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

Induction of E-cadherin endocytosis by loss of protein phosphatase 2A expression in human breast cancers

The cell-cell adhesion molecule E-cadherin is stabilized by linking intracellularly with the actin cytoskeleton through PP2A-mediated recruitment of IQGAP1 to Rac1-bound E-cadherin-catenins complex in nonmalignant HME cells. However, little is known about the dysfunction of E-cadherin by loss or reduced expression of PP2A in human breast cancer cells. We report here that both human breast cancer MDA-MB-231 and MCF-7 cells were deficient in expression of the PP2A-A protein and lost the IQGAP1 recruitment to Rac1-bound catenins. In MDA-MB-231 cells, E-cadherin was also deficient. Immunohistochemical analysis of the normal-carcinoma matched human breast tissue arrays revealed that PP2A-A was expressed in 96% of normal tissue specimens but not in 57% of carcinoma specimens. Expression of E-cadherin in MCF-7 cells was 1.5-fold higher than that in HME cells, however, 80% of E-cadherin was endocytosed and incompletely anchored to F-actin. Therefore, we propose that the dysfunction of E-cadherin due to its endocytosis may occur in some proportion of human breast carcinomas in which the PP2A-A protein is lost or significantly reduced.     

α–E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic

α–Epithelial catenin (E-catenin) is an important cell–cell adhesion protein. In this study, we show that α–E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in α–E-catenin^−/− keratinocytes, an effect that is reversed by expression of exogenous α–E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that α–E-catenin regulates traffic independently from its function in cell–cell adhesion. Although neither the integrity of dynactin–dynein complexes nor their association with vesicles is affected by α–E-catenin, α–E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of α–E-catenin is necessary for this regulation, we hypothesize that α–E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.