Molecular architecture of adherens junctions.

Adherens junctions are composed of a cadherin-catenin complex and its associated proteins. Recently, an increasing number of novel members of adherens junctions, including membrane and PDZ proteins, have been reported. Interactions among these components in adherens junctions seem to be dynamically regulated during the formation of adherens junction complexes in epithelial cells.

alpha-catenin-independent recruitment of ZO-1 to nectin-based cell-cell adhesion sites through afadin.

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.

Posttranscriptional regulation of alpha-catenin expression is required for Wnt signaling in L cells.

alpha-Catenin is an essential component of the cadherin-catenin cell-cell adhesion complex. An excess amount of alpha-catenin also affects the Wnt signaling pathway probably through its direct binding to beta-catenin. Here, we examined the molecular mechanisms of the posttranscriptional regulation of alpha-catenin expression. We constructed an expression vector with alpha-catenin cDNA lacking the 5'-untranslated sequence. In L cell transfectants stably expressing mRNA derived from this vector, the amount of exogenous alpha-catenin protein was about 10-fold higher than that of the endogenous protein. The expression level of the exogenously expressed alpha-catenin mRNA, however, was about 80% of that of endogenous molecule. Most of the endogenous and exogenous alpha-catenin protein in cadherin-negative cells was degraded 5 h after inhibition of protein synthesis. Although alpha-catenin contains the PEST sequence, various proteasome and calpain inhibitors did not affect the level of expression of endogenous alpha-catenin protein in L cells. Overexpressed alpha-catenin showed cytoplasmic localization, disturbed the nuclear localization of stabilized beta-catenin, and inhibited TCF-4-responsive transactivation after Wnt-3a treatment. These results suggested that the low-efficiency of translation and unidentified degradation mechanisms maintained the low levels of alpha-catenin expression in the cytoplasm as a necessary condition for the Wnt signaling pathway.

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs.

Regulation of Lef-mediated transcription and p53-dependent pathway by associating beta-catenin with CBP/p300.

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that beta-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of beta-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable beta-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of beta-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

Transglutaminase type 1 and its cross-linking activity are concentrated at adherens junctions in simple epithelial cells.

Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivo labeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions.

Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions.

In multicellular organisms, various compositionally distinct fluid compartments are established by epithelial and endothelial cellular sheets. For these cells to function as barriers, tight junctions (TJs) are considered to create a primary barrier for the diffusion of solutes through the paracellular pathway [1] [2] [3]. In ultrathin sections viewed under electron microscopy, TJs appear as a series of apparent fusions, involving the outer leaflets of plasma membranes of adjacent cells, to form the so-called kissing points of TJs, where the intercellular space is completely obliterated [4]. Claudins are a family of 16 proteins whose members have been identified as major integral membrane proteins localized exclusively at TJs [5] [6] [7] [8]. It remains unclear, however, whether claudins have the cell-adhesion activity that would explain the unusual intercellular adhesion at TJs. Using mouse L-fibroblast transfectants expressing various amounts of claudin-1, -2 or -3, we found that these claudins possess Ca(2+)-independent cell-adhesion activity. Using ultrathin-section electron microscopy, we observed many kissing points of TJs between adjacent transfectants. Furthermore, the cell-adhesion activity of occludin, another integral membrane protein localized at TJs [9] [10] [11], was negligible when compared with that of claudins. Thus, claudins are responsible for TJ-specific obliteration of the intercellular space.

Co-expression of E-cadherin and alpha-catenin molecules in colorectal cancer.

Immunohistochemical staining for epithelial (E)-cadherin and alpha-catenin was performed using frozen sections taken from fresh operative specimens, by the avidin-biotin-peroxidase complex method. Tumors were classified into three types according to the expression modality. Cancer cells with expression at the cell-cell boundaries were defined as normal; when the expression was positive, but not concentrated at the cell-cell boundaries, they were defined as cytoplasmic; and when the tumor showed no staining, they were defined as lost. The relationship between these three expression types and the clinicopathological features of colorectal cancer was investigated. In all 50 normal mucosa samples, E-cadherin and alpha-catenin were coexpressed normally. The expression type of E-cadherin and alpha-catenin was normal in 11 and 13 of the cancer tissue specimens, respectively, cytoplasmic in 26 and 29, respectively, and lost in 13 and 8, respectively. Cytoplasmic or lost expression was observed in cancer demonstrating an advanced clinical stage (E-cadherin, P = 0.0065; alpha-catenin, P = 0.0069), advanced tumor penetration (P = 0.0003, P = 0.0001), undifferentiated tumor histology (P = 0.0196, P = 0.0343), widespread lymph node involvement (P = 0.0204, P = 0.0340), and liver metastasis (P = 0.0063, P = 0.0299). In conclusion, the expression type of E-cadherin is significantly correlated to that of alpha-catenin, and the loss of their expression indicates the metastatic potentiality of colorectal cancer.

alpha-catenin-deficient F9 cells differentiate into signet ring cells.

It has been demonstrated that alpha-catenin is frequently lost in diffuse type adenocarcinomas. We have isolated alpha-catenin-deficient mouse teratocarcinoma F9 cells by gene targeting. Wild-type F9 cell aggregates cultured in the presence of retinoic acid differentiated into embryoid bodies with an outer layer of epithelial cells. In contrast, cell aggregates of alpha-catenin-deficient cells did not develop outer layers under the same conditions. The outer surface cells of alpha-catenin-deficient cell aggregates, however, differentiated into epithelial cells as determined by their expression of epithelial marker proteins. These differentiated cells scattered from aggregates and showed signet ring cell morphology, which is frequently observed in diffuse type adenocarcinomas. We have provided clear evidence that a single mutation in the alpha-catenin gene may be a direct cause not only of the scattered properties of cells but also of signet ring cell formation in diffuse type adenocarcinoma.

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

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

alpha-Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.

alphaE-catenin, a cadherin-associated protein, is required for tight junction (TJ) organization, but its role is poorly understood. We transfected an alphaE-catenin-deficient colon carcinoma line with a series of alphaE-catenin mutant constructs. The results showed that the amino acid 326-509 domain of this catenin was required to organize TJs, and its COOH-terminal domain was not essential for this process. The 326-509 internal domain was found to bind vinculin. When an NH2-terminal alphaE-catenin fragment, which is by itself unable to organize the TJ, was fused with the vinculin tail, this chimeric molecule could induce TJ assembly in the alphaE-catenin-deficient cells. In vinculin-null F9 cells, their apical junctional organization was impaired, and this phenotype was rescued by reexpression of vinculin. These results indicate that the alphaE-catenin-vinculin interaction plays a role in the assembly of the apical junctional complex in epithelia.

Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.

The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.

Aberrant E-cadherin and alpha-catenin expression in prostate cancer: correlation with patient survival.

E-cadherin maintains the normal differentiated phenotype in epithelial cells; this function is partly mediated by alpha-catenin, which links E-cadherin to the cell cytoskeleton. Dysfunction of E-cadherin in vitro and in vivo is associated with an invasive phenotype. However, the role of alpha-catenin is largely undetermined. We analyzed the expression of E-cadherin and alpha-catenin in prostate cancer to assess the relationship of abnormal expression to stage, grade and survival. E-cadherin expression was evaluated in 99 prostate cancers. In 79 of those specimens, alpha-catenin was also assessed. In benign prostatic epithelium, both E-cadherin and alpha-catenin were expressed uniformly at the cell membrane. Abnormal E-cadherin expression was found in 56% of cancer specimens, whereas alpha-catenin expression was abnormal in 42%. Abnormal expression of each molecule was significantly correlated with Gleason score (P < 0.0001) and the ratio of resection chippings infiltrated by tumor (P < 0.0001). E-cadherin expression was also associated with the extent of disease on the initial bone scan (P = 0.017). Univariate analysis showed significantly lower survival rate for patients with abnormal E-cadherin (P = 0.0003) or alpha-catenin expression (P = 0.031). Multivariate regression analysis showed that the prognostic value of E-cadherin was independent of tumor grade but not of metastasis. These results suggest that perturbation of cell-cell adhesion is involved in the progression of prostate cancer and that analysis of E-cadherin expression may be clinically useful.

Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with alpha catenin, but not to that with beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin was approximately 0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of approximately 10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

Suppression of invasive ability of highly metastatic rat prostate cancer by introduction of human chromosome 8.

BACKGROUND: Introduction of human chromosome 8 to a highly metastatic subline (AT6.2) from the Dunning R-3327 rat prostate cancer resulted in suppression of metastatic ability of the resultant microcell hybrids (AT6.2-8 clones) [12]. The present study has been performed to clarify which step of metastasis was suppressed in the microcell hybrids. METHODS: Northern blot analysis of E-cadherin and alpha-catenin, in vitro invasion assay, and intra-venous metastasis assay by injection of tumor cells into the lateral tail vein of nude mice were performed. RESULTS: No detectable expressions of either E-cadherin or alpha-catenin were found in either AT6.2 parental or AT6.2-8 microcell hybrid clones. In the invasion assay, invasiveness of AT6.2-8 hybrid clones was less than that of the AT6.2 parental clone. In the intravenous metastasis assay, no significant differences in the number of lung metastases were observed among these cell lines. CONCLUSIONS: Introduction of human chromosome 8 to AT6.2 cells shows suppression of invasiveness and no suppression of cell dissociation or process after entry into blood circulation. This suggests that human chromosome 8 contains suppressor gene(s) for the invasive ability of prostate cancer.

Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation.

We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift.

The elevation of tyrosine phosphorylation level is thought to induce the dysfunction of cadherin through the tyrosine phosphorylation of beta catenin. We evaluated this assumption using two cell lines. First, using temperature-sensitive v-src-transfected MDCK cells, we analyzed the modulation of cadherin-based cell adhesion by tyrosine phosphorylation. Cell aggregation and dissociation assays at nonpermissive and permissive temperatures indicated that elevation of the tyrosine phosphorylation does not totally affect the cell adhesion ability of cadherin but shifts it from a strong to a weak state. The tyrosine phosphorylation levels of beta catenin, ZO-1, ERM (ezrin/radixin/moesin), but not alpha catenin, vinculin, and alpha-actinin, were elevated in the weak state. To evaluate the involvement of the tyrosine phosphorylation of beta catenin in this shift of cadherin-based cell adhesion, we introduced v-src kinase into L fibroblasts expressing the cadherin-alpha catenin fusion protein, in which beta catenin is not involved in cell adhesion. The introduction of v-src kinase in these cells shifted their adhesion from a strong to a weak state. These findings indicated that the tyrosine phosphorylation of beta catenin is not required for the strong-to-weak state shift of cadherin-based cell adhesion, but that the tyrosine phosphorylation of other junctional proteins, ERM, ZO-1 or unidentified proteins is involved.

Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells.

Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF relationship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, alpha-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, alpha-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron microscopic level these spot-like AJ were seen as aggregates of small 'units' of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.