The extracellular domains of E- and N-cadherin determine the scattered punctate localization in epithelial cells and the cytoplasmic domains modulate the localization

Abstract

The accumulation of classical cadherins is essential for their function, but the mechanism is poorly understood. Hence, we investigated the accumulation of E- and N-cadherin and the formation of cell junctions in epithelial cells. Immunostaining revealed a scattered dot-like accumulation of E- and N-cadherin throughout the lateral membrane in MDCK II and other epithelial cells. Mutant E-cadherin lacking the beta-catenin binding site accumulated granularly at cell-cell contact sites and showed weak cell aggregation activity in cadherin-deficient epithelial cells, MIA PaCa2 cells. Mutant E-cadherin lacking the p120-catenin binding site exhibited scattered punctate accumulation and strong cell adhesion activity in MIA PaCa2 cells. Electron microscopy demonstrated that MIA PaCa2 transfectants of E-cadherin containing beta-catenin binding site formed adherens junction, whereas E-cadherin lacking the binding site did not. Mutant N-cadherins showed accumulation properties similar to those of corresponding mutant E-cadherins. Moreover, wild type and mutant N-cadherin lacking the p120-catenin binding site showed subapical accumulation in polarized DLD-1 cells, whereas mutant N-cadherin lacking beta-catenin binding site did not. These results indicate that the extracellular domains of E- and N-cadherin determines the basic localization pattern, whereas the cytoplasmic domains modulate it thereby affects the cell adhesion activity, subapical accumulation, and the formation of adherens junction.

p120-catenin Is Essential for N-cadherin-mediated Formation of Proper Junctional Structure, thereby Establishing Cell Polarity in Epithelial Cells

“…3 and Fig. 4C), as had been reported for the corresponding mutant of E-cadherin (Ozaki et al, 2010). The N-cadherin mutant lacking the p120-catenin-binding site (Ncad/Δp120) gave similar results (unpublished observation).…”

“…HA-tag was attached to the C-termini of these N-cadherin constructs. E-cadherin mutants corresponding to the above N-cadherin mutants were described previously (Ozaki et al, 2010). Amino acid sequences of cadherins are numbered from the N-terminus of the mature molecules in this study.…”

“…Immunofluorescence staining, electron microscopy, SDS-PAGE, and immunoblot analysis were carried out as described previously (Ozaki et al, 2010). Statistical analyses were performed by using Student's t-test.…”

“…However, we may have missed some important features of classical cadherins by these studies, since Ecadherin is mainly expressed in epithelial cells, not in fibroblasts. Hence, in the present study we used the epithelial cell line MIA PaCa-2 (MIA2), a pancreatic adenocarcinoma, which does not express classical cadherins, but can support the strong cell adhesion activity of classical cadherins when transfected with them (Fukumoto et al, 2008;Ozaki et al, 2010). In addition, we chose N-cadherin as a model classical cadherin and studied the properties of its cytoplasmic domain by mutagenesis in this study, since N-cadherin is not endocytosed much even in the absence of p120-cateninor β-catenin-binding site (Chen et al, 2003;Ozawa, 2003), thus providing a simpler experimental system for the study of function of the cytoplasmic domain than that of Ecadherin.…”

Epithelial DLD-1 Cells with Disrupted E-cadherin Gene Retain the Ability to Form Cell Junctions and Apico-basal Polarity

“…However, no such cell line is readily available, and the generation of an E-cadherin null cell line has been quite difficult, if not impossible. Indeed, E-cadherin-deficient epithelial cells such as A431D and MIA PaCa2 cells have been reported (Lewis et al, 1997;Ozaki et al, 2010), but they have not been used widely because of several difficulties such as the expression of other cadherins and no apico-basal polarity formation.…”