We characterized the role of the E-cadherin adhesion system in the formation of epithelial tight junctions using the calcium switch model. In MDCK cells cultured in low (micromolar) calcium levels, the tight junctional protein Zonula Occludens-1 (ZO-1) is distributed intracellularly in granular clusters, the larger of which codistribute with E-cadherin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to the cell surface, where it localized in regions rich in E-cadherin. Immunoprecipitation with ZO-1 antibodies of extracts from cells kept in low calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the association of ZO-1 with alpha-, beta-, and gamma-catenins. E-cadherin was not detected in the ZO-1 immunoprecipitates but it was found in beta-catenin immunoprecipitates that excluded ZO-1, suggesting that the binding of ZO-1 to catenins may weaken the interaction of these proteins with E-cadherin. Immunofluorescence and immunoelectron microscopy confirmed a close association of beta-catenin and ZO-1 at 0 and 2 h after Ca2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E-cadherin and formed a distinct, separate apical ring. The ZO-1-catenin complex was not detected in fully polarized monolayers. MDCK cells permanently transformed with Moloney sarcoma virus, which expresses low levels of E-cadherin, displayed clusters of cytoplasmic ZO-1 granules and very little of this protein at the cell surface. Upon transfection with E-cadherin into Moloney sarcoma virus-MDCK cells, ZO-1 redistributed to E-cadherin-rich lateral plasma membrane but later failed to segregate into mature tight junctions. Our experiments suggest that catenins participate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic transformation may block the formation of tight junctions, either by decreasing the levels of E-cadherin or by preventing a late event: the segregation of tight junction from the zonula adherens.

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