CATENINS AND ZONULA OCCLUDENS-1 FORM A COMPLEX DURING EARLY STAGES INTHE ASSEMBLY OF TIGHT JUNCTIONS

We characterized the role of the E-cadherin adhesion system in the for mation of epithelial tight junctions using the calcium switch model. I n MDCK cells cultured in low (micromolar) calcium levels, the tight ju nctional protein Zonula Occludens-1 (ZO-1) is distributed intracellula rly in granular clusters, the larger of which codistribute with E-cadh erin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to th e cell surface, where it localized in regions rich in E-cadherin. Immu noprecipitation with ZO-1 antibodies of extracts from cells kept in lo w calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the assoc iation 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-c atenin immunoprecipitates that excluded ZO-1, suggesting that the bind ing of ZO-1 to catenins may weaken the interaction of these proteins w ith E-cadherin. Immunofluorescence and immunoelectron microscopy confi rmed a close association of beta-catenin and ZO-1 at 0 and 2 h after C a2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E-cadherin an d formed a distinct, separate apical ring. The ZO-1-catenin complex wa s not detected in fully polarized monolayers. MDCK cells permanently t ransformed with Moloney sarcoma virus, which expresses low levels of E -cadherin, displayed clusters of cytoplasmic ZO-1 granules and very li ttle of this protein at the cell surface. Upon transfection with E-cad herin into Moloney sarcoma virus-MDCK cells, ZO-1 redistributed to E-c adherin-rich lateral plasma membrane but later failed to segregate int o mature tight junctions. Our experiments suggest that catenins partic ipate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic trans formation may block the formation of tight junctions, either by decrea sing the levels of E-cadherin or by preventing a late event: the segre gation of tight junction from the zonula adherens.