Rajasekaran A K, Hojo M, Huima T, Rodriguez-Boulan E
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021, USA.
J Cell Biol. 1996 Feb;132(3):451-63. doi: 10.1083/jcb.132.3.451.
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.
我们使用钙转换模型来研究E-钙黏蛋白黏附系统在上皮紧密连接形成中的作用。在低（微摩尔）钙水平培养的MDCK细胞中，紧密连接蛋白闭合蛋白-1（ZO-1）以颗粒簇的形式分布于细胞内，其中较大的颗粒与E-钙黏蛋白共分布。在转移至正常（1.8 mM）钙水平激活E-钙黏蛋白黏附两小时后，ZO-1显著重新分布至细胞表面，定位于富含E-钙黏蛋白的区域。用ZO-1抗体对处于低钙状态的细胞提取物以及转移至1.8 mM Ca2+两小时后的细胞提取物进行免疫沉淀，结果表明ZO-1与α-、β-和γ-连环蛋白存在关联。在ZO-1免疫沉淀物中未检测到E-钙黏蛋白，但在排除了ZO-1的β-连环蛋白免疫沉淀物中发现了E-钙黏蛋白，这表明ZO-1与连环蛋白的结合可能会削弱这些蛋白与E-钙黏蛋白的相互作用。免疫荧光和免疫电子显微镜证实了在Ca2+转换后0小时和2小时，β-连环蛋白与ZO-1紧密关联。在Ca2+转换48小时后，上皮细胞完全极化时，大部分ZO-1已与侧面的E-钙黏蛋白分离，并形成一个明显的、独立的顶端环。在完全极化的单层细胞中未检测到ZO-1-连环蛋白复合物。用莫洛尼肉瘤病毒永久转化的MDCK细胞，其E-钙黏蛋白表达水平较低，显示出细胞质ZO-1颗粒簇，且该蛋白在细胞表面含量极少。在用E-钙黏蛋白转染莫洛尼肉瘤病毒-MDCK细胞后，ZO-1重新分布至富含E-钙黏蛋白的侧面质膜，但随后未能分离形成成熟的紧密连接。我们的实验表明，连环蛋白在紧密连接形成早期参与了ZO-1从胞质溶胶向细胞表面的转运，并且肿瘤转化可能会通过降低E-钙黏蛋白水平或阻止一个后期事件：紧密连接与黏着带的分离，从而阻碍紧密连接的形成。
