Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions.
Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions.
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DOI：
10.1083/jcb.132.3.451
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发表时间：
1996-02
期刊：
影响因子：
--
通讯作者：
Rodriguez-Boulan E
中科院分区：
文献类型：
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作者：
Rajasekaran AK;Hojo M;Huima T;Rodriguez-Boulan E
我们利用钙开关模型表征了E-钙粘附素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ZO-1分布在细胞内的颗粒团中，其中较大的颗粒团与E-钙粘素共同分布。在通过转移到正常钙水平(1.8 mM)激活E-钙粘附素黏附2小时后，ZO-1显著地重新分布到细胞表面，在那里它定位于富含E-钙粘附素的区域。用ZO-1抗体免疫沉淀保持在低钙状态的细胞提取物，并在迁移到1.8 mM钙离子后2小时显示ZO-1与α-、β-和伽马-连环蛋白的关联。在ZO-1免疫沉淀物中未检测到E-钙粘蛋白，但在不含ZO-1的β-连环素免疫沉淀物中检测到E-钙粘蛋白，提示ZO-1与连环素的结合可能削弱了这些蛋白与E-钙粘蛋白的相互作用。免疫荧光和免疫电子显微镜证实，钙离子交换后0 h和2 h，β-连环蛋白与ZO-1密切相关。钙离子交换后48h，当上皮细胞完全极化时，大部分ZO-1已从外侧E-钙粘蛋白分离，并形成一个明显的、独立的顶环。在完全极化的单分子膜中未检测到ZO-1-连环蛋白复合体。用Moloney肉瘤病毒永久转化的MDCK细胞表达低水平的E-钙粘附素，在细胞表面显示出成簇的胞质ZO-1颗粒和极少的这种蛋白。将E-钙粘蛋白导入Moloney肉瘤病毒-MDCK细胞后，ZO-1重新分布到富含E-钙粘蛋白的侧质膜上，但后来未能分离成成熟的紧密连接。我们的实验表明，在紧密连接发育的早期，连接素参与了ZO-1从胞浆到细胞表面的动员，肿瘤转化可能通过降低E-钙粘附素的水平或通过防止后期事件：紧密连接与小带的分离而阻止紧密连接的形成。
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.