Morphological and functional analysis of tight junction by overexpression or suppression of tight junction plaque protein ZO-1

紧密连接的完整膜蛋白claudin-1的羧基端与紧密连接的斑块蛋白ZO-1结合，位于ZO-1羧基端一半的肌动蛋白结合区（ABR）与肌动蛋白丝结合。我们之前的报道表明，当在MDCK细胞中羧基端表达带有myc表位的claudin-1时，沿整个侧质膜形成异常的紧密连接链。在这项研究中，我们检测了1CL-ZO1Cmyc或1CL-ZO1CΔABRmyc是否导致MDCK细胞异常紧密连接链。1CL-ZO1Cmyc由claudin-1、含有ABR的羧基末端半段和myc表位组成。1CL-ZO1CΔABRmyc由claudin-1、无ABR的羧基末端一半和myc表位组成。在1CL-ZO1Cmyc-或1CL-ZO1CΔABRmyc-expressing MDCK细胞中，畸变链的频率都降低了，但它们仍然存在。这些结果表明，其他因素可能是必要的，以保持紧密的连接链，在最顶端区域的外侧膜。Claudin-1在大鼠附睾上皮的整个外侧膜上形成紧密连接链，这就提出了在外侧膜上存在异常紧密连接链的可能性。冻裂法发现附睾上皮外侧膜偶有异常链存在。这一结果表明，claudin-1可能作为一个单体存在，并作为一个蛋白质池在侧膜中发挥作用。我们报道了大鼠附睾中claudin-2和-10的免疫组织化学定位以及小鼠claudin转录本的RT-PCR情况。

The carboxyl terminus of claudin-1, an integral membrane protein at tight junctions, binds to ZO-1 which is a plaque protein at tight junctions, and the actin binding region (ABR) localized at the carboxyl terminal half of ZO-1 binds to actin filaments. Our previous report showed that aberrant tight junction strands were formed along entire lateral plasma membrane when claudin-1 with myc-epitope at its carboxyl terminus was expressed in MDCK cells. In this study, we examined whether 1CL-ZO1Cmyc or 1CL-ZO1CΔABRmyc caused aberrant tight junction strands in MDCK cells. 1CL-ZO1Cmyc consisted of claudin-1, carboxyl terminal half containing ABR, and myc epitope. 1CL-ZO1CΔABRmyc consisted of claudin-1, carboxyl terminal half without ABR, and myc epitope. The frequency of aberrant strands was reduced both in 1CL-ZO1Cmyc- or 1CL-ZO1CΔABRmyc-expressing MDCK cells, but they still existed. These results suggest that other factors may be necessary for maintaining tight junction strands at the most apical region of lateral membranes.Claudin-1 which forms tight junction strands localized along the entire lateral membrane of rat epididymal epithelium, and this raises the possibility that aberrant tight junction strands may exist along lateral membrane. We found using freeze fracture method that aberrant strands only occasionally existed in lateral membrane of epididymal epithelium. This result suggests that claudin-1 may exist as a monomer and function as a pool of protein in lateral membrane. We reported immunohistochemical localization of claudin-2 and -10 in rat epididymis and condition of RT-PCR for mouse claudin transcripts.