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的羧基末端与紧密连接的斑块蛋白ZO-1结合，位于ZO-1羧基末端的肌动蛋白结合区(ABR)与肌动蛋白细丝结合。我们以前的报道表明，当Claudin-1在其羧基末端带有myc表位的Claudin-1在MDCK细胞中表达时，沿着整个侧质膜形成了异常的紧密连接链。在这项研究中，我们检测了1CL-ZO1Cmyc或1CL-ZO1CABRmyc是否引起Δ细胞的异常紧密连接链。1CL-ZO1Cmyc由claudin-1、含有ABR的羧基末端半部分和myc表位组成。1CL-ZO1CΔABRmyc由Claudin-1、不含ABR的羧基末端部分和Myc表位组成。在表达1CL-ZO1Cmyc和1CL-ZO1CABRmyc的Δ细胞中，异常链的频率均降低，但它们仍然存在。这些结果提示，在附属膜最顶端区域维持紧密连接链可能还需要其他因素。Claudin-1形成紧密连接链，分布于大鼠附精上皮的整个侧膜，这增加了沿侧膜存在异常紧密连接链的可能性。用冷冻断裂法观察发现，附精上皮外侧膜中仅偶见有异常条索。这一结果表明，Claudin-1可能以单体的形式存在，并在侧膜上作为蛋白质池发挥作用。我们报道了Claudin-2和Claudin-10在大鼠附睾中的免疫组织化学定位和RT-PCR检测小鼠Claudin转录本的条件。

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.