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

通过过表达或抑制紧密连接斑蛋白 ZO-1 对紧密连接进行形态学和功能分析

基本信息

批准号：
16590146
负责人：
INAI Tetsuichiro
金额：
$ 2.11万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590146/
关键词：
tight junction claudin ZO-1 actin MDCK cell freeze fracture epididymis RT-PCR MDCK permeability Cx43

项目摘要

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.

紧密连接处的整合膜蛋白claudin-1的羧基末端与紧密连接处的斑块蛋白ZO-1结合，位于ZO-1羧基末端一半的肌动蛋白结合区（ABR）与肌动蛋白丝结合。我们以前的报道表明，当在MDCK细胞中表达羧基端带有myc抗原表位的claudin-1时，沿着沿着整个侧质膜形成异常的紧密连接链。在这项研究中，我们研究了1CL-ZO 1Cmyc或1CL-ZO 1C ΔABRmyc是否引起MDCK细胞中的异常紧密连接链。1CL-ZO 1Cmyc由claudin-1、含有ABR的羧基端半部分和myc表位组成。1CL-ZO 1C ΔABRmyc由claudin-1、羧基端不含ABR的一半和myc表位组成。在表达1CL-ZO 1Cmyc或1CL-ZO 1C Δ ABRmyc的MDCK细胞中，异常链的频率均降低，但它们仍然存在。这些结果表明，在侧膜最顶端区域，紧密连接链的形成可能需要其他因素的参与，Claudin-1沿着整个侧膜分布，这增加了侧膜沿着存在异常紧密连接链的可能性。冷冻断裂法发现附睾上皮侧膜上偶见异常股线。这一结果表明，claudin-1可能作为单体存在于侧膜中，并作为蛋白库发挥作用。本文报道了claudin-2和claudin-10在大鼠附睾中的免疫组织化学定位及小鼠claudin转录本的RT-PCR条件。