Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.
Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.
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通过表达突变型紧密连接膜蛋白，细胞旁通透性和跨上皮电阻的功能性分离以及顶端-基底外侧膜内扩散屏障的破坏。
DOI：
10.1083/jcb.134.4.1031
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发表时间：
1996-08
影响因子：
7.8
通讯作者：
Matter, K
中科院分区：
文献类型：
--
作者：
Balda, M S;Whitney, J A;Flores, C;Gonzalez, S;Cereijido, M;Matter, K
紧密连接是连接上皮层中单个细胞的最顶端的细胞间连接，被认为形成限制细胞旁和膜内扩散的密封。为了分析紧密连接的功能，我们产生了稳定的MDCK菌株2细胞系，表达全长或COOH末端截短的鸡闭合蛋白，唯一已知的紧密连接的跨膜成分。共聚焦免疫荧光和免疫电子显微镜表明，突变occludin被纳入紧密连接，但与全长鸡occludin相反，表现出不连续的连接染色模式，也破坏了由内源性occludin形成的连续连接环。这种重新安排occludin是没有明显的变化，在交界处的形态所看到的薄切片电子显微镜，也没有明显的不连续性观察到的交界股冷冻断裂。然而，野生型和突变occludin的表达诱导增加跨上皮电阻（TER）。与TER相反，特别是COOH末端截短的occludin的表达导致小分子量示踪剂的细胞旁通量增加数倍。由于大小或不同类型的阳离子的选择性是不变的，野生型和突变occludin的表达似乎已经激活了现有的机制，允许选择性的细胞旁通量的存在下，电密封的紧密连接。Occludin也参与形成的顶端/基底外侧膜内扩散屏障，因为COOH-末端截短occludin的表达被发现，使MDCK细胞不能维持荧光脂质在一个特定的标记细胞表面域。
Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.