Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, Tsukita S
Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
J Cell Biol. 1998 Apr 20;141(2):397-408. doi: 10.1083/jcb.141.2.397.
Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.
闭合蛋白是紧密连接（TJ）中唯一已知的整合膜蛋白，目前认为它直接参与紧密连接的屏障和围栏功能。通过靶向破坏闭合蛋白基因的两个等位基因，生成了缺乏闭合蛋白的胚胎干细胞（ES细胞）。当这些细胞进行悬浮培养时，它们聚集形成简单的，然后是囊性胚状体（EBs），其形成时间进程与野生型ES细胞形成EBs的时间进程相同。免疫荧光显微镜和超薄切片电子显微镜显示，极化上皮细胞（内脏内胚层样）不仅从野生型ES细胞，而且从缺乏闭合蛋白的ES细胞中分化出来，勾勒出EBs。冷冻断裂分析表明，野生型和缺乏闭合蛋白的上皮细胞之间，TJ链的数量或形态没有显著差异。此外，紧密连接相关的外周膜蛋白闭合蛋白相关蛋白1（ZO-1）在缺乏闭合蛋白的上皮细胞中仍仅集中在TJ处。与这些形态学观察结果高度一致，缺乏闭合蛋白的上皮细胞中的TJ作为低分子量示踪剂通过细胞旁途径扩散的主要屏障发挥作用。这些发现表明，目前还存在尚未鉴定的TJ整合膜蛋白，它们可以形成链状结构，募集ZO-1，并在没有闭合蛋白的情况下发挥屏障作用。
