Structure and function of occludin in tight junctions : comparison with connexin gap junctions

通过靶向破坏occiudin基因的两个等位基因来产生occiudin缺陷的胚胎干（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（EB），其时间过程与野生型ES细胞形成EB的时间过程相同。免疫荧光显微镜和透射电镜显示，极化上皮（内脏内胚层样）细胞分化描绘EB不仅从野生型，但也从oceludin缺陷的ES细胞。冷冻断裂分析表明野生型和闭合蛋白缺陷的上皮细胞之间的TI链的数量或形态没有显着差异。这些发现表明，有尚未确定的TJ膜整合蛋白（S），可以形成链结构。因此，我们重新检查了从鸡肝中分离的连接部分，从其中首次鉴定出occiudin。在该馏分的众多组分中， 关于我们 通过4 M胍-HCl提取以及超声处理，然后逐步蔗糖密度梯度离心，仅检测到一条约22 kD的宽银染带。从宽带的下半部分和上半部分获得两个不同的肽序列，并且数据库的相似性搜索允许我们分离编码相关小鼠22-kD蛋白质的两个全长cDNA，分别亲水性分析表明，这两个孔四个跨膜结构域，虽然他们没有表现出任何序列相似性occludin。免疫荧光和免疫电子显微镜显示，这两种蛋白质标记的FLAG或GFP的目标，并纳入TJ链本身。我们将它们分别命名为“claudin-1”和“claudin-2”。虽然紧密连接蛋白与TI的精确结构/功能关系仍然难以捉摸，但这些发现表明具有四个推定跨膜结构域的多个完整膜蛋白，闭合蛋白和紧密连接蛋白，构成TJ链。少

Occiudin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occiudin 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 oceludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TI strands between wild-type and occludin-deficient epithelial cells. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures. We therefore re-examined the isolated junction fraction from chicken liver, from which occiudin was first identified. Among numerous components of this fraction, … More only a broad silver-stained band around 22 kD was detected with the occludin band through 4 M guanidine-HCI extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of data bases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 a.a., respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TI still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands. Less