Role of Rab13 in regulating cell polarity and adhesion

紧密连接(TJ)是位于细胞间隙顶端的连续的环状带状结构，它们勾勒出上皮细胞质膜顶区和基外区之间的边界。TJS由完整的TJ蛋白和TJ斑块蛋白组成。包括occludin和claudins在内的整合型TJ蛋白介导细胞间的黏附并形成物理的细胞间屏障，而TJ斑块蛋白聚集整合型TJ蛋白并形成各种支架、信号和囊泡运输蛋白的组织平台。Rab13是一种存在于上皮细胞的TJ斑块蛋白，与功能性TJ的组装有关。我们先前证明了Rab13特异性地介导了occludin的内吞循环。在本研究中，我们鉴定了Mical-L2(与CASL-like 2相互作用的分子)是一种新的Rab13结合蛋白，并将其重新命名为JRAb(Junctional Rab13-binding Protein)。免疫沉淀和免疫荧光显微镜显示JRAb通过其C末端与GTP结合形式的Rab13特异性结合，该末端含有一个卷曲的结构域，并定位于上皮细胞MTD-1A的TJs。循环实验表明，缺乏Rab13结合域的JRAb突变体(JRAB-N)特异性地抑制occludin的内吞循环，但不抑制转铁蛋白受体。钙离子开关实验进一步表明，JRAB-N和Rab13 Q67L可抑制阻滞素在质膜上的募集，抑制跨上皮电阻的形成，并抑制细胞旁扩散屏障的形成。在MTD-1A细胞中，肌动蛋白解聚时，JRAb从TJ中移位，在成纤维细胞NIH3T3中，JRAb沿应力纤维分布。这些结果表明，JRAb通过将Rab13连接到肌动蛋白细胞骨架而介导闭合蛋白的内吞循环和功能性TJ的形成。

Tight junctions (TJs) are continuous, circumferential belt-like structures located at the apical end of the intercellular space, where they delineate the boundaries between the apical and basolateral domains of the plasma membrane of epithelial cells. TJs are composed of integral TJ proteins and TJ plaque proteins. While integral TJ proteins including occludin and claudins mediate cell-cell adhesion and create physical intercellular barrier, TJ plaque proteins cluster integral TJ proteins and form an organizing platform for a variety of scaffolding, signaling, and vesicular transport proteins. Rab13 is identified as a TJ plaque protein in epithelial cells and is implicated in the assembly of functional TJs. We previously demonstrated that Rab13 specifically mediated the endocytic recycling of occludin. In the present study, we have identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein and renamed it as JRAB (junctional Rab13-binding protein). Immunoprecipitation and immunofluorescence microscopy showed that JRAB specifically bound to the GTP-bound form of Rab13 via its C-terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a JRAB mutant lacking the Rab13-binding domain (JRAB-N) specifically inhibited the endocytic recycling of occludin, but not transferrin receptor. Ca^<2+>-switch assay further revealed that JRAB-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. JRAB was displaced from TJs upon actin depolymerization in MTD-1A cells and was distributed along stress fibers in fibroblastic NIH3T3 cells. These results suggested that JRAB mediated the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton.