Role of Rab13 in regulating cell polarity and adhesion

紧密连接（TJs）是位于细胞间隙顶端的连续的环形带状结构，在那里它们描绘上皮细胞质膜的顶端和基底侧域之间的边界。TJ由完整的TJ蛋白和TJ斑块蛋白组成。虽然完整的TJ蛋白，包括occludin和claudins介导细胞-细胞粘附，并创建物理细胞间屏障，TJ斑块蛋白集群完整的TJ蛋白，并形成一个组织平台的各种支架，信号，和囊泡运输蛋白。Rab 13被鉴定为上皮细胞中的TJ斑块蛋白，并参与功能性TJ的组装。我们先前证明Rab 13特异性介导occludin的内吞再循环。在本研究中，我们已经确定MICAL-L2（分子与CasL-like 2相互作用）作为一种新的Rab 13结合蛋白，并将其重新命名为JRAB（Junctional Rab 13-binding protein）。免疫沉淀和免疫荧光显微镜显示，JRAB特异性结合GTP结合形式的Rab 13通过其C-末端，其中包含一个卷曲螺旋结构域，并定位在TJ上皮MTD-1A细胞。循环实验表明，缺乏Rab 13结合结构域的JRAB突变体（JRAB-N）特异性抑制occludin的内吞循环，但不转铁蛋白受体。Ca^<2+>-开关测定进一步揭示JRAB-N和Rab 13 Q67 L抑制闭合蛋白向质膜的募集、跨上皮电阻的形成和细胞旁扩散屏障的形成。在MTD-1A细胞中，JRAB在肌动蛋白解聚后从TJ中移位，并在成纤维细胞NIH 3 T3细胞中沿着应力纤维分布。这些结果表明，JRAB介导的occludin的内吞回收和功能的TJ的形成，通过连接Rab 13肌动蛋白细胞骨架。

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.