JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin
JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin
复制标题
DOI：
10.1091/mbc.e05-09-0826
复制
发表时间：
2006-05-01
影响因子：
3.3
通讯作者：
Sasaki, T
中科院分区：
文献类型：
--
作者：
Terai, T;Nishimura, N;Sasaki, T
紧密连接（TJ）的动态周转是上皮细胞形态发生过程中上皮-间充质转化和/或间充质-上皮转化所必需的。我们先前证明Rab 13特异性介导occludin的内吞再循环。在这里，我们确定MICAL-L2（与CasL样2相互作用的分子）作为一种新的Rab 13结合蛋白。免疫沉淀和免疫荧光显微镜显示，MICAL-L2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，缺失Rab 13结合结构域的MICAL-L2突变体（MICAL-L2-N）特异性抑制闭合蛋白的内吞循环，但不抑制转铁蛋白受体。调用开关测定进一步揭示MICAL-L2-N以及Rab 13 Q67 L抑制闭合蛋白向质膜的募集、跨上皮电阻的发展以及细胞旁扩散屏障的形成。MICAL-L2在肌动蛋白解聚后从TJ中被置换，并分别沿着Call-depleted MTD-1A和成纤维细胞NIH 3 T3细胞中的辐射肌动蛋白电缆和应力纤维分布。这些结果表明，MICAL-L2介导的occludin的内吞回收和功能性TJ的形成，通过连接Rab 13肌动蛋白细胞骨架。我们将MICAL-L2重命名为JRAB（Junctional Rabl 3-binding protein）。
The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 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 MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Call switch assay further revealed that MICAL-L2-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. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Call-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rabl3-binding protein).