细胞极性调控因子LET-413/Scrib在囊泡循环运输中的调控功能研究

Clathrin-independent endocytic recycling is tightly regulated by RAB-10 and essential for various processes including cell polarity establishment and maintenance, cell migration and cytokinesis. The functional implication of small GTPase RAB-10 in recycling transport is mainly mediated by the effectors. Nevertheless, the activating mechanism of RAB-10 in endocytic recycling trafficking has not been fully evaluated in vivo. By utilizing C. elegans intestinal epithelia as the model system, our preliminary results demonstrate that, in let-413 mutant, CIE cargo hTAC accumulates in enlarged vesicles and RAB-10 localizes diffusively in cytosol, implicating LET-413/Scrib in the clathrin-independent endocytic recycling process. To better understand LET-413 regulatory mechanisms in recycling, we propose to analyze the subcellular localization and the cargo specificity of LET-413. Using epistatic analysis we will determine the genetic relationship of LET-413 with RAB-5 and RAB-10. Then, we will assay if LET-413 interacts with RAB-10 GEF protein DENN-4 directly, whether or not LET-413 plays a vital role in DENN-4 mediated RAB-10 activation. Furthermore, we will assay the potential physical interaction of LET-413 with RAB-5 and RAB-10, and evaluate the functional necessity of LET-413 in RAB-5 to RAB-10 cascade during endocytic recycling. Our proposed studies are expected to explore the recycling regulation mechanisms of LET-413 in whole animal, defining mechanistic details of RAB-10-mediated recycling regulatory network. Our studies will be of broad interest to the scientific community of cellular development and immune response etc..

RAB-10调控非笼形蛋白依赖内吞 (CIE) 膜蛋白囊泡循环运输，与细胞极性建立、细胞迁移等过程密切相关。RAB-10功能主要由其激活态RAB-10(GTP) 招募效应因子介导，当前对RAB-10激活调控机制仍缺乏深入了解。通过线虫在体循环运输研究系统，我们发现细胞极性调控因子LET-413/Scrib参与调控CIE膜蛋白循环，let-413突变细胞中hTAC异常累积，RAB-10定位弥散。本课题将研究LET-413亚细胞定位机制及膜蛋白调控特异性，阐明LET-413与DENN-4(RAB-10-GEF)间相互作用及其功能效应，解析RAB-10激活调控机制，并进一步探讨LET-413在RAB-5、RAB-10间Rab级联推进中的功能意义。本项目将系统研究整体动物中LET-413循环调控机理，加深对RAB-10调控网络的理解，为细胞发育、免疫应答等循环运输相关生物学过程的研究提供科学思路。

RAB-10/Rab10是极性上皮细胞中调控内吞循环运输的主要基因。其对微丝在内吞体上的捆绑成束以及PI(4,5)P2水平的调节都是必需的。为了更好地理解RAB-10活性的调节过程，我们寻找了与RAB-10非活性状态相互作用的蛋白。由此鉴定出LET-413（Scrib/Erbin在秀丽隐杆线虫中的同源物）是一种新型的RAB-10(GDP)结合伴侣。我们研究了LET-413在线虫肠细胞内调控RAB-10活性作用的具体机制，结果显示LET-413是RAB-5的效应因子，与内吞体上的RAB-10共定位，并且LET-413与RAB-10的共定位是RAB-5依赖性的。值得注意的是，LET-413增强了DENN-4与RAB-10(GDP)的相互作用并促进了DENN-4作为鸟嘌呤核苷酸交换因子对RAB-10的激活。LET-413缺失导致RAB-10效应因子TBC-2和CNT-1的细胞质弥散分布。最后，我们证明RAB-10或LET-413的缺失导致细胞侧膜异常过度伸展。我们的研究表明，LET-413是DENN-4介导RAB-10活化所必需，且LET-413参与的RAB-5与RAB-10级联有助于保持秀丽线虫肠上皮细胞的膜完整性