Endocytosis and Recycling in C. elegans and Mammals

描述（由申请人提供）：细胞和组织在很大程度上通过严格调节蛋白质和膜运输来建立和维持其独特的结构。这个过程的一个关键方面是内吞循环，即内化大分子从内体选择性地返回细胞表面。了解内吞循环对细胞生物学至关重要，并与包括癌症和II型糖尿病在内的许多生物医学领域具有广泛的相关性。我们的一般方法是利用秀丽隐杆线虫遗传学的强大特征来表征体内循环过程所需的蛋白质。然后，我们将这些发现扩展到哺乳动物细胞和相关秀丽隐杆线虫蛋白及其哺乳动物同源物的体外分析。在这些研究中，我们主要集中在我们开创的一个系统上，秀丽隐杆线虫的肠道，一个非常简单的模型，可以方便地分析完整极化上皮内的内吞膜运输途径。在之前的授权期内，我们对在之前的筛选中鉴定的RME-1/EHD家族蛋白如何在内体膜管化和裂变中起作用有了新的认识，并鉴定了在这一过程中与RME-1一起起作用的新蛋白。我们还鉴定并鉴定了几种新的小GTPase rabb -10/Rab10的效应物，我们之前发现这种蛋白是蠕虫肠和极化哺乳动物MDCK细胞基底侧循环的主要调节因子。我们提出了三个新的目标来进一步阐明内吞循环的分子机制。我们计划破译在受体循环过程中调节早期内核GTPase rabb -5的新型抑制级联。新的数据表明，通过回收内核体蛋白RAB-10、CED-10/Rac和AMPH-1/Amphiphysin进行调节。我们还将阐明新发现的rabb -10效应物EHBP-1与膜、细胞骨架以及rabb -10的明显反馈调节的分子联系。最后，我们重点了解了RAB-10途径中一个新的参与者，即ARF6效应物UNC-16/ JIP3的内体调控的分子基础。我们将UNC-16连接到该途径中的ARF-GAP和一个新的Rab-GTPase调节因子。这里提出的实验应该为内吞循环如何工作提供重要的新见解。考虑到从蠕虫到哺乳动物的这种途径的高度系统发育保守性，以及我们在人类细胞中的平行分析，我们期望我们的工作将为跨物种相关的关键保守元件提供广泛的见解。这项工作对于了解疾病的病因和确定疾病干预的治疗靶点具有重要意义。

DESCRIPTION (provided by applicant): Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. One key aspect of this process is endocytic recycling, the selective return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to cell biology and has broad relevance to many areas of biomedicine including cancer and type II diabetes. Our general approach has been to exploit powerful features of C. elegans genetics to characterize proteins that are required for the recycling process in vivo. We then extend these findings to mammalian cells and to in vitro analysis of the relevant C. elegans proteins and their mammalian homologs. For many of these studies we focused on a system that we pioneered, the C. elegans intestine, a very simple model that allows facile analysis of endocytic membrane transport pathways within intact polarized epithelia. During the previous granting period we gained new understanding of how RME-1/EHD family proteins, identified in our previous screens, function in endosomal membrane tubulation and fission, and identified new proteins that function with RME-1 in this process. We also identified and characterized several new effectors for the small GTPase RAB-10/Rab10, a protein that we previously showed is a master regulator of basolateral recycling in the worm intestine and polarized mammalian MDCK cells. We propose three new aims to further elucidate the molecular mechanisms underlying endocytic recycling. We plan to decipher a novel inhibitory cascade regulating early endosome GTPase RAB-5 during receptor recycling. New data indicates regulation by recycling endosome proteins RAB-10, CED-10/Rac, and AMPH-1/Amphiphysin. We will also elucidate the molecular links of newly identified RAB-10 effector EHBP-1 to membranes, the cytoskeleton, and apparent feedback regulation of RAB-10. Finally, we focus on understanding the molecular basis of endosomal regulation by a new player in the RAB-10 pathway, putative ARF6 effector UNC-16/ JIP3. We connect UNC-16 to an ARF-GAP in this pathway and to a new Rab-GTPase regulator. The experiments proposed here should provide significant new insights into how endocytic recycling works. Given the high level of phylogenetic conservation of such pathways from worms to mammals, and our parallel analysis in human cells, we expect that our work will provide extensive insight into key conserved elements relevant across species. This work is important for understanding disease etiology and in identifying therapeutic targets for disease intervention.