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蛋白质和膜转运的严格调节来建立和维持其独特的结构。该过程的一个关键方面是内吞再循环，即内化的大分子从内体选择性返回细胞表面。了解内吞再循环对细胞生物学具有根本的重要性，并且与包括癌症和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C的强大功能。elegans遗传学来表征体内回收过程所需的蛋白质。然后，我们将这些发现扩展到哺乳动物细胞和相关的C。线虫蛋白质及其哺乳动物同源物。对于许多这些研究，我们集中在一个系统，我们开创了，C。elegans肠，一个非常简单的模型，允许在完整的极化上皮细胞内的内吞膜转运途径的分析。在之前的授权期间，我们对在我们之前的筛选中鉴定的RME-1/EHD家族蛋白如何在内体膜微管和裂变中起作用有了新的理解，并鉴定了在此过程中与RME-1起作用的新蛋白。我们还确定和表征了几种新的效应物的小GTTRAB-10/Rab 10，我们以前显示的蛋白质是一个主调节器的底外侧循环在蠕虫肠和极化哺乳动物MDCK细胞。我们提出了三个新目标来进一步阐明内吞再循环的分子机制。我们计划破译一种新的抑制级联调节早期内体GTdR RAB-5在受体再循环。新的数据表明，通过再循环内体蛋白RAB-10，CED-10/Rac和AMPH-1/Amphiphysin进行调节。我们还将阐明新发现的RAB-10效应器EHBP-1的分子联系的膜，细胞骨架，和RAB-10的明显的反馈调节。最后，我们专注于了解RAB-10通路中的新参与者，推定的ARF 6效应子EAR-16/JIP 3的内体调控的分子基础。我们在这条通路中将ARF-16连接到ARF-GAP，并连接到新的Rab-GTT调节剂。这里提出的实验应该为内吞再循环如何工作提供重要的新见解。鉴于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，我们希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病因和确定疾病干预的治疗靶点非常重要。 公共卫生相关性：我们的研究重点是内化的大分子从内体返回细胞表面（内吞再循环），这是一个基本的细胞生物学过程，对生物医学的许多领域都具有根本的重要性。例如，内吞再循环是葡萄糖转运蛋白（Glut 4）从内体到质膜的胰岛素刺激运动中的关键控制点，这是II型糖尿病中出错的过程。更好地了解内吞再循环功能将是非常重要的，在确定治疗目标，以打击这种和其他疾病。

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. PUBLIC HEALTH RELEVANCE: Our research focuses on the return of internalized macromolecules to the cell surface from endosomes (endocytic recycling), a basic cell biological process that is of fundamental importance to many areas of biomedicine. For instance, endocytic recycling is a key control point in the insulin-stimulated movement of glucose transporters (Glut4) from endosomes to the plasma membrane, a process that goes awry in type II diabetes. A better understanding of how endocytic recycling functions will be profoundly important in identifying therapeutic targets to combat this and other diseases.