Endocytosis and Recycling in C. elegans and Mammals

描述(申请人提供)：细胞和组织在很大程度上通过对蛋白质和膜运输的严格调控来建立和维持其独特的结构。这一过程的一个关键方面是内吞循环，即内化的大分子从内吞体内选择性地返回到细胞表面。了解细胞内循环对细胞生物学具有重要意义，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。我们的总体方法是利用线虫遗传学的强大功能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，并对相关线虫蛋白及其哺乳动物同源物进行体外分析。对于这些研究中的许多，我们专注于我们首创的系统，线虫肠道，一个非常简单的模型，可以方便地分析完整的极化上皮内的细胞膜运输途径。在之前的授予期间，我们对先前筛选中鉴定的RME-1/EHD家族蛋白如何在内膜管形成和分裂中发挥作用有了新的理解，并鉴定了与RME-1一起在这一过程中发挥作用的新蛋白。我们还鉴定了几个新的小GTP酶RAB-10/Rab10的效应物，我们之前已经证明，RAB-10/Rab10是蠕虫肠道和极化哺乳动物MDCK细胞基侧循环的主要调节蛋白。我们提出了三个新的目标，以进一步阐明内吞循环的分子机制。我们计划破译一种新的抑制级联反应，在受体循环过程中调节早期内切体GTP酶RAB-5。新的数据表明，通过循环内体蛋白RAB-10、CED-10/RAC和Amph-1/AmphiPhysiin进行调节。我们还将阐明新发现的RAB-10效应器EHBP-1与膜、细胞骨架的分子联系以及RAB-10的表观反馈调节。最后，我们着重于了解RAB-10途径中一个新的成员，可能的ARF6效应分子UNC-16/JIP3对内体调节的分子基础。我们将UNC-16连接到该途径中的一个ARF-GAP和一个新的Rab-GTPase调节子。这里提出的实验应该会为细胞内循环是如何工作的提供重要的新见解。鉴于从蠕虫到哺乳动物的这种途径的高度系统发育保守，以及我们在人类细胞中的平行分析，我们预计我们的工作将为跨物种相关的关键保守元件提供广泛的洞察。这项工作对于理解疾病病因和确定疾病干预的治疗靶点非常重要。

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.