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，并与生物医学的许多领域具有广泛的相关性。细胞内循环对维持细胞极性尤为重要，细胞极性是上皮组织的一个基本特征。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。在之前的授权期内，我们对RME-1/EHD1家族蛋白质如何在循环中发挥作用有了新的理解，这些蛋白质在我们之前的筛查中被鉴定出来。我们还发现了与RME-1功能相关的新蛋白(ALX-1/Alix、SDPN-1/Syndapin和ARF-6/Arf6)。我们进一步建立了线虫肠道作为阐明极化上皮细胞内膜转运途径的模型，并首次表明在蠕虫肠道和极化哺乳动物MDCK细胞中，小GTP酶RAB-10/Rab10是基侧循环所必需的。为了从机制上了解RAB-10是如何驱动循环的，我们确定了五种蛋白质，它们与RAB-10在活跃的、GTP结合的构象中特定地相互作用。这些新蛋白可能是RAB-10的效应器，直接介导RAB-10驱动的转运。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。首先，我们建议测试我们新发现的RAB-10结合蛋白是RAB-10效应器这一假设的关键预测。这将通过更好地定义它们与RAB-10的物理相互作用，确定每个候选效应器被敲除时对回收的影响，以及测试这些蛋白质的工程相互作用缺陷形式修复被剔除动物的回收缺陷的能力来实现。其次，我们建议测试线虫Alix和Syndapin的同源物模型，该模型是我们在前一次授予期间开发的，表明它们通过招募和激活内体上的肌动蛋白调节器来控制循环。最后，我们建议利用一个新分离的针对线虫Arf6直系同源基因的敲除，来测试这个关键的GTP酶在极化上皮细胞内转运中的作用，并了解它在这个复杂的环境中是如何调节的。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这类途径的高度系统发育保守性，我们的工作应该为人类细胞中的同等途径提供广泛的预测性洞察。我们的研究重点是控制内吞循环的分子机制--内化的大分子从内小体返回到细胞表面。了解细胞内循环对于生物医学的许多领域都是至关重要的。例如，在胰岛素刺激下葡萄糖转运体(GLUT4)从内体到脂肪和肌肉细胞质膜的运动中，内吞循环是一个关键的控制点。回收活动的失败被认为是II型糖尿病的主要原因，II型糖尿病最近在美国达到了流行的程度。更好地了解内吞细胞循环的功能对于确定抗击这种疾病和其他疾病的治疗靶点将是极其重要的。

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. Endocytic recycling is particularly critical to the maintenance of cell polarity, a defining and essential feature of epithelial tissues. 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. During the previous granting period we gained new understanding of how RME-1/Ehd1 family proteins, identified in our previous screens, function in recycling. We also identified new proteins (ALX-1/Alix, SDPN-1/Syndapin, and ARF-6/Arf6) that function with RME-1 in this process. We went on to establish the C. elegans intestine as a model for elucidating endocytic membrane transport pathways in polarized epithelia, and showed for the first time that the small GTPase RAB-10/Rab10 is required for basolateral recycling in the worm intestine and polarized mammalian MDCK cells. To gain mechanistic insight into how RAB-10 drives recycling we identified five proteins that specifically interact with RAB-10 in the active, GTP-bound, conformation. These new proteins are likely RAB-10 effectors, directly mediating RAB-10 driven transport. We propose three new aims to further elucidate the molecular mechanisms underlying endocytic recycling. First we propose to test key predictions of the hypothesis that our newly identified RAB-10 binding proteins are RAB-10 effectors. This will be accomplished by better defining their physical interactions with RAB-10, determining the effects on recycling when each candidate effector is knocked out, and testing the ability of engineered interaction defective forms of these proteins to rescue recycling defects in knockout animals. Second we propose to test a model for C. elegans orthologs of Alix and Syndapin, that we developed during the prior granting period, suggesting that they control recycling through the recruitment and activation of actin regulators on endosomes. Finally we propose to leverage a newly isolated knockout for the only C. elegans Arf6 ortholog, to test the role of this key GTPase in endocytic transport in polarized epithelia, and to understand how it is regulated in this complex environment. 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, our work should provide extensive predictive insight into equivalent pathways in human cells. Our research focuses on the molecular mechanisms controlling endocytic recycling - the return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling 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 of adipose and muscle cells. Failure in this recycling event is thought to be a major cause of type II diabetes, a disease that has recently reached epidemic proportions in the United States. A better understanding of how endocytic recycling functions will be profoundly important in identifying therapeutic targets to combat this and other diseases.