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，并且与生物医学的许多领域具有广泛的相关性。内吞再循环对于维持细胞极性是特别关键的，细胞极性是上皮组织的定义和基本特征。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。在上一个授权期间，我们对RME-1/Ehd 1家族蛋白质如何在我们以前的筛选中鉴定出在回收中起作用有了新的理解。我们还鉴定了在此过程中与RME-1起作用的新蛋白质（ALX-1/阿利克斯、SDPN-1/Syndapin和ARF-6/Arf 6）。我们继续建立了C。elegans肠作为阐明极化上皮细胞中内吞膜转运途径的模型，并首次表明小GTd-RAB-10/Rab 10是蠕虫肠和极化哺乳动物MDCK细胞中基底外侧再循环所需的。为了深入了解RAB-10如何驱动再循环，我们鉴定了五种蛋白质，它们在活性GTP结合构象中与RAB-10特异性相互作用。这些新蛋白质可能是RAB-10效应物，直接介导RAB-10驱动的转运。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。首先，我们建议测试我们新鉴定的RAB-10结合蛋白是RAB-10效应子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，确定当每个候选效应物被敲除时对再循环的影响，以及测试这些蛋白质的工程化相互作用缺陷形式拯救敲除动物中的再循环缺陷的能力来实现。第二，我们提出了一个模型来测试C。elegans直系同源物的阿利克斯和Syndapin，我们在先前的授权期间开发的，这表明他们控制再循环通过招募和激活肌动蛋白调节器的内涵体。最后，我们建议利用一个新分离的敲除唯一的C。elegans Arf 6直系同源物，以测试这种关键的GT3在极化上皮细胞内吞转运中的作用，并了解它在这种复杂环境中是如何调节的。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，我们的工作应该提供广泛的预测洞察人类细胞中的等效途径。我们的研究重点是控制内吞再循环的分子机制-内化的大分子从内体返回细胞表面。了解内吞再循环对生物医学的许多领域具有根本的重要性。例如，内吞再循环是胰岛素刺激的葡萄糖转运蛋白（Glut 4）从内体到脂肪和肌肉细胞的质膜的运动中的关键控制点。这种回收活动的失败被认为是II型糖尿病的主要原因，这种疾病最近在美国达到了流行病的程度。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. 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.