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具有重要的基础意义，并与生物医学的许多领域具有广泛的相关性。细胞极性是上皮组织的基本特征，而内吞循环对细胞极性的维持尤为重要。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。在之前的批准期间，我们对在之前的筛选中鉴定的RME-1/Ehd1家族蛋白如何在回收中发挥作用有了新的了解。我们还发现了新的蛋白（ALX-1/Alix、SDPN-1/Syndapin和ARF-6/Arf6）在这一过程中与RME-1一起发挥作用。我们继续建立秀丽隐杆线虫肠道作为模型来阐明极化上皮的内噬膜运输途径，并首次表明小GTPase rabb -10/Rab10在蠕虫肠道和极化哺乳动物MDCK细胞的基底侧循环中是必需的。为了深入了解rabb -10驱动循环的机制，我们鉴定了五种与rabb -10在活性、gtp结合构象中特异性相互作用的蛋白质。这些新蛋白可能是rabb -10效应物，直接介导rabb -10驱动的运输。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。首先，我们提议测试我们新发现的rabb -10结合蛋白是rabb -10效应物的假设的关键预测。这将通过更好地定义它们与RAB-10的物理相互作用来实现，确定每个候选效应物被敲除时对循环的影响，并测试这些蛋白的工程相互作用缺陷形式挽救敲除动物中循环缺陷的能力。其次，我们提议测试一个线虫的Alix和Syndapin的同源物模型，我们在之前的授权期间开发，表明它们通过内体上的肌动蛋白调节因子的招募和激活来控制循环。最后，我们提出利用新分离的唯一的线虫Arf6同源基因敲除，来测试这个关键的GTPase在极化上皮内吞运输中的作用，并了解它是如何在这种复杂的环境中被调节的。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。鉴于这种途径从蠕虫到哺乳动物的高度系统发育保守性，我们的工作应该为人类细胞中的等效途径提供广泛的预测性见解。我们的研究重点是控制内吞循环的分子机制-内化大分子从内体返回细胞表面。了解内吞循环对生物医学的许多领域都具有重要的基础意义。例如，内吞循环是胰岛素刺激下葡萄糖转运体（Glut4）从内体向脂肪和肌肉细胞质膜运动的关键控制点。这种循环利用的失败被认为是II型糖尿病的主要原因，这种疾病最近在美国已经达到流行病的程度。更好地了解内吞循环的功能将对确定治疗靶点以对抗这种疾病和其他疾病具有深远的重要意义。

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. 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.