Membrane Traffic 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;内化的大分子从内体选择性返回细胞表面。再循环内体也有助于大自噬，即细胞在不利条件下生存所需的细胞“自食”过程。因此，了解内吞再循环过程及其在细胞生理学中的作用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。在上一个授予期间，我们对早期作用的Rab GTSARB-5如何被其GAP TBC-2下调有了新的理解。我们发现，三个再循环调节剂直接与TBC-2相互作用，并有助于TBC-2内体募集。这些包括RAB-10，一种在RAB-5之后在内吞再循环中起作用的关键GT酶; Rho GT酶CED-10/Rac 1，我们也显示调节再循环，以及F-BAR蛋白AMPH-1/两性霉素。这种新的途径代表了终止RAB-5活性所需的抑制性级联反应，因为货物从早期内体过渡到再循环内体。我们还阐明了另一种RAB-10效应子EHBP-1与膜和细胞骨架的分子联系，调节内体的管状结构。在新的初步工作中，我们已经确定了RAB-10 GTdR与外囊膜束缚复合物的关键新联系，并提出测试RAB-10在回收和自噬过程中如何影响外囊的机制模型。我们进一步确定了另一个关键的再循环调节因子，F-BAR蛋白SDPN-1/ Syndapin，并确定了新的SDPN-1相互作用的合作伙伴，重要的再循环功能。我们提出了广泛的新的体内和体外分析，以确定如何Syndapin家族蛋白质的功能的内涵体。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。

 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. Recycling endosomes also contribute to macroautophagy, the process of cellular "self-eating" required for cell survival under adverse conditions. Thus understanding the endocytic recycling process and its role in cellular physiology 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 the early acting Rab GTPase RAB-5 is down regulated by its GAP TBC-2. We found that three recycling regulators directly interact with TBC-2 and contribute to TBC-2 endosomal recruitment. These include RAB-10, a key GTPase acting after RAB-5 in endocytic recycling; the Rho GTPase CED-10/Rac1 that we also show regulates recycling, and the F-BAR protein AMPH-1/ amphiphysin. This novel pathway represents an inhibitory cascade required to terminate the activity of RAB-5 as cargo transitions from early to recycling endosomes. We also elucidated molecular links of another RAB-10 effector EHBP-1 to membranes and the cytoskeleton, regulating the tubular architecture of endosomes. In new preliminary work we have identified key new links of the RAB-10 GTPase to the exocyst membrane tethering complex and propose to test mechanistic models for how RAB-10 influences exocyst during recycling and autophagy. We further identify another key recycling regulator, F-BAR protein SDPN-1/ Syndapin, and identify new SDPN-1 interaction partners important for recycling function. We propose extensive new in vivo and in vitro analysis to define how Syndapin family protein functions on endosomes. 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.