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；内化的大分子从内吞体内选择性地返回到细胞表面。循环内吞体内也有助于宏观自噬，这是细胞在不利条件下生存所需的细胞“自噬”过程。因此，了解细胞内循环过程及其在细胞生理学中的作用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，我们对早期作用的Rab GTP酶RAb-5是如何被其GAP TBC-2下调的有了新的理解。我们发现，三种循环调节因子直接与TBC-2相互作用，促进TBC-2内体募集。其中包括RAB-10，一个在RAB-5之后在细胞内循环中起作用的关键GTP酶；Rho GTP酶CED-10/rac1，我们还展示了调节循环的Rho GTP酶，以及F-bar蛋白Amph-1/两面体蛋白。这一新的途径代表了一种抑制级联反应，当货物从早期的内吞体内过渡到循环内体时，需要终止RAB-5的活性。我们还阐明了另一个RAB-10效应器EHBP-1与膜和细胞骨架的分子连接，调节内小体的管状结构。在新的前期工作中，我们已经确定了RAB-10 GTP酶与胞囊膜系留复合体的关键新联系，并提议测试RAB-10在循环和自噬过程中如何影响胞囊的机制模型。我们进一步确定了另一个关键的循环调节因子，F-bar蛋白SDPN-1/Syndapin，并确定了对循环功能重要的新的SDPN-1相互作用伙伴。我们提出了广泛的新的体内和体外分析，以确定Syndapin家族蛋白如何在内体上发挥作用。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. 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.