The Clathrin Coated Vesicle Cycle

 描述（由申请人提供）：尽管网格蛋白介导的内吞作用（CME）是迄今为止最好理解的进入细胞的途径，但关于内吞机制的组分如何协调工作以分选货物、使膜变形并夹断网格蛋白包被的囊泡（CCV），仍有许多内容有待了解。CME在营养吸收中起作用，调节表面受体和转运蛋白的表达，并控制受体酪氨酸激酶和GPCR的信号传导活性。在突触处，CME在空间和时间上都受到高度调节，以在神经传递期间实现突触囊泡的快速有效再循环。长期以来被认为是一个组成性过程，我们现在认识到，CME在非神经元细胞也受到高度调控。与Gaudenz Danuser合作，我的实验室应用定量活细胞全内反射荧光显微镜来分析网格蛋白包被的凹坑（CCP）的动态行为，并量化CCV的启动，成熟和释放速率。这些研究表明，很大一部分新生的CCP未能成熟并流产。在过去的8年里，这一发现和其他发现使我们提出存在一个内吞检查点，作为早期CCP组装的保真度监测器。控制通过该检查点的过渡的关键调控事件发生在新生CCP成核后的早期阶段。我们最近证明，招募内吞辅助蛋白（EAP）通过与衔接蛋白复合物，AP 2，以及发动蛋白-2的早期招募新生CCP的相互作用是CCP成熟的关键决定因素。我们最近还发现，CCP成熟受损的非神经元细胞，动员发动蛋白-1绕过内吞检查点，加速CME。本提案的目标1和2将分别剖析和计算模拟AP 2复合物和发动蛋白在这些关键早期事件中的作用，以阐明确保快速有效的CME的新机制。虽然内吞机制的许多组成部分是已知的，并已被分组为模块的基础上，其招聘到CCP的时间层次。很少有EAP的功能特点，这些时间模块是否反映功能的相互作用仍然是未知的。潜在的冗余或协同功能相互作用可以通过遗传相互作用来鉴定，虽然已经进行了几种基于siRNA的筛选来鉴定内吞机制的组分，但值得注意的是，这些筛选甚至未能鉴定已知的EAP，这可能是由于先前的筛选无法检测部分效应、功能冗余和/或如我们最近发现的CME的稳健性和可塑性。在目标3中，我们提出了一种新的方法来筛选CME和早期内体运输的缺陷，并进行大规模平行的全基因组shRNA筛选和后续上位性作图，以确定内吞机制组件之间的功能关系。这些研究将为决定CME效率和稳健性的因素提供前所未有的新见解。

 DESCRIPTION (provided by applicant): Although clathrin-mediated endocytosis (CME) is by far the best understood pathway for entry into the cell, much remains to be learned regarding how components of the endocytic machinery work coordinately to sort cargo, deform the membrane and pinch off clathrin coated vesicles (CCVs). CME functions in nutrient uptake, to regulate the expression of surface receptors and transporters and to control the signaling activity of receptor tyrosine kinases and GPCRs. At the synapse CME is highly regulated in both space and time to accomplish rapid and efficient recycling of synaptic vesicles during neurotransmission. Long considered a constitutive process, we now appreciate that CME in nonneuronal cells is also highly regulated. In collaboration with Gaudenz Danuser, my lab has applied quantitative live cell total internal reflection fluorescence microscopy to analyze the dynamic behaviors of clathrin coated pit (CCPs) and to quantify rates of initiation, maturation and the release of CCVs. These studies have revealed that a large fraction of nascent CCPs fail to mature and are aborted. This and other findings over the past 8 years, have led us to propose the existence of an endocytic checkpoint that serves as a fidelity monitor for early CCP assembly. Key regulatory events that govern transition through this checkpoint occur at early stages following nucleation of nascent CCPs. We recently demonstrated that the recruitment of endocytic accessory proteins (EAPs) through interactions with the adaptor protein complex, AP2, as well as the early recruitment of dynamin-2 to nascent CCPs is critical determinants of CCP maturation. We also recently discovered that nonneuronal cells with impaired CCP maturation, enlist dynamin-1 to bypass the endocytic checkpoint and accelerate CME. Aims 1 and 2 of this proposal will dissect and computationally model the roles of AP2 complexes and dynamin, respectively, in these critical early events to elucidate novel mechanisms that ensure rapid and efficient CME. While many components of the endocytic machinery are known, and have been grouped into modules based on the temporal hierarchy of their recruitment to CCPs. Few EAPs have been functionally characterized and whether these temporal modules reflect functional interactions remains unknown. Potential redundant or synergistic functional interactions can be identified through genetic interactions and while several siRNA based screens have been conducted to identify components of the endocytic machinery, remarkably, these have failed to identify even known EAPs, potentially due to the inability of previous screens to detect partial effects, functional redundancy and/or, as we recently discovered, the robustness and plasticity of CME. In Aim 3, we propose a new approach to screen for defects in CME and early endosomal trafficking and to conduct a massively parallel, genome-wide shRNA screen and follow-up epistasis mapping to identify functional relationships between components of the endocytic machinery. Together these studies will provide unprecedented new insight into factors that determine CME efficiency and robustness.