Endocytic Functions for the Mammalian Actin Cytoskeleton

描述（由申请人提供）：通过将创新的尖端技术与现有方法相结合，拟议的研究将揭示哺乳动物细胞中网格蛋白介导的内吞作用（CME）的基本机制原理。在基因组编辑的细胞中以内源性水平表达荧光蛋白融合体将允许比先前可能实现的更忠实地报告内吞动力学。因此，RNAi、物理和小分子扰动的影响将比以前更灵敏地检测和更有力地分析。这项研究的预期成果是了解几十种蛋白质的协调活动如何用于内吞囊泡形成的机械化学过程。由于将分析多种蛋白质，因此将揭示内吞系统的整体设计原则。三个目标将被解决：1。内吞蛋白募集和囊泡形成的时空动力学：使用基因组编辑的稳定细胞系，在天然水平上表达五种不同内吞蛋白-荧光蛋白融合的成对组合，实时成像和分析软件将用于确定精确的募集概况，为功能，机制，调节和系统逻辑提供强大的见解。将对数据进行数学建模，并生成功能研究的假设。数学建模还将探讨脂质在膜弯曲和断裂力的产生中发挥积极作用的假设。2.体内内吞蛋白功能的阐明：基因组编辑的细胞系的实时成像将灵敏地测试功能扰动对CME的影响。内吞蛋白在其生物学背景下的功能将使用RNAi和小分子抑制剂来阐明。将测试已知的内吞蛋白和在生物信息学筛选中鉴定的三种新的内吞蛋白的功能。实时观察的超微结构基础将通过电子显微镜来揭示。化学遗传策略将通过基因组编辑来改进，以阐明网格蛋白轻链在体内的功能。3.货物、实物和 内吞动力学的发育参数：将检验内吞货物负荷和膜张力影响CME动力学的假设。使用不同组织来源的基因组编辑的细胞系和干细胞，将测试CME在发育过程中针对不同生理状态进行微调和修改的假设。

DESCRIPTION (provided by applicant): By combining innovative, cutting edge technologies with established approaches, the proposed research will uncover fundamental mechanistic principles governing clathrin-mediated endocytosis (CME) in mammalian cells. Expression of fluorescent protein fusions at endogenous levels in genome-edited cells will allow more faithful reporting of endocytic dynamics than could have previously been achieved. As a result, effects of RNAi, physical and small molecule perturbations will be more sensitively detected and more powerfully analyzed than was previously possible. The expected outcome of this research is an understanding of how coordinated activities of dozens of proteins are harnessed for the mechanochemical process of endocytic vesicle formation. Because multiple proteins will be analyzed, holistic design principles for the endocytic system will be revealed. Three aims will be addressed: 1. Spatio-temporal dynamics of endocytic protein recruitment and vesicle formation: Using genome-edited, stable cell lines expressing pair-wise combinations of five different endocytic protein-fluorescent protein fusions at native levels, real-time imaging and analytical software will be used to determine precise recruitment profiles, providing powerful insights into function, mechanism, regulation and system logic. The data will be modeled mathematically and will generate hypotheses for functional studies. Mathematical modeling will also explore the hypothesis that lipids play an active role in generation of membrane-bending and scission forces. 2. Elucidation of endocytic protein functions in vivo: Real-time imaging of genome-edited cell lines will sensitively test the impact of function perturbations on CME. Functions of endocytic proteins in their biological context will be elucidated using RNAi and small molecule inhibitors. Functions of known endocytic proteins and three novel endocytic proteins identified in a bioinformatic screen will be tested. The ultrastructural underpinnings of real-time observations will be revealed by electron microscopy. Chemical-genetic strategies will be improved by genome editing to elucidate clathrin light chain function in vivo. 3. Impact of cargo, physical and developmental parameters on endocytic dynamics: The hypotheses that endocytic cargo load and membrane tension affect CME dynamics will be tested. Using genome-edited cell lines of varied tissue origin and stem cells, the hypothesis that CME is fine-tuned and modified developmentally for distinct physiological states will be tested.