Actin assembly and clathrin-mediated endocytosis in yeast and mammals

 描述（由申请人提供）：提出了关于网格蛋白介导的内吞作用（CME）和CME期间肌动蛋白力产生的机制和调节及其对芽殖酵母和哺乳动物细胞中细胞功能的至关重要性的研究。肌动蛋白在无数过程中发挥作用，包括细胞运动、细胞器运输、粘附、收缩性、细胞形状、细胞极性以及维持膜张力和细胞机械刚性。在肌动蛋白机制和组装调节的知识方面存在重大差距。在芽殖酵母的研究中，有两个关于肌动蛋白调控和功能的关键问题将被解决：（1）细胞周期如何调控肌动蛋白电缆组装？(2)1型肌球蛋白和Arp2/3复合体是如何共同作用产生使膜弯曲的力的？对于以前的研究，最近的观察，由Clb2/Cdk1 fimmunoglobulin磷酸化是至关重要的细胞周期调控肌动蛋白组装将被利用，以发展一个机制的理解，肌动蛋白组装是如何在细胞周期中进行调节。对于后者的研究，深入的生物化学，生物物理，遗传和细胞生物学的方法将结合起来，以确定如何1型肌球蛋白有助于部队生产的Arp2/3核肌动蛋白网络在CME。CME负责通过质膜的渗透性屏障从细胞环境中摄取分子，因此对于确定细胞如何对其周围环境做出反应至关重要。许多介导CME的蛋白质和脂质已经被鉴定，并且它们的功能在生物化学和细胞中被确定。荧光标记的CME蛋白的活细胞成像揭示了约60种CME蛋白的复杂的募集时间和顺序。然而，货物捕获如何与囊泡形成协调，如何实现正确的蛋白质募集顺序和时机，哪些事件和分子在途径中起关键作用，以及力如何使膜弯曲并驱动囊泡断裂，尚未完全了解。以下关键问题将在芽殖酵母和哺乳动物细胞中得到解决：CME位点的起始和成熟是如何调节的？什么活动是CME囊泡形成所必需的？检查点是否监控CME？什么生物物理原理支配着CME？肌动蛋白的内吞功能是什么？它们是如何被调节的？化学和物理参数如何影响CME动力学和效率？CME在细胞分化过程中如何变化？哺乳动物细胞研究将在80多个稳定的组织培养物和干细胞系上进行，这些细胞系使用基因组编辑以天然内源性水平表达CME蛋白作为荧光蛋白融合物。细胞分化对CME动力学和效率的影响将在基因组编辑的干细胞中进行。由于CME蛋白在结构和功能上高度保守，从酵母和哺乳动物的研究中学到的原理将相互补充和相互告知，并提供一个全面的机制理解，而这两者都不能单独产生。

 DESCRIPTION (provided by applicant): Studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME, and their critical importance to cell function in both budding yeast and mammalian cells, are proposed. Actin functions in countless processes including cell motility, organelle transport, adhesion, contractility, cell shape, cell polarity, and maintenance of membrane tension and cell mechanical rigidity. Significant gaps exist in knowledge of actin mechanisms and assembly regulation. Two key questions concerning actin regulation and function will be addressed in studies of budding yeast: (1) How does the cell cycle regulate actin cable assembly? (2) How do type 1 myosin and the Arp2/3 complex work together to create forces that generate membrane curvature? For the former studies, recent observation that fimbrin phosphorylation by Clb2/Cdk1 is crucial for cell cycle regulation of actin assembly will be leveraged to develop a mechanistic understanding of how actin assembly is regulated in the cell cycle. For the latter studies, in- depth biochemical, biophysical, genetic, and cell biological approaches will be combined to determine how type 1 myosins contribute to force production by Arp2/3-nucleated actin networks during CME. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane, and therefore, is crucial for determining how cells respond to their surroundings. Many proteins and lipids that mediate CME have been identified, and their functions determined biochemically and in cells. Live cell imaging of fluorescently labeled CME proteins has revealed the intricate recruitment timing and order for some 60 CME proteins. However, how cargo capture is coordinated with vesicle formation, how correct protein recruitment order and timing are achieved, which events and molecules play critical roles in the pathway, and how forces curve the membrane and drive vesicle scission, are not fully understood. The following key questions will be addressed in budding yeast and mammalian cells: How are CME site initiation and maturation regulated? What activities are essential for CME vesicle formation? Does a checkpoint monitor CME? What biophysical principles govern CME? What are actin's endocytic functions and how are they regulated? How do chemical and physical parameters affect CME dynamics and efficiency? How does CME change during cellular differentiation? Mammalian cell studies will be conducted on over 80 stable tissue culture and stem cell lines generated using genome editing to express CME proteins as fluorescent protein fusions at native, endogenous levels. Effects of cell differentiation on CME dynamics and efficiency will be conducted in the genome-edited stem cells. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and mammals will each complement and inform the other and provide a comprehensive mechanistic understanding that neither alone could generate.