Actin assembly and clathrin-mediated endocytosis in yeast and mammals

酵母和哺乳动物中肌动蛋白组装和网格蛋白介导的内吞作用

• 批准号: 9071612
• 负责人: DAVID G DRUBIN
• 金额: $ 96.71万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071612
• 关键词: Actins; Address; Adhesions; Affect; Biochemical; Biological; Biophysics; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cell Line; Cell Polarity; Cell Shape; Cell membrane; Cell physiology; Cells; Chemicals; Chimeric Proteins; Cholesterol Homeostasis; Clathrin; Complement; Complex; Endocytosis; Environment; Event; Fimbrin; Generations; Genetic; Growth; Hearing; Knowledge; Label; Learning; Lipids; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Mechanics; Mediating; Membrane; Monitor; Myosin ATPase; Normal Cell; Organelles; Pathway interactions; Permeability; Phenotype; Phosphorylation; Play; Process; Production; Proteins; Regulation; Role; Saccharomycetales; Signal Transduction; Site; Stem cells; Structure; Time; Vesicle; Work; Yeasts; blood pressure regulation; cell motility; genome editing; live cell imaging; pathogen; prevent; public health relevance; tissue/cell culture; uptake

 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.

 描述(由申请人提供)：建议研究在胞内胞吞过程中网状蛋白介导的内吞作用(CME)和肌动蛋白作用力的产生的机制和调节，以及它们对萌芽酵母和哺乳动物细胞功能的关键作用。肌动蛋白在细胞运动、细胞器运输、黏附、收缩、细胞形态、细胞极性、维持细胞膜张力和细胞机械刚性等过程中发挥着重要作用。在肌动蛋白机制和组装调控方面的知识存在着显著的差距。有关肌动蛋白调节和功能的两个关键问题将在发芽酵母的研究中解决：(1)细胞周期如何调节肌动蛋白电缆组装？(2)1型肌球蛋白和Arp2/3复合体如何共同作用来产生产生膜曲率的力？对于前一项研究，最近观察到Clb2/CDK1对肌动蛋白组装的细胞周期调控至关重要，这一观察结果将被用来发展对肌动蛋白组装如何在细胞周期中调控的机械性理解。对于后一项研究，将结合深入的生化、生物物理、遗传学和细胞生物学方法来确定1型肌球蛋白如何在CME期间通过Arp2/3核肌动蛋白网络促进FORCE的产生。CME负责通过质膜的通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环境做出反应至关重要。许多介导CME的蛋白质和脂类已被鉴定，并通过生化和细胞内确定它们的功能。荧光标记的CME蛋白的活细胞成像揭示了大约60个CME蛋白复杂的招募时间和顺序。然而，货物捕获如何与囊泡形成相协调，如何实现正确的蛋白质招募顺序和时机，哪些事件和分子在该途径中发挥关键作用，以及力如何弯曲细胞膜和驱动囊泡断裂，还没有完全弄清楚。以下关键问题将在萌芽酵母和哺乳动物细胞中解决：CME位点的启动和成熟是如何调节的？什么活动对CME囊泡的形成是必不可少的？检查点是否监控CME？什么生物物理学原理支配着CME？肌动蛋白的内吞功能是什么？它们是如何调节的？化学和物理参数如何影响CME的动力学和效率？细胞分化过程中CME是如何变化的？哺乳动物细胞研究将在80多个稳定的组织培养和干细胞系上进行，这些细胞系使用基因组编辑来表达CME蛋白，作为天然、内源性水平的荧光蛋白融合。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蛋白质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影响，并提供一个单独无法产生的全面机制理解。