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: 9980927
• 负责人: DAVID G DRUBIN
• 金额: $ 104.93万
• 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/9980927
• 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 Factor; Knowledge; Label; Lipids; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Mechanics; Mediating; Membrane; Monitor; Myosin S-1; Normal Cell; Organelles; Pathologic; Pathway interactions; Permeability; Phenotype; Phosphorylation; Play; Process; Production; Proteins; Regulation; Role; Saccharomycetales; Signal Transduction; Site; Structure; Vesicle; Work; Yeasts; blood pressure regulation; cell motility; fluorescence imaging; genome editing; live cell imaging; normal hearing; pathogen; prevent; public health relevance; recruit; stem cells; tissue 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（通过应用提供）：提出了有关网格蛋白介导的内吞作用（CME）和肌动蛋白在CME期间产生肌动蛋白力的研究的研究，以及它们对在萌芽酵母和哺乳动物细胞中细胞功能的关键重要性。肌动蛋白在无数过程中起作用，包括细胞运动，细胞器传输，粘合剂，收缩力，细胞形状，细胞极性以及膜张力和细胞机械刚性的维持。在肌动蛋白机制和组装调节的知识中存在很大的差距。关于肌动蛋白调节和功能的两个关键问题将在萌芽的酵母研究中解决：（1）细胞周期如何调节肌动蛋白电缆的组装？ （2）1型肌球蛋白和ARP2/3复合物如何共同创造产生膜货币的力？对于以前的研究，最近观察到，CLB2/CDK1的Fimbrin磷酸化对于肌动蛋白组装的细胞周期调节至关重要，将利用对肌动蛋白在细胞周期中如何调节肌动蛋白组装的机械理解。在后来的研究中，将合并深入的生化，生物物理，遗传和细胞生物学方法，以确定CME期间ARP2/3-核酸肌动蛋白网络对1型肌动物的迫使产生的贡献。 CME负责通过质膜的渗透性屏障从细胞的环境中吸收分子，因此对于确定细胞对周围环境的反应至关重要。已经鉴定出许多介导CME的蛋白质和脂质，并且它们的功能在生化和细胞中确定。荧光标记的CME蛋白的活细胞成像揭示了大约60 cme蛋白的复杂募集时间和顺序。但是，如何与蔬菜形成协调货物，如何实现正确的蛋白质募集顺序和时间，哪些事件和分子在途径中起着关键作用，以及力量如何弯曲膜和驱动场地科学，尚不完全了解。以下关键问题将在萌芽的酵母和哺乳动物细胞中解决：CME站点计划如何调节？哪些活动对于CME场地组成至关重要？检查点是否监视CME？哪些生物物理原理控制CME？什么是肌动蛋白的内吞功能，如何调节？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对使用基因组编辑产生的80多个稳定的组织培养和干细胞系进行，以表达CME蛋白作为天然内源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蛋白在结构和功能中高度构成，因此从酵母和哺乳动物研究中学到的原则将分别完成并告知彼此，并提供全面的机械理解，即两者都无法产生。