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: 10166490
• 负责人: DAVID G DRUBIN
• 金额: $ 102.81万
• 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 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10166490
• 关键词: Actins; Address; Adhesions; Affect; Biochemical; Biochemical Reaction; COVID-19; Cell Line; Cell Polarity; Cell membrane; Cell physiology; Cells; Chimeric Proteins; Clathrin; Complement; Cytoskeleton; Development Polarity; Endocytosis; Environment; Event; Evolution; Excision; Generations; Human; Label; Lipids; Mammals; Measures; Mediating; Membrane; Membrane Proteins; Normal Cell; Organelles; Pathologic; Pathway interactions; Permeability; Play; Process; Production; Proteins; Regulation; Role; Route; Saccharomycetales; Site; Structure; Vesicle; Yeasts; cell motility; cell type; empowered; fluorescence imaging; genome editing; human stem cells; human tissue; pandemic disease; reaction rate; recruit; stem cells; tissue culture; uptake

PROJECT SUMMARY Proposed are complementary studies on the mechanisms and regulation of clathrin-mediated endocytosis (CME) and actin force generation during CME in budding yeast and human stem cells. CME is responsible for uptake of molecules from a cell's environment through the permeability barrier of the plasma membrane and for selective removal of plasma membrane proteins. It is also one of the main routes for COVID-19 to enter cells. Therefore, this process is crucial for determining how cells respond to their surroundings and has heightened translational significance. Many proteins and lipids that mediate CME have been identified and their functions determined biochemically and in living cells. Imaging of fluorescently labeled CME proteins in live cells 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 human stem cells: 1) How does membrane curvature affect biochemical reaction rates? 2) How does CME become specialized for different cell types during differentiation? 3) How does a checkpoint sense cargo and regulate CME progress? and, 4) How does actin assemble at CME sites and how does its ultrastructure contribute to CME force production and adapt to increased membrane tension? Yeast studies will be empowered by a rich legacy in the lab of elucidating actin assembly and force production mechanisms. Human cell studies will be empowered by over 120 stable human tissue culture and stem cell lines generated using genome editing to express CME and actin cytoskeleton proteins as fluorescent protein fusions at native, endogenous levels. Because CME proteins are highly conserved in structure and function, principles learned from studies of yeast and humans will complement and inform each other. Together, these studies will provide a comprehensive mechanistic understanding that could not be achieved by studies in only one cell type. Because the actin cytoskeleton has been adapted by evolution for diverse, essential activities including cell motility, organelle transport, adhesion, and cell polarity development, what is learned will apply broadly for many cellular processes and will join the growing armamentarium of possible defensive measures against the pandemic.

项目总结 建议对笼蛋白介导的内吞作用的机制和调节进行补充研究。 在萌芽酵母和人类干细胞的CME过程中，肌动蛋白和肌动蛋白力的产生。芝加哥商品交易所负责 通过细胞膜的通透性屏障从细胞环境中摄取分子 用于选择性去除质膜蛋白。也是新冠肺炎进入的主要渠道之一 细胞。因此，这一过程对于确定细胞如何对周围环境做出反应并具有 更高的翻译意义。许多介导CME的蛋白质和脂质已经被鉴定出来，并 它们的功能由生物化学和活细胞决定。荧光标记CME蛋白在体内的成像 活细胞揭示了大约60种CME蛋白复杂的招募时间和顺序。然而，如何 货物捕获与囊泡形成相协调，蛋白质补充顺序和时机如何正确 已实现，哪些事件和分子在该途径中起关键作用，以及作用力如何使膜弯曲 以及驱动囊泡断裂，目前还没有完全了解。以下关键问题将在萌芽中得到解决 酵母和人类干细胞：1)膜曲率如何影响生化反应速率？2)如何 在分化过程中，CME对不同类型的细胞是否特化？3)检查点是如何感觉到的 货运和监管CME进展？以及，4)肌动蛋白是如何在CME部位组装的，它是如何 超微结构有助于CME力的产生，并适应膜张力的增加？酵母菌研究将 通过在实验室中阐明肌动蛋白组装和力量产生机制的丰富遗产来增强能力。 人类细胞研究将由120多个稳定的人类组织培养和干细胞系提供支持 利用基因组编辑将CME和肌动蛋白细胞骨架蛋白表达为天然的荧光蛋白融合， 内源性水平。因为cme蛋白质在结构和功能上高度保守，所以我们了解到 来自酵母和人类的研究将相辅相成，相互通报。总而言之，这些研究将提供 仅通过对一种细胞类型的研究不能实现的全面的机制理解。 因为肌动蛋白细胞骨架已经通过进化适应了包括细胞在内的各种基本活动。 运动性、细胞器运输、黏附和细胞极性的发展，所学的知识将广泛应用于 许多细胞过程，并将加入越来越多的可能的防御措施，以防止 大流行。