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: 10676743
• 负责人: DAVID G DRUBIN
• 金额: $ 102.85万
• 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/10676743
• 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; Cytoskeletal Proteins; Cytoskeleton; Development Polarity; Endocytosis; Environment; Event; Evolution; Excision; Generations; Human; Label; Learning; 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; empowerment; fluorescence imaging; genome editing; human stem cells; human tissue; pandemic disease; reaction rate; recruit; stem cells; tissue/cell 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.

项目摘要 提出的是关于网格蛋白介导的内吞作用机理和调节的补充研究 （CME）在发芽酵母和人类干细胞中CME期间的肌动蛋白力产生。 CME负责 通过质膜的渗透性屏障从细胞环境中吸收分子， 用于选择性去除质膜蛋白。它也是Covid-19进入的主要路线之一 细胞。因此，此过程对于确定细胞如何应对周围环境至关重要 提高了翻译意义。已经确定了许多介导CME的蛋白质和脂质，并且 它们的功能在生化和活细胞中确定。成像荧光标记的CME蛋白 活细胞揭示了大约60个CME蛋白的复杂募集时间和顺序。但是，如何 货物捕获与囊泡形成协调，蛋白质募集顺序和时机的正确性 达到的，哪些事件和分子在途径中起着关键作用，以及力如何弯曲膜 和驱动囊泡分裂，尚不完全了解。以下关键问题将在萌芽中解决 酵母和人类干细胞：1）膜曲率如何影响生化反应率？ 2）如何 CME在分化过程中是否专门针对不同的细胞类型？ 3）检查点如何感觉 货物并规范CME的进展？ 4）肌动蛋白如何在CME站点组装及其如何组装 超微结构有助于CME力产生并适应增加的膜张力？酵母研究将 在阐明肌动蛋白组装和力生产机制的实验室中，有丰富的遗产赋予能力。 人类细胞研究将受到超过120个稳定的人体组织培养和干细胞系的能力 使用基因组编辑表达CME和肌动蛋白细胞骨架蛋白作为天然的荧光蛋白融合 内源性水平。因为CME蛋白在结构和功能上是高度保守的，所以学到了原则 从酵母和人类的研究中，将补充并互相告知。这些研究将共同​​提供 仅在一种细胞类型的研究中无法实现的全面机械理解。 因为肌动蛋白的细胞骨架已通过进化调整为多种多样的活动，包括细胞 运动，细胞器传输，粘附和细胞极性发展，所学的知识将广泛适用于 许多蜂窝过程，将加入不断增长的防御措施对抗 大流行。

项目成果

Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation.

• DOI: 10.1083/jcb.202002160
• 发表时间: 2020-11-02
• 期刊: The Journal of cell biology
• 作者: Pedersen RTA;Hassinger JE;Marchando P;Drubin DG
• 通讯作者: Drubin DG

Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.

• DOI: 10.1083/jcb.201710084
• 发表时间: 2018-09-03
• 期刊: The Journal of cell biology
• 作者: Dambournet D;Sochacki KA;Cheng AT;Akamatsu M;Taraska JW;Hockemeyer D;Drubin DG
• 通讯作者: Drubin DG

Load adaptation by endocytic actin networks.

• DOI: 10.1091/mbc.e21-11-0589
• 发表时间: 2022-05-15
• 期刊: MOLECULAR BIOLOGY OF THE CELL
• 影响因子: 3.3
• 作者: Kaplan, Charlotte;Kenny, Sam J.;Chen, Xuyan;Schoeneberg, Johannes;Sitarska, Ewa;Diz-Munoz, Alba;Akamatsu, Matthew;Xu, Ke;Drubin, David G.
• 通讯作者: Drubin, David G.

Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

出芽和裂殖酵母中网格蛋白介导的内吞作用的直接比较揭示了保守和可进化的特征

• DOI: 10.7554/elife.50749
• 期刊: eLife
• 影响因子: 7.7
• 作者: Yidi Sun;Johnannes Schoeneberg;Shirley Chen;Tommy Jiang;Charlotte Kaplan;Thomas D. Pollard;David G. Drubin
• 通讯作者: David G. Drubin

Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis.

• DOI: 10.1016/j.celrep.2015.08.048
• 发表时间: 2015-09-29
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Hong SH;Cortesio CL;Drubin DG
• 通讯作者: Drubin DG