Actin assembly and clathrin-mediated endocytosis in yeast and mammals

项目摘要 建议补充研究的机制和调控网格蛋白介导的内吞作用 (CME)以及芽殖酵母和人类干细胞中CME期间肌动蛋白力的产生。CME负责 通过质膜的渗透屏障从细胞环境中摄取分子， 用于选择性去除质膜蛋白。这也是COVID-19进入的主要途径之一 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至关重要， 翻译的重要性。已经鉴定了许多介导CME的蛋白质和脂质， 它们的功能由生物化学和活细胞决定。荧光标记的CME蛋白质的成像 活细胞揭示了大约60种CME蛋白质复杂的募集时间和顺序。但如何 货物捕获与囊泡形成协调，如何正确的蛋白质招募顺序和时机 实现，哪些事件和分子在途径中发挥关键作用，以及力如何弯曲膜 和驱动囊泡断裂的机制还不完全清楚。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速率？2)如何 CME在分化过程中是否对不同的细胞类型变得特化？3)检查点如何感知 货物和规范CME进展？以及，4）肌动蛋白如何在CME位点组装， 超微结构有助于CME力的产生和适应膜张力的增加？酵母研究将 在阐明肌动蛋白组装和力产生机制的实验室中获得了丰富的遗产。 人类细胞研究将通过产生120多个稳定的人类组织培养和干细胞系来实现 使用基因组编辑在天然条件下将CME和肌动蛋白细胞骨架蛋白表达为荧光蛋白融合体， 内源性水平。由于CME蛋白在结构和功能上高度保守， 从酵母和人类的研究将相互补充和相互告知。这些研究将提供 一个全面的机制的理解，不能实现的研究，只有一种细胞类型。 由于肌动蛋白细胞骨架已经通过进化适应了各种各样的基本活动，包括细胞 运动，细胞器运输，粘附和细胞极性发展，所学到的将广泛适用于 许多细胞过程，并将加入日益增长的可能的防御措施， 流行病

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.