Regulation of the adaptive actin response by force-dependent bonds

通过力依赖性键调节适应性肌动蛋白反应

• 批准号: 10537442
• 负责人: Leanna Marie Owen
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10537442
• 关键词: Actin-Binding Protein; Actins; Affinity; Binding; Biological Assay; Biophysics; C-terminal; Cell Adhesion; Cell membrane; Cells; Clathrin; Cytoskeleton; Dissociation; Encapsulated; Endocytosis; Environment; Equilibrium; Extracellular Matrix; F-Actin; Generations; Genetic Screening; Homologous Gene; Human; Huntington Interacting Protein 1-Related; Intuition; Kinetics; Mammalian Cell; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Microfilaments; Minus End of the Actin Filament; Molecular; Molecular Genetics; Motor; Muscle Contraction; Myosin ATPase; Pattern; Physiological; Polymers; Proteins; Receptor Signaling; Regulation; Reporting; Role; Structure; Surface; System; Talin; Testing; Tissues; Ursidae Family; Vesicle; Work; Yeasts; base; cell motility; in silico; insight; laser tweezer; mechanical force; mechanical load; mutant; optical traps; polymerization; response; simulation; transmission process

Abstract Force transmission through the actin cytoskeleton is fundamental to how cells sense the geometric and mechanical constraints of their environments, move through tissues, remodel the extracellular matrix, and regulate signaling receptors at the plasma membrane (PM) to determine cell fate. In systems such as the leading edge of migrating cells or in clathrin-mediated endocytosis (CME), polymerizing actin pushes against a membrane to generate protrusive force. The dynamics, structure, and force generation of actin are regulated by mechanics and actin binding proteins (ABPs) that bundle, branch, break, soften, stiffen, polymerize, tether, or move actin filaments. Aside from myosin motors, how mechanical force regulates the affinity of ABPs has seldom been investigated. When ABPs are mechanically anchored in the cell, force at the ABP-actin interface regulates the lifetime of the ABP-actin bond, which I refer to hereafter as the force-dependent actin dissociation rate (FDADR). While intuition suggests the lifetimes of molecular bonds should shorten when the molecules are pulled apart (a “slip bond”)1, a surprising majority of recently characterized ABPs involved in cell adhesion form “catch bonds” with actin that increase in lifetime (sometimes >100-fold2) as force increases2–5. These bonds are highly tuned to the direction of force applied relative to the actin filament polarity2,4,6 with the most extreme reported example being the asymmetric catch bond formed by talin's ABS3 domain2. The functional impact of the FDADR of these ABPs is not known. Due to actin's importance in generating and transmitting mechanical force, equilibrium bulk measurements of ABP-actin interactions provide an incomplete picture of how ABPs contribute to actin cytoskeleton structure, function, and regulation. During CME the PM is bent to encapsulate membrane-bound cargoes. When PM tension is high, actin polymerization force is required to bend the membrane and pull the nascent vesicle into the cell7. Actin at the CME pit “adapts” to PM tension by localizing to the surface of the pit preferentially in conditions of elevated PM tension (i.e., precisely only when it is required for CME completion)8. I will test the hypothesis that the THATCH actin binding domains of CME adapter HIP1R forms an asymmetric catch bond like homolog talin ABS3. I will discover mutants with altered binding in a yeast molecular-genetic screen and characterize their FDADR. I will develop a stochastic simulation to uncover the role of the FDADR in actin network structure and function during CME. I hypothesize that HIP1R's FDADR is tuned to selectively bind actin filaments that bear mechanical load, thus supporting endocytosis over a range of membrane tensions amidst dense cortical filamentous actin. Mutant HIP1R THATCH with altered FDADR will be expressed in mammalian cells, and their impact on CME and actin organization will be determined and compared to simulations, thus relating FDADR to actin structure and function.

摘要 通过肌动蛋白细胞骨架的力传递是细胞感知几何和 环境的机械约束，在组织中移动，重塑细胞外基质，以及 调节质膜上的信号受体以决定细胞的命运。在诸如领先的 在迁移细胞的边缘或在网状蛋白介导的内吞作用(CME)中，聚合的肌动蛋白推动 膜能产生突出力。肌动蛋白的动力学、结构和力的产生是由 机械和肌动蛋白结合蛋白(ABPs)，可以捆绑、分支、断裂、软化、僵硬、聚合、系留或 移动肌动蛋白细丝。除了肌球蛋白马达，机械力如何调节ABPs的亲和力还很少。 被调查过了。当ABPs机械地固定在细胞内时，ABP-肌动蛋白界面上的力调节 ABP-肌动蛋白键的寿命，以下我将其称为与力相关的肌动蛋白解离率 (FDADR)。虽然直觉表明，当分子 撕裂(一种“滑移粘结”)1，令人惊讶的是，最近被描述为ABPs的大多数参与了细胞黏附形式 与肌动蛋白结合，随着作用力增加2-5，生命期延长(有时为100倍)。这些结合是 高度调整到相对于肌动蛋白细丝极化施加的力的方向2，4，6，最极端 报道的例子是Talin的ABS3结构域形成的不对称捕获键2。对功能的影响 这些ABP的FDADR尚不清楚。由于肌动蛋白在机械机械的产生和传递中的重要性 ABP-肌动蛋白相互作用的力，平衡体积测量提供了一幅关于ABPS如何 有助于肌动蛋白细胞骨架的结构、功能和调节。 在CME过程中，PM被弯曲以封装被膜结合的货物。当PM张力较高时，肌动蛋白 需要聚合力来弯曲细胞膜并将新生的囊泡拉入细胞7。肌动蛋白在 在PM升高的情况下，CME凹坑通过优先定位到凹坑表面来“适应”PM张力 张力(即，仅当完成CME所需时)8.我将检验这样一个假设，即茅草 CME接头HIP1R的肌动蛋白结合域形成一个不对称的捕捉键，类似于ABS3的同系物talin。这就做 在酵母分子遗传学筛选中发现结合改变的突变体，并对其FDADR进行表征。这就做 开发一个随机模拟，以揭示FDADR在肌动蛋白网络结构和功能中的作用 CME。我假设HIP1R的FDADR被调整为选择性地结合承受机械负荷的肌动蛋白细丝， 从而在致密的皮质丝状肌动蛋白中间的一系列膜张力上支持内吞作用。突变型 改变FDADR的HIP1R茅草将在哺乳动物细胞中表达，及其对CME和肌动蛋白的影响 将确定组织并与模拟进行比较，从而将FDADR与肌动蛋白结构和 功能。