Regulation of the adaptive actin response by force-dependent bonds

摘要 通过肌动蛋白细胞骨架的力传递是细胞如何感知几何和生物信息的基础。 它们的环境的机械约束，通过组织移动，重塑细胞外基质， 调节质膜（PM）上的信号受体以决定细胞命运。在诸如领先的 在迁移细胞的边缘或网格蛋白介导的内吞作用（CME）中，聚合肌动蛋白推压细胞边缘， 膜以产生膨胀力。肌动蛋白的动力学、结构和力的产生受 力学和肌动蛋白结合蛋白（ABP），其束、分支、断裂、软化、伸展、束缚、或 移动肌动蛋白丝。除了肌球蛋白马达，机械力如何调节ABPs的亲和力很少 被调查了当ABP机械锚定在细胞中时，ABP-肌动蛋白界面的力调节 ABP-actin键的寿命，我在下文中称之为力依赖性肌动蛋白解离速率 （FDADR）。虽然直觉表明，当分子被破坏时，分子键的寿命会缩短， 拉开（"滑动键"）1，令人惊讶的是，最近表征的大多数涉及细胞粘附形式的ABP 与肌动蛋白的"捕获键"随着力的增加而增加寿命（有时> 100倍2）2 - 5。这些债券 高度调谐到相对于肌动蛋白丝极性施加的力的方向2，4，6与最极端的 报道的例子是由talin的ABS3结构域2形成的不对称捕获键。的功能影响 这些ABP的FDADR未知。由于肌动蛋白在产生和传递机械信号中的重要性， 力，ABP-actin相互作用的平衡体积测量提供了ABPs如何 有助于肌动蛋白细胞骨架的结构，功能和调节。 在CME期间，PM被弯曲以封装膜结合的货物。当PM张力高时， 需要聚合力来使膜弯曲并将新生囊泡拉入细胞7。肌动蛋白在 CME凹坑"适应" PM张力，在PM升高的条件下优先定位于凹坑表面 张力（即，确切地说，仅当需要完成CME时）8。我将检验THATCH的假设 CME衔接子HIP1R的肌动蛋白结合结构域形成不对称的捕获键，类似同源物talin ABS 3。我会 在酵母分子遗传筛选中发现具有改变的结合的突变体，并表征其FDADR。我会 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丝， 从而支持在致密皮质丝状肌动蛋白中的膜张力范围内的内吞作用。突变体 FDADR改变的HIP1 R THATCH将在哺乳动物细胞中表达，及其对CME和肌动蛋白的影响 组织将被确定，并与模拟进行比较，从而将FDADR与肌动蛋白结构联系起来， 功能

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.