Structural Mechanisms of Cytoskeletal Force-Sensing

项目摘要 体内的细胞感知来自其局部环境的线索，这些线索通过一种 协调一系列的分子事件称为信号。信号传导对于告诉细胞它是否 应该生长和分裂，迁移到身体的不同部位，或者在完成其功能后自杀。 或者受到了无法弥补的伤害通常，在患病的情况下，信号传递过程会被发现不正确 细胞例如，癌细胞的分裂和迁移失去了控制，并忽略了应该让它们留在体内的线索。 check.信号有多种形式。特异性分子结合并激活细胞中的同源受体蛋白， 被称为“化学信号”，这是广泛理解的。物理力和细胞的刚性 环境也会引起特定的细胞行为，但我们对蛋白质如何在特定的环境中产生作用的理解相对较差。 传递这些“机械信号”。成功的药物中有很大一部分靶向蛋白质分子， 在化学信号中起作用。许多这样的治疗方法的发展是由确定 受体蛋白和分子之间相互作用的详细三维化学结构 激活它们，促进药物的设计，精确地干预这些过程。尽管 重要的是，治疗靶向机械信号传导的努力受到限制。长期目标是 一个研究项目是可视化力如何调节机械信号的三维结构 蛋白质来激活它们，以促进阻止这些变化的药物的开发。 该提案特别关注于理解细胞聚合物（“细丝”）如何由 肌动蛋白协调机械信号。细胞含有许多由肌动蛋白丝组成的网络， 肌球蛋白分子马达蛋白和数百种其他结合伴侣，它们共同产生和 传递不同的力量。我们假设，特定类型的力导致不同的物理重排， 肌动蛋白丝，可以通过直接结合相互作用被细胞中的其他蛋白质检测到。我们将 鉴定以力敏感方式结合肌动蛋白的蛋白质（目标1），特别关注描绘 赋予力敏感性的蛋白质的精确区域。接下来我们将观察侧向弯曲力 (Aim 2）和肌球蛋白马达蛋白（Aim 3）产生的纵向拉伸和压缩力影响肌动蛋白 细丝结构，假设这些力机制产生不同的重排， 被有约束力的伴侣所歧视。为了实现这些目标，我们正在开发样品制备和 计算图像分析方法来可视化肌动蛋白聚合物的三维结构， 冷冻电子显微镜（cryo-EM）的机械力的存在。除了提供基本的见解外， 细胞如何通过蛋白质结构的变化来感知力，我们的研究将指导 将精确的分子干预发展成由肌动蛋白控制的机械信号传导过程。

PROJECT SUMMARY Cells in the body perceive cues from their local environment, which control cellular behavior through a coordinated series of molecular events known as signaling. Signaling is critically important for telling a cell if it should grow and divide, migrate to a different part of the body, or commit suicide if it has completed its function or been irreparably damaged. Frequently, signaling processes are found to be working incorrectly in diseased cells. For instance, cancer cells divide and migrate out of control and ignore cues which should keep them in check. Signals come in multiple forms. Specific molecules bind and activate cognate receptor proteins in the cell, known as “chemical signaling”, which is broadly well-understood. Physical forces and the rigidity of a cell’s environment also elicit specific cell behaviors, but we have a comparatively poor understanding of how proteins transmit these “mechanical signals”. A significant fraction of successful drugs target protein molecules which operate in chemical signaling. The development of many such treatments was stimulated by determining the detailed three-dimensional chemical structures of the interactions between receptor proteins and the molecules which activate them, facilitating the design of drugs which precisely intervene in these processes. Despite its importance, efforts to therapeutically target mechanical signaling have been limited. The long-term goal of this research project is to visualize how forces modulate the three-dimensional structure of mechanical signaling proteins to activate them, in order to facilitate the development of drugs that block these changes. This proposal is specifically focused on understanding how cellular polymers (“filaments”) composed of the protein actin coordinate mechanical signaling. The cell contains many networks composed of actin filaments, myosin molecular motor proteins, and hundreds of other binding partners, which collectively generate and transmit diverse forces. We hypothesize that specific types of forces cause distinct physical rearrangements in actin filaments, which can be detected by other proteins in the cell through direct binding interactions. We will identify proteins which bind actin in a force-sensitive manner (Aim 1), focusing specifically on delineating the precise regions of the proteins which confer force-sensitivity. We will next visualize how side-wise bending forces (Aim 2) and length-wise tensile and compressive forces generated by myosin motor proteins (Aim 3) impact actin filament structure, hypothesizing these force regimes produce distinct rearrangements which can be discriminated by binding partners. In pursuit of these Aims, we are developing sample preparation and computational image analysis approaches to visualize the three-dimensional structure of actin polymers in the presence of mechanical forces with cryo-electron microscopy (cryo-EM). In addition to providing basic insights into how forces are perceived by cells through changes in protein structure, our studies will guide the development of precise molecular interventions into mechanical signaling processes governed by actin.