Structural Mechanisms of Cytoskeletal Force-Sensing

项目总结 身体中的细胞从它们的局部环境中感知线索，这些线索通过 被称为信号的一系列协调的分子事件。信令对于告诉细胞是否 应该生长和分裂，迁移到身体的不同部位，或者如果它已经完成了它的功能就自杀 或者受到了不可挽回的损害。通常，在患病的患者中发现信令过程工作不正常 细胞。例如，癌细胞分裂和迁移失控，忽视了应该让它们留在体内的线索。 检查完毕。信号有多种形式。特定分子结合并激活细胞中的同源受体蛋白， 被称为“化学信号”，它被广泛地理解。物理力和细胞的刚性 环境也会导致特定的细胞行为，但我们对蛋白质是如何 传递这些“机械信号”。相当一部分成功的药物针对的是蛋白质分子 在化学信号中工作。许多这样的治疗方法的发展是通过确定 受体蛋白与分子相互作用的详细三维化学结构 激活它们，促进精确干预这些过程的药物的设计。尽管它的 重要的是，治疗靶向机械信号转导的努力有限。这样做的长期目标是 研究项目是可视化地展示力如何调制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。 这项建议特别侧重于理解细胞聚合物(“细丝”)是如何由 肌动蛋白协调机械信号的传递。该细胞含有许多由肌动蛋白细丝组成的网络， 肌球蛋白分子马达蛋白和数百个其他结合伙伴，共同产生和 传递不同的力量。我们假设，特定类型的力会导致不同的物理重排 肌动蛋白细丝，可被细胞中的其他蛋白质通过直接结合相互作用检测到。我们会 确定以力敏感的方式结合肌动蛋白的蛋白质(目标1)，特别侧重于描绘 赋予力敏感的蛋白质的精确区域。接下来，我们将可视化横向弯曲力是如何 (目标2)和肌球蛋白马达蛋白产生的沿长度方向的拉力和压缩力(目标3)冲击肌动蛋白 细丝结构，假设这些力机制会产生不同的重排，这可能是 受到有约束力的合作伙伴的歧视。为了实现这些目标，我们正在发展样品制备和 计算机图像分析方法可视化肌动蛋白聚合物的三维结构 用低温电子显微镜(Cryo-EM)观察机械力的存在。除了提供基本的见解之外 关于细胞如何通过蛋白质结构的变化感知作用力，我们的研究将指导 发展精确的分子干预进入由肌动蛋白控制的机械信号传递过程。

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.