Structure and function of nonmuscle myosins

项目摘要 真核细胞的一个标志是它们的迁移、分裂、附着和对环境提示做出反应的能力。 非肌肉肌球蛋白-2(NM2)马达在这些基本细胞的许多方面发挥着重要作用 形成与肌动蛋白细丝相互作用的短双极细丝的过程。NM2电机是二进制开关 其根据蜂窝上下文在非活动状态和活动状态之间改变。NM2电机的精确控制 活性对其作为肌动蛋白细胞骨架主调节因子的细胞功能至关重要。由于以下原因而导致的异常监管 NM2类似基因的突变导致了包括血液和神经疾病在内的一系列疾病， 心脏病、耳聋、肾炎和癌症。因此需要针对NM2的治疗方法，但缺乏基本的 对NM2并列基因的结构和调控的了解是其发展的瓶颈。我们的目标是 对NM2电机如何产生力有详细的结构和机械方面的理解 各种细胞功能。使用创新和跨学科的技术，包括最先进的冷冻- 电子显微镜、X射线结晶学、稳态动力学、体外运动分析和高分辨率 在荧光显微镜下，我们将系统地剖析NM2的激活和调节机制。至 要做到这一点，在目标1中，我们将确定NM2电机的ATPase循环中的主要结构状态来解释 酶的功能。在目标2中，我们将确定一个高分辨率的冷冻-EM结构的非活动状态完全- 长度NM2来解释其分子结构。在目标3中，我们会研究废除 在细胞内NM2细丝的动态上形成非活性状态的能力。总体而言，我们的研究将提供一个 对NM2的结构、功能和调控有更深的理解。重要的是，这种知识将会进步 我们对NM2在细胞中的新功能的理解，从而为未来的发展奠定了基础 NM2特异性疗法。

Project Summary A hallmark of eukaryotic cells is their ability to migrate, divide, adhere and respond to environmental cues. Nonmuscle myosin-2 (NM2) motors play an essential role in many aspects of these fundamental cellular processes by forming short bipolar filaments that interact with actin filaments. NM2 motors are binary switches that alter between inactive and active states depending on the cellular context. The precise control of NM2 motor activity is critical for its cellular function as master regulator of the actin cytoskeleton. Aberrant regulation due to mutations in NM2 paralogs contribute to a whole host of diseases including blood and neurological disorders, heart diseases, deafness, nephritis, and cancers. NM2-specific therapies are thus needed, yet the lack of basic knowledge about the structure and regulation of NM2 paralogs is a bottleneck to their development. We aim to develop a detailed structural and mechanistic understanding of how force generation by NM2 motors drive various cellular functions. Using innovative and interdisciplinary techniques including the state-of-the-art cryo- electron microscopy, X-ray crystallography, steady-state kinetics, in vitro motility assays and high-resolution fluorescence microscopy, we will systematically dissect the mechanisms of activation and regulation of NM2. To achieve this, in Aim 1, we will determine the major structural states in the ATPase cycle of NM2 motors to explain enzyme function. In Aim 2, we will determine a high-resolution cryo-EM structure of the inactive state of full- length NM2 to explain its molecular architecture. In Aim 3, we will study the consequences of abolishing the ability to form an inactive state on the dynamics of NM2 filaments in cells. Collectively, our studies will provide a deeper understanding of the structure, function and regulation of NM2. Importantly, this knowledge will advance our understanding of emergent NM2 functions in cells and thus, lay the foundation for future development of NM2-specific therapeutics.