Mechanoregulation of Ciliary Motility

纤毛运动的机械调节

• 批准号: 10180150
• 负责人: Alan Brown
• 金额: $ 41.41万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10180150
• 关键词: Algae; Animal Model; Bacteria; Behavior; Biogenesis; Biological Models; Blood Circulation; Brain; Cells; Cerebrospinal Fluid; Chemicals; Chlamydomonas reinhardtii; Chronic; Cilia; Cilium Microtubule; Communication; Complement; Complex; Cryoelectron Microscopy; Data; Defect; Disease; Docking; Dynein ATPase; Electrons; Epithelial; Etiology; Excision; Genes; Human; Immunoprecipitation; Individual; Infertility; Lead; Link; Liquid substance; Literature; Locomotion; Lung diseases; Machine Learning; Maps; Mass Spectrum Analysis; Mechanics; Mediating; Microtubules; Modeling; Molecular; Molecular Machines; Motor; Movement; Mucous body substance; Mutagenesis; Mutation; Nature; Oocytes; Organ; Organelles; Organism; Paralysed; Pathogenicity; Pathway interactions; Pattern; Proteomics; Publishing; Radial; Regulation; Resolution; Respiratory System; Sampling; Side; Signal Transduction; Speed; Structure; Surface; Testing; Work; base; biological systems; cell motility; ciliopathy; cilium motility; crosslink; driving force; extracellular; fluid flow; insight; nexin; novel; particle; pathogen; reconstruction; respiratory virus; sperm cell

Project Summary Mechanoregulation is a fundamental mechanism for the control of dynamic and multiscale biological systems. A mechanoregulatory network is responsible for the motility of cilia by converting the action of thousands of individual dynein motors bound to doublet microtubules in the cilium into a single waveform. This waveform has evolved to efficiently displace fluid, allowing either cell self-propulsion or the transport of extracellular liquid over epithelial surfaces. Ciliary motility in humans is therefore essential for the movement of sperm cells, the removal of bacteria and viruses from the respiratory tract, and the circulation of cerebrospinal fluid in the brain. Cilia are also used by protozoan pathogens for movement, contributing to their pathogenicity. The biflagellate alga, Chlamydomonas reinhardtii, has become the model system for studying the relationship between cilia ultrastructure and ciliary motilty. Using this organism, we have determined single-particle electron cryomicroscopy (cryo-EM) structures of the bases of dyneins and mechanoregulatory complexes natively bound to doublet microtubules. These structures map the interconnected network of microtubules, mechanoregulators, and dynein motors in unparalleled atomic detail. The structures reveal the mechanisms that dock mechanoregulators to doublet microtubules and generate new hypotheses for how they control dynein behavior. These preliminary structural studies provide a unique opportunity to better understand the structure, function and assembly pathway of the largest mechanoregulator, the radial spoke. In aim 1, I propose to elucidate the complete structure of a native radial spoke using cryo-EM and cross-linking mass spectrometry. Structural information will resolve how its 20+ unique subunits interact and function together to respond to both mechanical and chemical signals. Due to the high conservation of radial-spoke subunits among organisms, our structure will provide insights into the etiology of ciliopathy-causing mutations in humans. In aim 2, I propose to test hypotheses that have arisen from our “on-doublet” structures using an interdisciplinary combination of structure-guided mutagenesis, waveform analysis by high-speed microcinematography, and structural characterization using electron cryotomography. This work will provide experimental evidence for the fundamental molecular mechanisms that control ciliary motility. In aim 3, I propose to use a proteomic and structural approach to determine the mechanisms of radial-spoke assembly. This work will test our current structure-based model of assembly and has the potential to identify the first radial-spoke biogenesis factors. Collectively, these studies will provide unprecedented mechanistic insight into the mechanoregulatory pathways that control ciliary motility and promises to open new avenues for the treatment of ciliopathies.

项目摘要 机械调节是控制动态多尺度生物系统的一种基本机制。 一个机械调节网络负责纤毛的运动，通过转换数千个 单个动力蛋白发动机与纤毛中的双微管结合成一个单一的波形。该波形 已经进化到可以有效地排出液体，允许细胞自我推进 或胞外液体的运输 在上皮性表面。因此，人类的纤毛运动对精子细胞的运动是必不可少的 清除呼吸道中的细菌和病毒，以及脑脊液在大脑中的循环。 纤毛也被原生动物病原体用于运动，有助于它们的致病性。 双鞭毛类 莱茵衣藻已成为研究纤毛关系的模式系统 超微结构和纤毛运动。利用这种有机体，我们已经确定了单粒子电子 动力蛋白和机械调节复合体天然碱基的低温显微镜(Cryo-EM)结构 结合在双重微管上。这些结构映射了相互连接的微管网络， 机械调节器和动力蛋白马达以无与伦比的原子细节。这些结构揭示了这些机制 将机械调节器对接成双微管，并产生关于它们如何控制的新假说 动力系统的行为。这些初步的结构研究提供了一个独特的机会来更好地理解 最大的机械调节器--径向辐条的结构、功能和装配路径。在目标1中，我 提出用低温EM和交联质谱来阐明天然径向辐条的完整结构 光谱分析。结构信息将解析其20多个独特的亚基如何相互作用和共同发挥作用 对机械信号和化学信号都有反应。由于径向辐条亚基的高度保守性 在生物体中，我们的结构将提供对导致纤毛疾病突变的病因学的见解 人类。在目标2中，我建议使用一个 多学科结合的结构导向诱变、高速波形分析 微电影摄影术，以及使用电子冷冻断层摄影术的结构表征。这项工作将提供 控制纤毛运动的基本分子机制的实验证据。在目标3中，我 建议使用蛋白质组学和结构学方法来确定径向辐条组装的机制。 这项工作将测试我们目前基于结构的组装模型，并有可能确定第一个 径向-辐条生物发生因素。总的来说，这些研究将提供前所未有的机械洞察力 控制纤毛运动的机械调节途径，并有望开辟新的途径 纤毛病的治疗。