Mechanoregulation of Ciliary Motility

项目摘要 机械调节是控制动态和多尺度生物系统的基本机制。 一个机械调节网络负责纤毛的运动性，通过转换数千个 单个动力蛋白马达与纤毛中的双微管结合成单个波形。该波形 已经进化到可以有效地排出液体， 或者细胞外液的运输 覆盖在上皮表面上。因此，人类的纤毛运动对于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，以及脑脊髓液的循环。 纤毛也被原生动物病原体用于运动，有助于其致病性。 双鞭毛藻 莱茵衣藻（Chlamyeliumreinhardtii）已成为研究纤毛之间关系的模式系统 超微结构和纤毛运动。利用这种生物，我们已经确定了单粒子电子 低温显微镜（cryo-EM）结构的基础动力蛋白和机械调节复合物天然 结合在双微管上这些结构映射了微管的互连网络， 机械调节器和动力蛋白发动机在无与伦比的原子细节。这些结构揭示了 将机械调节因子与双态微管对接， 动力蛋白行为这些初步的结构研究提供了一个独特的机会，更好地了解 最大的机械调节器--径向轮辐的结构、功能和装配路径。在目标1中，我 我建议阐明完整的结构，一个原生径向辐条使用冷冻电镜和交联质量 光谱法结构信息将解决其20多个独特的亚基如何相互作用和共同发挥作用， 对机械和化学信号都有反应。由于辐射辐子单元的高度保守性 在生物体中，我们的结构将提供对引起纤毛病变突变的病因学的见解， 人类在目标2中，我建议使用一个 结构导向突变的跨学科组合，高速波形分析 显微电影术和使用电子冷冻断层扫描的结构表征。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的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装配模型，并有可能确定第一个 辐射辐生物成因因子总的来说，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，并有望为纤毛运动开辟新的途径。 治疗纤毛病。

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.