Structural characterization of single, double and triple-headed axonemal dyneins

项目摘要 运动纤毛在生育、先天免疫和胚胎发育中起重要作用。运动纤毛的跳动 是由一个不同的家族的ATP依赖马达称为轴丝动力蛋白。轴丝动力蛋白是 在纤毛轴丝内以重复的模式拴系到双线微管上， 它们包含的运动域（或头部）的数量及其在轴丝中的位置。外动力蛋白 臂（ODA）是双头或三头复合物，每24 nm重复一次，而内部动力蛋白臂 (IDA)是双头或单头的，每96 nm重复一次。它们的不同位置，周期性， 亚基组成表现为不同的活动：ODA决定节拍频率，而IDA决定节拍频率。 确定波形的幅度。尽管它们对纤毛运动和人类的健康具有根本的重要性， 然而，尽管在生理学上，对大的轴丝动力蛋白家族的结构和机制知之甚少。在这 建议，我们计划利用单粒子电子低温显微镜（cryo-EM）的最新进展，以确定 所有主要类别的轴丝动力蛋白的结构。为了捕获轴丝动力蛋白在它们活跃的微管中- 结合态，我们已经开发出分离和确定天然动力蛋白的高分辨率结构的方法， 来自三种生物体的结合的双线微管-双鞭毛模式生物衣原体 reinhardtii，Bos taurus和人类。C. reinhardtii将用于结构，生化和遗传研究 三头官方发展援助（目标1）和双头和单头国际开发援助（目标2）。预计这些结构将 揭示了将轴丝动力蛋白停靠在双重微管的机制，结构重排， 它们在动力性中风期间经历，它们通过钙和微管曲率的调节，以及功能性的 动力蛋白间相互作用在产生纤毛波形中的相关性。单头和多头结构 轴丝动力蛋白将提供深入了解其特异质亚基和一般功能的功能， 指导轴丝动力蛋白家族进化的原则。动力蛋白束缚双态的结构 来自人类和牛的微管（目的3）将揭示它们与藻类轴丝动力蛋白的差异（对于 例如双头和三头ODA之间的比较），并帮助解释纤毛病变的病因- 导致突变。轴丝动力蛋白的突变是原发性纤毛运动障碍（PCD）的主要原因， 目前无法治愈的遗传性疾病，其特征在于新生儿呼吸窘迫，慢性气道感染， 和不孕症。因此，对轴丝动力蛋白结构的了解将具有重要意义。 这对确定可用于治疗纤毛缺陷的关键机制具有重要意义。

Project Summary Motile cilia play essential roles in fertility, innate immunity, and embryonic development. The beat of motile cilia is powered by a diverse family of ATP-dependent motors called axonemal dyneins. Axonemal dyneins are tethered in repeating patterns to doublet microtubules within the ciliary axoneme and are classified by the number of motor domains (or heads) that they contain and their position within the axoneme. Outer dynein arms (ODA) are either double or triple-headed complexes that repeat every 24 nm, whereas inner dynein arms (IDA) are either double or single-headed and repeat every 96 nm. Their different positions, periodicities, and subunit compositions manifest as different activities: the ODA determines the beat frequency, whereas the IDA determines the amplitude of the waveform. Despite their fundamental importance to ciliary motility and human physiology, little is known about the structures and mechanisms of the large axonemal dynein family. In this proposal, we plan to exploit recent advances in single-particle electron cryomicroscopy (cryo-EM) to determine structures of all major classes of axonemal dynein. To capture axonemal dyneins in their active, microtubule- bound states we have developed methods to isolate and determine high-resolution structures of native dynein- bound doublet microtubules from three organisms - the biflagellate model organism Chlamydomonas reinhardtii, Bos taurus and humans. C. reinhardtii will be used for structural, biochemical, and genetic studies of a triple-headed ODA (Aim 1) and double and single-headed IDAs (Aim 2). These structures are expected to reveal the mechanisms that dock axonemal dyneins to the doublet microtubule, the structural rearrangements they undergo during the powerstroke, their regulation by calcium and microtubule curvature, and the functional relevance of inter-dynein interactions in generating the ciliary waveform. Structures of single and multi-headed axonemal dyneins will provide insights into the functions of their idiosyncratic subunits and the general principles that have guided evolution of the axonemal dynein family. Structures of dynein-bound doublet microtubules from humans and cows (Aim 3) will reveal their differences with algal axonemal dyneins (for example the comparison between double and triple-headed ODAs) and help explain the etiology of ciliopathy- causing mutations. Mutations in axonemal dyneins are the leading cause of primary ciliary dyskinesis (PCD), a currently incurable inherited disease characterized by neonatal respiratory distress, chronic airway infections, and infertility. Advances in understanding the structures of axonemal dyneins will therefore have important implications for the identification of key mechanisms that can targeted for therapy of defective cilia.