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

项目摘要 游动纤毛在生育、先天免疫和胚胎发育中起着重要作用。活动的纤毛的节拍 是由一系列不同的被称为轴线动力蛋白的ATP依赖的马达驱动的。轴线动力蛋白是 在纤毛轴丝内以重复的模式拴在双微管上，并按 它们包含的运动域(或头)的数量以及它们在轴丝中的位置。外部动力系统 臂(Oda)是每24纳米重复一次的双头或三头复合体，而内部动力蛋白臂 (IDA)为双头或单头，每96 nm重复一次。他们不同的职位，不同的周期性和 亚基组成表现为不同的活动：oda决定节拍频率，而ida 确定波形的幅度。尽管它们对纤毛运动和人类 在生理学方面，人们对大轴内动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冷冻显微镜(Cryo-EM)的最新进展来确定 轴丝动力蛋白的所有主要类别的结构。将轴丝动力蛋白捕获到它们活跃的微管中- 束缚态我们已经开发出方法来分离和确定天然动力蛋白的高分辨率结构- 三种生物--双鞭毛类模式生物衣藻的结合双微管 莱恩哈迪尔牛、金牛座和人类。将用于结构、生物化学和遗传学研究。 三头官方发展援助(目标1)和双头和单头发展援助(目标2)。这些结构预计将 揭示轴线动力蛋白对接到二联体微管的机制，结构重排 在卒中期间，它们经历了钙离子和微管弯曲的调节，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。单头和多头的结构 轴丝动力蛋白将提供对其特殊亚单位和一般 指导轴丝动力蛋白家族进化的原则。动力蛋白结合双重态的结构 来自人和牛的微管(目标3)将揭示它们与藻类轴线动力蛋白的差异(对于 例如，双头和三头ODA的比较)，并有助于解释纤毛疾病的病因- 导致突变。轴丝动力蛋白的突变是导致原发性睫状体运动障碍(PCD)的主要原因。 目前无法治愈的遗传病的特征是新生儿呼吸窘迫，慢性呼吸道感染， 和不孕不育。因此，了解轴膜动力蛋白结构的进展将具有重要意义 对识别可靶向治疗纤毛缺陷的关键机制的启示。

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.