Molecular Mechanisms of Ciliary Motility

描述（由申请人提供）：本项目的总体目标是了解如何调节分子马达动力蛋白以产生运动纤毛和鞭毛的高节拍频率和复杂波形特征。在哺乳动物中，运动的纤毛/鞭毛是精子推进、清除呼吸道和中耳碎片、脑脊液循环以及确定发育过程中左右身体计划所必需的。因此，纤毛运动缺陷导致生育能力受损、呼吸窘迫、脑积水、中耳炎和/或左右体轴随机化。虽然在了解动力马达的发力特性方面取得了重大进展，但如何在空间和时间上控制微管滑动仍然是该领域最大的未解问题之一。大量证据表明，中心装置和径向辐条是控制微管滑动和纤毛跳动的信号转导网络的主要组成部分。我们实验室和其他实验室的重大努力已经确定了这些结构的组成，然而，将这些组成部分整合到对纤毛运动的更广泛的机制理解中是缺乏的。在这项建议中，我们为弥合这一差距迈出了根本的一步。在目标1和2的建议，我们将利用我们的发现，两个径向辐条（RS1和RS2）的重复辐条对是异质组成。在Aim 1中，我们对RS1和RS2的微管结合接头的鉴定为我们提供了确定特异性辐条相关蛋白的靶向和锚定机制的机会，这些蛋白可能建立了96 nm轴突重复。在Aim 2中，我们检验了这对中的每个辐条控制特定动力蛋白臂亚型的活动的假设。我们发现RS2的适配器CSC是WT运动所必需的，并与动力蛋白调节复合体和特定的内动力蛋白臂异构体接触，这一发现支持了这一观点。在目标3中，我们关注径向辐条-中心装置的相互作用。这个目标是建立在我们发现的与中央装置的C1微管相关的复合物的基础上的，这是野生型运动所必需的。我们将结合遗传学，功能分析和生物物理/计算方法来确定中心对投影和径向辐条之间的关键相互作用，并测试这些结构之间的物理相互作用如何调节微管滑动和纤毛跳动的假设。Aim 3的实验可能会揭示生物系统中纳米尺度摩擦力作用的新原理，但这对细胞功能有深远的影响。我们的联合研究将通过解决关于大型、高度保守的大分子组装之间的空间和时间控制相互作用如何调节动力蛋白驱动的微管滑动的基本问题，弥合我们对纤毛运动理解的主要差距。这些研究也将对微管相关电机的更广泛领域产生影响。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how the molecular motor dynein is regulated to produce the high beat frequency and complex waveforms characteristic of motile cilia and flagella. In mammals, motile cilia / flagella are required for sperm propulsion, removal of debris from the respiratory tract and middle ear, circulation of cerebrospinal fluid, and for determination of the left-right body plan during development. As a consequence, defects in ciliary motility result in impaired fertility, respirator distress, hydrocephalus, otitis media, and/or randomization of the left-right body axis. While significant progress has been made in understanding the force generating properties of the dynein motors, how microtubule sliding is controlled both spatially and temporally remains one of the biggest unanswered questions in the field. Substantial evidence indicates that the central apparatus and radial spokes are major components of a signal transduction network which controls microtubule sliding and ciliary beating. Significant efforts from our lab and others have established the composition of these structures, yet, integrating these components into a broader mechanistic understanding of ciliary motility is lacking. In this proposal, we take a fundamental step towards bridging this gap. In Aims 1 and 2 of the proposal we will capitalize on our discovery that the two radial spokes (RS1 and RS2) of the repeating spoke pairs are heterogeneous in composition. In Aim 1 our identification of the microtubule binding adaptors for RS1 and RS2 provide us with the opportunity to define a mechanism for the targeting and anchoring of specific spoke associated proteins that likely establish the 96 nm axonemal repeat. In Aim 2 we test the hypothesis that each spoke in the pair controls the activity of specific dynein arm subforms. This Aim is supported by our discovery that the adaptor for RS2, the CSC, is required for WT motility and makes contact with the dynein regulatory complex and specific inner dynein arm isoforms. In Aim 3 we focus on radial spoke - central apparatus interactions. This Aim is founded on our discovery of complexes associated with the C1 microtubule of the central apparatus that are essential for wild-type motility. We will combine genetics, functional assays and biophysical /computational approaches to identify key interactions between the central pair projections and radial spokes and to test hypotheses about how physical interactions between these structures modulate microtubule sliding and ciliary beating. Experiments in Aim 3 will likely reveal new principles for the frictional forces acting ata nanoscopic scale in biological systems but which have profound consequences on cell function. Our combined studies will bridge major gaps in our understanding of ciliary motility by addressing fundamental question about how spatially and temporally controlled interactions between large, highly conserved, macromolecular assemblies regulate dynein-driven microtubule sliding. These studies will also have an impact on the more general field of microtubule-associated motors.