Molecular Mechanisms of Ciliary Motility

描述（由申请人提供）：本项目的总体目标是了解分子马达动力蛋白是如何调节的，以产生运动纤毛和鞭毛的高拍频和复杂波形特征。在哺乳动物中，运动纤毛/鞭毛是精子推进、从呼吸道和中耳清除碎片、脑脊液循环以及在发育期间确定左右身体平面所必需的。因此，纤毛运动缺陷导致生育能力受损、呼吸困难、脑积水、中耳炎和/或左右体轴随机化。虽然在理解动力蛋白马达的力产生特性方面取得了重大进展，但微管滑动在空间和时间上如何控制仍然是该领域最大的未回答的问题之一。大量的证据表明，中央器和辐射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。我们实验室和其他人的重大努力已经建立了这些结构的组成，然而，将这些成分整合到更广泛的纤毛运动机制的理解是缺乏的。在这项建议中，我们为弥合这一差距迈出了根本性的一步。在该提案的目标1和2中，我们将利用我们的发现，即重复辐条对的两个径向辐条（RS 1和RS 2）在组成上是异质的。在目标1中，我们对RS 1和RS 2的微管结合衔接子的鉴定为我们提供了定义可能建立96 nm轴丝重复的特定辐条相关蛋白的靶向和锚定机制的机会。在目标2中，我们测试的假设，在对每个发言控制特定的动力蛋白臂子形式的活动。我们发现RS 2的适配器CSC是WT运动所需的，并与动力蛋白调节复合物和特定的内部动力蛋白臂同种型接触，这一发现支持了这一目的。在目标3中，我们专注于径向轮辐中心装置的相互作用。这一目标是建立在我们发现的复合物与C1微管的中央装置是必不可少的野生型运动。我们将结合联合收割机遗传学，功能测定和生物物理/计算方法，以确定中央对预测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。目标3中的实验可能会揭示生物系统中纳米尺度摩擦力的新原理，但这对细胞功能有深远的影响。我们的综合研究将弥合我们对纤毛运动的理解的主要差距，通过解决有关如何在空间和时间上控制大的，高度保守的，大分子组装体之间的相互作用调节动力蛋白驱动的微管滑动的基本问题。这些研究也将对更广泛的微管相关马达领域产生影响。

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.