Molecular Mechanisms of Ciliary Motility

纤毛运动的分子机制

• 批准号: 9329299
• 负责人: Elizabeth F Smith
• 金额: $ 30.78万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329299
• 关键词: Address; Animal Model; Binding; Biochemical; Biological Assay; Biophysics; Blood Circulation; Calcium; Cell physiology; Cerebral Ventricles; Cerebrospinal Fluid; Characteristics; Chlamydomonas; Collaborations; Complex; Data; Defect; Development; Distress; Dynein ATPase; Embryo; Epithelial; Excision; Family; Fertility; Flagella; Frequencies; Friction; Genetic; Goals; Head; Heterogeneity; Human; Hydrocephalus; Impairment; Individual; Infertility; Left; Liquid substance; Lung; Mammals; Mechanical Ventilators; Mediating; Microtubules; Molecular; Molecular Motors; Motor; Mucociliary Clearance; Organelles; Otitis Media; Phenotype; Play; Positioning Attribute; Primary Ciliary Dyskinesias; Property; Protein Isoforms; Proteins; Radial; Randomized; Regulation; Respiratory System; Respiratory distress; Respiratory tract structure; Role; Signal Transduction; Slide; Sperm Motility; Structure; Surface; Testing; Tomogram; arm; biological systems; cell motility; ciliopathy; cilium motility; design; developmental disease; experimental study; genetic approach; insight; interest; macromolecular assembly; middle ear; mutant; pathogen; public health relevance; reconstitution; respiratory; sperm cell; stress disorder

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.

描述(申请人提供)：这个项目的总体目标是了解分子马达动力蛋白是如何被调节的，以产生具有运动纤毛和鞭毛特征的高拍频和复杂波形。在哺乳动物中，运动的纤毛/鞭毛是精子推进、清除呼吸道和中耳的碎片、脑脊液循环以及在发育过程中确定左右身体平面所必需的。其结果是，纤毛运动缺陷会导致生育能力受损、呼吸机窘迫、脑积水、中耳炎和/或左右身体轴的随机化。虽然在理解动力蛋白马达的力产生特性方面已经取得了重大进展，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悬而未决的问题之一。大量证据表明，中枢器和放射状辐条是控制微管滑动和纤毛搏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大量工作已经确定了这些结构的组成，然而，将这些组成整合到对纤毛运动的更广泛的机械理解中是缺乏的。在这项提案中，我们朝着弥合这一差距迈出了根本的一步。在提案的目标1和2中，我们将利用我们的发现，即重复轮辐对的两个径向轮辐(RS1和RS2)在组成上是不同的。在目标1中，我们对RS1和RS2微管结合接头的鉴定为我们提供了一种机会，以定义一种靶向和锚定特定轮辐相关蛋白的机制，这些蛋白可能建立96 nm的轴丝重复。在目标2中，我们测试了这样的假设，即该对中的每个辐条控制特定动力蛋白臂亚型的活动。我们的发现支持这一目标，即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部动力蛋白臂异构体接触。在目标3中，我们将重点放在径向辐条-中央机构的相互作用上。这一目的是建立在我们发现的与中央装置的C1微管有关的复合体上，这些复合体对野生型运动是必不可少的。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，并测试关于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说。目标3中的实验可能会揭示在纳米尺度上作用于生物系统的摩擦力的新原理，但这些摩擦力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的、高度保守的大分子组件之间的空间和时间控制的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。这些研究还将对更广泛的微管相关马达领域产生影响。

项目成果

Microtubule binding protein PACRG plays a role in regulating specific ciliary dyneins during microtubule sliding.

微管结合蛋白PACRG在微管滑动过程中调节特定睫状动力蛋白方面起作用。

• DOI: 10.1002/cm.21340
• 发表时间: 2016-12
• 期刊: Cytoskeleton (Hoboken, N.J.)
• 作者: Mizuno K;Dymek EE;Smith EF
• 通讯作者: Smith EF