REGULATION OF DYNEIN-DRIVEN FLAGELLAR MOTILITY

动力蛋白驱动的鞭毛运动的调节

• 批准号: 7072316
• 负责人: PINFEN YANG
• 金额: $ 24.26万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-10 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072316
• 关键词: Chlamydomonas; SDS polyacrylamide gel electrophoresis; biological signal transduction; biosensor device; calcium binding protein; calmodulin; cilium /flagellum motility; cytoskeleton; dynein ATPase; electron microscopy; enzyme activity; high performance liquid chromatography; intermolecular interaction; light microscopy; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; nucleotides; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to elucidate the control mechanism of motile cilia and flagella, and the focus, founded on new data, is on the radial spoke structure and calcium control of the dynein-driven motility. The broad significance of this work is best illustrated by the congenital syndrome, primary cilia dyskinesia. Noted symptoms include situs inversus, infertility, severe chronic infection of respiratory tract and hydrocephaly. Elucidating the control mechanism is essential for understanding the roles of these organelles in diverse cell types and for averting defective motility. The important questions include how the dynein motor activity is coordinated and how calcium and cyclic nueleotides modulate the dynein-driven motility. Independent lines of evidence indicate that radial spoke play a vital role in control of dynein motors and based on structural analysis and informative Chlamydomonas mutants, the radial spokes operate as mechano-chemical transducers to control dynein via a network of kinases, phosphatases and calcium sensors. Among the key molecules are two constitutive spoke proteins, RSP2 and calmodulin, that are essential for motility. Calmodulin, the prototypical calcium sensor located in spoke, is involved in calcium-induced motility changes but the mechanism is not known. RSP2, a recently cloned phosphoprotein, contains two calmodulin-binding motifs and binds calmodulin in a calcium-dependent manner. Most intriguing, RSP2 and isolated spokes display kinase activity. The simplest hypothesis is that RSP2/calmodulin complex mediates calcium control of motility by changing the physical and enzymatic properties of the radial spokes. Three aims are designed to test this hypothesis. [1] Assess mutant constructs of recombinant RSP2, defective in calmodulin-binding and phosphotransfering domain in a RSP2 mutant (pf24). The mutant constructs are expected to rescue spoke assembly but fail to rescue calcium control of motility. [2] Measure the effect of calcium on kinase activity of isolated radial spokes and phosphorylation of RSP2. Predictably, spoke kinase activity is calcium sensitive. [3] Define radial spoke structure using new electron microscopic approaches, and define the location and molecular interactions of calmodulin in the spoke. Predictably calcium binding will change spoke structure. These experiments directly test the hypothesis and address the fundamental mechanism of control of ciliary and flagellar motility. The results will also have broad impact on how dynein-driven motility is controlled and how kinases and calcium sensors are anchored in the microtubule cytoskeleton.

描述(申请人提供)：这项建议的长期目标是阐明运动纤毛和鞭毛的控制机制，重点是基于新数据的径向辐条结构和动力蛋白驱动的运动的钙控制。这项工作的广泛意义最好地说明了先天性综合征，即原发性纤毛运动障碍。明显的症状包括内翻、不孕不育、严重的慢性呼吸道感染和脑积水。阐明调控机制对于了解这些细胞器在不同细胞类型中的作用和避免运动障碍至关重要。重要的问题包括动力蛋白运动活动是如何协调的，钙和环核苷酸是如何调节动力蛋白驱动的运动的。独立的证据表明，径向辐条在动力蛋白发动机的控制中起着至关重要的作用，基于结构分析和信息丰富的衣藻突变体，径向辐条作为机械力化学换能器通过一系列激酶、磷酸酶和钙传感器网络来控制动力蛋白。在关键分子中，有两个构成轮辐的蛋白质，RSP2和钙调蛋白，它们对运动性是必不可少的。钙调素是位于辐条的典型钙感受器，参与钙诱导的运动性改变，但其机制尚不清楚。RSP2是新近克隆的一种磷酸化蛋白，它含有两个钙调素结合基序，并以钙依赖的方式与钙调素结合。最耐人寻味的是，RSP2和孤立的辐条显示出激酶活性。最简单的假设是，RSP2/钙调蛋白复合体通过改变径向辐条的物理和酶性质来调节钙离子对运动的控制。我们设计了三个目标来检验这一假设。[1]评估在RSP2突变体(Pf24)中存在钙调蛋白结合和磷转移结构域缺陷的重组RSP2的突变结构。突变的结构有望挽救轮辐组装，但未能挽救运动的钙控制。[2]测定钙对离体径向辐条的蛋白激酶活性和RSP2磷酸化的影响。可以预见，辐条激酶的活性是钙敏感的。[3]使用新的电子显微镜方法确定径向辐条结构，并确定钙调蛋白在辐条中的位置和分子相互作用。可以预见，钙结合会改变轮辐结构。这些实验直接验证了这一假说，并解决了纤毛和鞭毛运动控制的基本机制。研究结果还将对如何控制动力蛋白驱动的运动以及如何将激酶和钙传感器锚定在微管细胞骨架上产生广泛影响。