REGULATION OF DYNEIN-DRIVEN FLAGELLAR MOTILITY

动力蛋白驱动的鞭毛运动的调节

• 批准号: 6756490
• 负责人: PINFEN YANG
• 金额: $ 25.38万
• 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/6756490
• 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（由申请人提供）：该提案的长期目标是阐明运动纤毛和鞭毛的控制机制，基于新数据的重点是动力蛋白驱动运动的径向辐条结构和钙控制。先天性综合征——原发性纤毛运动障碍最能说明这项工作的广泛意义。值得注意的症状包括反位、不孕、严重的呼吸道慢性感染和脑积水。阐明控制机制对于理解这些细胞器在不同细胞类型中的作用和避免运动缺陷至关重要。重要的问题包括动力蛋白运动活动如何协调以及钙和环状核苷酸如何调节动力蛋白驱动的运动。独立的证据表明，径向辐条在动力蛋白马达的控制中发挥着至关重要的作用，并且基于结构分析和信息丰富的衣藻突变体，径向辐条作为机械化学传感器，通过激酶、磷酸酶和钙传感器网络控制动力蛋白。关键分子中有两种组成性辐条蛋白：RSP2 和钙调蛋白，它们对于运动至关重要。钙调蛋白是位于辐条中的典型钙传感器，参与钙诱导的运动变化，但其机制尚不清楚。 RSP2 是一种最近克隆的磷蛋白，含有两个钙调蛋白结合基序，并以钙依赖性方式结合钙调蛋白。最有趣的是，RSP2 和分离的辐条显示出激酶活性。最简单的假设是 RSP2/钙调蛋白复合物通过改变径向辐条的物理和酶性质来介导钙对运动的控制。设计了三个目标来检验这一假设。 [1] 评估重组 RSP2 的突变体构建体，RSP2 突变体 (pf24) 中钙调蛋白结合和磷酸转移结构域有缺陷。突变体结构有望挽救辐条组装，但无法挽救钙对运动的控制。 [2] 测量钙对孤立径向辐条激酶活性和 RSP2 磷酸化的影响。可以预见的是，辐条激酶活性对钙敏感。 [3] 使用新的电子显微镜方法定义径向辐条结构，并定义辐条中钙调蛋白的位置和分子相互作用。可以预见的是，钙结合将改变辐条结构。这些实验直接检验了这一假设并解决了控制纤毛和鞭毛运动的基本机制。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中产生广泛的影响。