REGULATION OF DYNEIN-DRIVEN FLAGELLAR MOTILITY

动力蛋白驱动的鞭毛运动的调节

• 批准号: 7366854
• 负责人: PINFEN YANG
• 金额: $ 1.22万
• 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/7366854
• 关键词: A kinase anchoring protein; Accounting; Address; Binding; Binding Sites; Biochemical; Biological; Calcium; Calcium Binding; Calmodulin; Cell division; Cell physiology; Chemical Models; Chemicals; Chlamydomonas; Chronic; Cilia; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Decompression Sickness; Dynein ATPase; Dyskinetic syndrome; Electron Microscopy; Electrons; Embryonic Development; Enzymes; FGF9 gene; Flagella; Goals; Hydrocephalus; Infertility; Intracellular Transport; Location; Mass Spectrum Analysis; Measures; Mediating; Microscopic; Microtubules; Modeling; Molecular; Motor; Motor Activity; Mutate; Negative Staining; Organelles; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Principal Investigator; Procedures; Property; Proteins; Publishing; Radial; Reagent; Recombinants; Regulation; Respiratory Tract Infections; Role; Sales; Signal Transduction; Situs Inversus; Slide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Symptoms; Syndrome; System; Testing; Transducers; Work; Yang; base; cell motility; cell type; design; gametocyte activating factor; hexokinase; in vivo; intracellular protein transport; mutant; particle; programs; protein localization location; research study; response; sensor

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 meehano-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/ealmodulin 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] Defme 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.

该提案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，研究的是径向辐条结构和动力蛋白驱动运动的钙控制。广义的 先天性综合征——原发性纤毛运动障碍最能说明这项工作的重要性。注意到的症状 包括内脏反位、不孕症、严重慢性呼吸道感染和脑积水。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缺陷至关重要 动力。重要的问题包括动力蛋白运动活动如何协调以及钙和循环如何 核苷酸调节动力蛋白驱动的运动。独立的证据表明径向辐条起着至关重要的作用 在控制动力蛋白马达中的作用，并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、磷酸酶和钙网络控制动力蛋白 传感器。关键分子中有两种组成性轮辐蛋白：RSP2 和钙调蛋白，它们对于 动力。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蛋白 基序并以钙依赖性方式结合钙调蛋白。最有趣的是，RSP2 和分离的辐条显示激酶 活动。最简单的假设是 RSP2/钙调蛋白复合物通过改变钙离子对运动的控制 径向辐条的物理和酶学特性。设计了三个目标来检验这一假设。 [1] 评估 重组 RSP2 的突变构建体，RSP2 中的钙调蛋白结合和磷酸转移结构域有缺陷 突变体（pf24）。突变体结构有望挽救辐条组装，但无法挽救钙控制 动力。 [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] 使用新型电子定义径向辐条结构 微观方法，并确定辐条中钙调蛋白的位置和分子相互作用。可以预见的是 钙结合会改变辐条结构。这些实验直接检验假设并解决 控制纤毛和鞭毛运动的基本机制。结果还将对如何产生广泛影响 控制动力蛋白驱动的运动以及激酶和钙传感器如何锚定在微管细胞骨架中。