REGULATION OF DYNEIN-DRIVEN FLAGELLAR MOTILITY

本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， 基于新的数据，是关于径向辐条结构和钙控制的动力蛋白驱动的运动。广大 这项工作的重要性最好地由先天性综合征，原发性纤毛运动障碍来说明。注意症状 包括内脏逆位、不孕症、严重慢性呼吸道感染和脑积水。阐明控制 这一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性细胞分化至关重要。 能动性重要的问题包括动力蛋白运动活动是如何协调的，钙和循环是如何相互作用的。 核苷酸调节动力蛋白驱动的运动性。独立线的证据表明，径向发言发挥了至关重要的 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原体突变体，径向 辐条作为机械化学传感器，通过激酶、磷酸酶和钙离子网络控制动力蛋白 传感器.关键分子中有两种组成性轮辐蛋白：RSP 2和钙调蛋白，它们对于 能动性钙调素是位于轮辐上的典型钙感受器，参与钙诱导的运动 变化，但机制尚不清楚。RSP 2是最近克隆的一种磷蛋白，含有两个钙调素结合蛋白， 基序并以钙依赖性方式结合钙调蛋白。最有趣的是，RSP 2和孤立的辐条显示激酶 活动最简单的假设是RSP 2/ealmodulin复合物通过改变钙离子浓度来介导钙离子对运动的控制。 径向辐条的物理和酶性质。三个目标旨在检验这一假设。[1]评估 RSP 2中钙调蛋白结合和磷酸转移结构域缺陷的重组RSP 2突变体构建体 突变体（pf 24）。预期突变体构建体挽救轮辐装配，但未能挽救细胞的钙控制。 能动性[2]测量钙对分离的放射状辐条的激酶活性和RSP 2的磷酸化的影响。 可以预见的是，轮辐激酶活性是钙敏感的。[3]利用新电子定义辐射状轮辐结构 微观的方法，并定义的位置和分子间的相互作用的钙调素在辐条。预见 钙结合将改变轮辐结构。这些实验直接验证了假设，并解决了 控制纤毛和鞭毛运动的基本机制。这些结果也将对如何实现这一目标产生广泛影响。 动力蛋白驱动的运动性是受控的，以及激酶和钙传感器如何锚定在微管细胞骨架中。

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.