Calcium regulation of flagellar motility

描述(申请人提供)：这个项目的长期目标是确定分子马达动力蛋白被调节的机制，以产生具有击打真核生物纤毛和鞭毛特征的复杂波形。这项拟议的研究特别旨在了解钙的变化如何调节纤毛和鞭毛弯曲的大小和形状，以控制运动。一些研究表明，鞭毛中央装置是调节动力蛋白活性以调制波形的信号转导途径的关键组成部分。这一建议的目标建立在这样的假设基础上，即中央装置局部控制钙传感器来调节动力蛋白的活性，并且钙调蛋白和轴膜钙调蛋白依赖的激酶介导钙信号。拟议的实验旨在验证这一假说，并确定参与钙调蛋白介导的信号转导途径的轴丝成分。这个 具体目标是：1)识别与中枢装置相关的钙调蛋白结合蛋白；2)表征与轴丝相关的钙调蛋白依赖蛋白；以及3)确定附着在特定双线微管亚群上的特定动力蛋白亚型的活性是否优先受到钙离子变化的调节。单细胞绿藻，莱茵衣藻，是这些研究的首选生物体，因为它是唯一提供运动性突变的系统，几乎没有限制的生化方法材料，以及独特的体外功能分析。 衣藻中许多编码鞭毛蛋白的基因与人类基因组和EST数据库中的基因具有很高的序列相似性。因此，在衣藻中获得的信息将直接适用于高等真核生物，并可能为导致包括卡特根氏综合征在内的原发纤毛运动障碍的缺陷提供洞察。研究动力蛋白对鞭毛波形的调控也将影响我们对某些发育过程的理解。例如，发育中的胚胎中的节状纤毛与形态梯度的产生有关，而形态梯度是导致左右不对称的原因。这一结果解释了约50%的静止纤毛综合征患者也有内翻位的观察。有趣的是，在发育过程中利用的结节纤毛并不组装一个中央装置，并且与在同一生物体中发现的上皮细胞的纤毛不同的波形跳动。这一观察进一步说明了控制适合特定细胞类型的纤毛和鞭毛波形的重要性。

DESCRIPTION (provided by applicant): The long-range goal of this project is to determine the mechanism by which the molecular motor dynein is regulated to produce the complex waveforms characteristic of beating eukaryotic cilia and flagella. The proposed research is specifically aimed at understanding how changes in calcium modulate the size and shape of ciliary and flagellar bends to control motility. Several studies have indicated that the flagellar central apparatus is a key component of a signal transduction pathway that regulates dynein activity to modulate waveform. The objectives of this proposal are founded on the hypothesis that the central apparatus locally controls a calcium sensor to regulate dynein activity, and that calmodulin and an axonemal calmodulin dependent kinase mediate the calcium signal. The proposed experiments are designed to test this hypothesis and to identify axoneme components involved in the calmodulin-mediated signal transduction pathway. The Specific Aims are: 1) to identify calmodulin-binding proteins associated with the central apparatus; 2) to characterize calmodulin dependent kinases associated with the axoneme; and 3) to determine if the activities of particular dynein subforms attached to specific subsets of doublet microtubules are preferentially modulated by changes in calcium. The unicellular green alga, Chlamydomonas reinhardtii, is the organism of choice for these studies as it is the only system that offers motility mutants, virtually unlimited material for biochemical approaches, and unique in vitro functional assays. Many of the genes encoding flagellar proteins in Chlamydomonas show high sequence similarity with genes in the human genome and EST databases. Therefore, the information obtained in Chlamydomonas will be directly applicable to higher eukaryotes and may provide insight into defects that result in primary cilia dyskinesia including Kartegener's syndrome. Studies of dynein regulation and control of flagellar waveform will also impact upon our understanding of certain developmental processes. For example, the nodal cilia in developing embryos have been implicated in the production of a morphogen gradient responsible for generating left-right asymmetry. This result explains the observation that about 50% of patients with immotile-cilia syndrome also have situs inversus. Interestingly, nodal cilia utilized during development do not assemble a central apparatus and beat with a different waveform than cilia of epithelial cells found in the same organism. This observation further illustrates the importance of controlling ciliary and flagellar waveforms appropriate for particular cell types.