CA2+:SECOND MESSENGER FOR CONTROL OF FLAGELLAR MOVEMENT

这项研究计划涉及钙离子作为一种 控制鞭毛搏动的第二信使。 拟议 实验将使用衣原体的光行为反应， reinhardtii作为一个模型系统的具体目标：i）分离 具有轴丝缺陷的突变体，该缺陷损害了它们对 光刺激; ii）确定轴丝行为的哪个方面具有 通过研究人类的生理机能， ATP再活化的脱膜突变细胞和分离的轴丝;和iii） 为了鉴定鞭毛轴丝中与Ca 2+有关的蛋白质， 在拍对称性的依赖性变化，通过比较蛋白质的组成， 来自野生型的分离鞭毛与来自突变细胞系的分离鞭毛 表现出有缺陷的光诱导行为。 缺陷型突变细胞系 在他们的光行为反应将被选择，然后筛选， 生理和行为测试，以确定哪一步在感官 任何突变都会改变转导途径。 的突变细胞 重新激活异常，表明他们有内在的缺陷， 运动机械，将从原来的收集选择， 光行为突变体 从这些突变体中纯化的鞭毛轴丝 将通过一维和二维凝胶电泳进行检查， 随后，电泳图案将被转移到 硝酸纤维素片研究磷酸化和Ca 2+结合特性 突变体和野生型轴丝的蛋白质。 这些研究的数据将 与完整的野生型的Ca 2+结合和磷酸化相比， 突变轴丝。 磷酸化对细胞再活化的影响 将研究轴丝以确定是否涉及磷酸化 Ca 2+依赖的拍对称性变化。 这些研究应确定 控制趋光行为的特定轴丝成分， 恐惧反应，产生关于纤毛或 鞭毛是从根本上控制，并进一步我们的理解， 其他涉及钙离子作为第二信使的过程。

This research plan is concerned with the function of calcium ions as a second messenger in the control of flagellar beat. The proposed experiments will use the photobehavioral responses of Chlamydomonas reinhardtii as a model system with the specific goals: i) to isolate mutants having axonemal defects that impair their ability to respond to photostimulation; ii) to determine which aspect of axonemal behavior has been altered through mutation by examining the physiology of ATP-reactivated, demembranated mutant cells and isolated axonemes; and iii) to identify proteins in the flagellar axoneme that are involved in Ca2+ dependent changes in beat symmetry by comparing the protein composition of isolated flagella from wild-type with those from mutant cell lines exhibiting defective light-induced behaviors. Mutant cell lines defective in their photobehavioral responses will be selected and then screened by physiological and behavioral test to determine which step in the sensory transduction pathway has been altered by any mutation. Mutant cells that reactivate abnormally, indicating that they have inherent defects in their motile machinery, will be selected from the original collection of photobehavioral mutants. Flagellar axonemes purified from these mutants will be examined by one- and two-dimensional gel electrophoresis, and subsequently the electrophoretic patterns will be transferred to nitrocellulose sheets to study phosphorylation and Ca2+ binding properties of proteins of muant and wild-type axonemes. Data from these studies will be compared to Ca2+ binding and phosphorylation of intact wild-type and mutant axonemes. The effects of phosphorylation on reactivation of axonemes will be investigated to determine if phosphorylation is involved in Ca2+ dependent changes in beat symmetry. These studies should identify specific axonemal components that govern phototactic behavior and the photophobic response, yield information on how beat patterns of cilia or flagella are fundamentally controlled, and further our understanding of other processes that involve calcium ions as a second messenger.