CA2+:SECOND MESSENGER FOR CONTROL OF FLAGELLAR MOVEMENT

这项研究计划涉及钙离子作为一种 控制鞭毛节拍的第二信使。建议数 实验将利用衣藻的光行为反应 RENHARTITI作为模式系统具有特定的目标：i)隔离 具有轴丝缺陷的突变体削弱了它们对 光刺激；ii)确定轴丝行为的哪一方面 通过检测细胞的生理学变化而改变 ATP重新激活的、去膜的突变细胞和分离的轴丝；以及 确定鞭毛轴丝中与钙离子有关的蛋白质 通过比较节拍对称性的蛋白质组成 从野生型和突变型细胞系中分离鞭毛 表现出有缺陷的光诱导行为的。缺陷突变细胞系 在他们的光行为反应中将被选择，然后通过 生理和行为测试，以确定感官的哪一步 任何突变都会改变转导途径。突变细胞 异常重新激活，表明它们在其 移动机械，将从原始收藏中挑选出来 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法进行检测，以及 随后，将电泳图转移到 硝酸纤维素膜用于研究磷酸化和钙结合特性 突变型和野生型轴丝的蛋白质。来自这些研究的数据将 与完整野生型和磷酸化的钙结合和磷酸化进行比较 突变型轴丝。磷酸化对肌动蛋白再激活的影响 将对轴丝进行研究，以确定是否涉及磷酸化 在钙离子依赖下，搏动对称性改变。这些研究应该确定 控制趋光行为的特定轴丝成分和 畏光反应，产生有关纤毛或纤毛的节拍模式的信息 鞭毛是从根本上控制的，并进一步加深了我们对 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.