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.
