The role of calcium ions as a second messenger in the control of flagellar beat will be examined. The major objective of the proposed experiments is to identify, using biochemical procedures, calcium- binding proteins in the flagellar axoneme that may be involved in controlling the shape of flagellar bends. Biochemical fractionation of axonemes isolated from Chlamydomonas reinhardtii will be carried out to determine the sub-axonemal distribution of calmodulin (CaM), which has been implicated in the control of flagellar beat. The distribution of CaM-binding proteins in the axoneme will be monitored by gel overlay procedures using 125-I-CaM or biotinylated CaM and avidin-peroxidase. Outer doublet microtubules will be prepared from axonemes to determine whether these structures contain CaM, CaM-binding proteins, or other calcium binding proteins of interest. Finally, polyclonal antisera directed against a specific CaM-binding protein will be prepared and used in immunolabelling studies to examine the distribution of this "candidate" protein wthin the axoneme and the outer doublet microtubules. These studies should clarify the mechanisms by which calcium alters flagellar waveform by identifying key calcium-binding proteins that may control flagellar shape, as well as generally broaden our understanding of processes that involve calcium as a second messenger.
