This proposal deals with the regulation of the beating motion of cilia in the protozoan Paramecium tetraurelia. The structure of cilia and flagella has been conserved in evolution; these organelles are very similar, whether found in protozoans, ciliated epithelia, or mammalian sperm. Ca ion is known to regulate the motion of cilia and flagella, but recent evidence points to a role for cyclic AMP (cAMP) as well. The proposed experiments are designed to determine the mechanism by which Ca and cAMP act together to regulate the frequency and direction of the ciliary beat. P. tetraurelia is used as test organism; with it, genetics, electrophysiology, microinjection, and large scale culturing for biochemical studies are convenient. Five general approaches are proposed: 1) Proteins responsible for cAMP synthesis (adenylate cyclase), for cAMP-dependent and Ca-dependent phosphorylation of proteins (protein kinases), for ATP-dependent axonemal motion (dyneins) and for Ca-dependent modulation of dyneins and cyclase (probably calmodulin) will be purified, characterized, and used to elicit monospecific antibodies. 2) The swimming speed and direction of permeable """"""""models"""""""", activated by ATP addition, will be quantitated, and the effects of cAMP, Ca, inhibitors of protein kinases, and antagonists of calmodulin will be determined. Protein factors which restore cAMP sensitivity to insensitive models will be isolated and characterized. 3) Patterns of ciliary protein phosphorylation in permeable models will be studied after phosphorylation with labeled ATP in the presence of cAMP, Ca, or both, and correlations between specific phosphorylations and changes in ciliary motion will be sought. 4) permeant photoactivable cAMP derivatives will be used to induce sudden changes in intracellular cAMP, and the resulting behavioral changes will be quantitated. 5) Mutants defectie in ciliary regulation will be isolated and compared with wild-type cells with respect to levels of cyclic nucleotide-dependent enzymes, endogenous substrates for protein phosphorylation, and behavior of permeable models in response to cAMP and Ca. Wild-type extracts will be examined for their ability to restore normal function and cAMP sensitivity to mutants, by direct addition to permeable models or by microinjection into living cells. The long-range goal of this research is to define in molecular terms the steps and mechanisms that lie along the path from perception of a sensory stimulus to the ciliary response to that stimulus.
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