The broad objective is to understand how calcium, an ubiquitous second messenger in eukaryotic cells, regulates dynein-driven microtubule sliding responsible for ciliary and flagellar motion. The rationale is to investigate both the ciliary membrane """"""""software"""""""" mediating calcium influx, and the axonemal """"""""hardware"""""""" that responds to the calcium signal. We will take advantage of the unique experimental virtues of giant cilia of ctenophores - comb plates and macrocilia - which exhibit different and well-documented types of calcium-regulated motor responses.
The specific aims of the study are: (1) To map the sites of voltage-gated calcium entry across the ciliary membrane and possible calcium fluxes within the cilia during activation of beating (macrocilia) and reversal of beat direction (comb plates). We will use microiontophoresis techniques, fluorescent calcium indicators, aequorin, vibrating calcium-sensitive electrodes, and whole-cilium electrophysiological recording methods. (2) To determine the site(s) and mechanism(s) of action of calcium within the axoneme. We will use new calcium-sensitive ATP-reactivated demembranated ciliary models, calcium-sensitive microtubule sliding disintegration, specific inhibitors, SDS PAGE, immunoblots, phosphoprotein assays, and immunogold electron microscopic labelling. In addition, we will characterize uniformly-oriented arrays of dynein arms as substrates for in vitro motility assays of dynein subunit function and microtubule translocation. (3) To visualize the molecular morphology of the mechanochemical cycle of dynein, and the pattern of dynein arm activity within motile axonemes. We will use rapid-freezing of isolated reactivated axonemal """"""""crystals"""""""" (macrocilia) under known physiological and motile conditions, followed by deep-etch and freeze-substitution electron microscopy. These studies will advance understanding of second messenger control of cell motility in a system which provides unique experimental advantages not available elsewhere. The findings will be relevant to human health problems involving microtubule-based cell movements (cancer) and intracellular vesicular transport (neurological deficiencies), mucociliary clearance (respiratory diseases), gamete transport in oviducts and sperm motility (fertility), and related health concerns.
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