The long term goal of this research is to understand the structure and function of the phototransductive compartment of photoreceptor cells. The proposed plan is to study the actin cytoskeleton and the calpain system in this compartment. Both appear to play critical roles in photoreceptor structure and function. Biochemical and cell biological procedures will be used. I. Actin-binding proteins in the phototransductive compartment. The goal of this section is to address the regulation and function of two actin-binding proteins: one from mammalian rod outer segments, the other from Drosophila rhabdomeres. In photoreceptor outer segments, the actin~cytoskeleton plays an essential role in disk membrane renewal. It also appears to provide a binding site for guanylate cyclase, and thus may help regulate the recovery of the cell to its dark-adapted state. Binding-studies will be used to determine the nature of the binding between f-actin and guanylate cyclase, and its effect on guanylate cyclase activity. The subcellular localization of guanylate cyclase will be determined with respect to its binding to f-actin under different lighting conditions. An actin cytoskeleton is also central to the structure of rhabdomeres. The ninaC proteins have a myosin-like domain and appear to associate with the actin cytoskeleton. Biochemical approaches are proposed to determine whether the proteins are indeed functional myosins. In addition, the photoreceptor ultrastructure of flies, with deletion and site-specific mutations in the ninaC gene, will be examined to help elucidate ninaC function. II. Calpain in rod outer segments.
The aim of this section is to understand the regulation and function of calpain in rod outer segments. This calcium-activated neutral protease has been implicated in the organization of the rod outer segment actin cytoskeleton and in the regulation of rhodopsin. The calpain system in rod outer segments will be characterized better by identifying proteins that inhibit or activate the enzyme, proteins that bind (and thus help target) the enzyme to the cytoskeleton, and proteins that are in situ substrates. In particular, experiments will test whether any of the actin cytoskeletal elements are proteolysed by calpain in situ, and whether inhibition of calpain activity affects disk membrane morphogenesis. The selective proteolysis of arrestin by calpain will be studied further by identifying the sites of cleavage, the subcellular distribution of truncated arrestin, and the effects of this truncation on rhodopsin function.
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