The primary objective of this research is to understand how motile processes in photoreceptors and RPE cells are regulated by light, circadian, and morphogenetic signals, ie. to identify all steps in the regulatory pathways which lead from the physiological signals ultimately to the appropriate effector systems (ie. microtubule- or actin-based cytoskeletons) responsible for producing movement. This work emphasizes use of teleost retinomotor movements as a model system since powerful quantitative assays are available which permit analysis of the effects of extra- and intra-cellular regulators on these movements. Information gained from these retinomotor studies will be used to further our understanding of other photoreceptor and RPE motile processes crucial to retinal function in all species, such as phagocytosis and morphogenesis. Work in the last grant period showed that the extracellular messenger dopamine mimics light onset by inducing light-adaptive retinomotor movements in photoreceptors and RPE, and that intracellular pathways involving specific interplays of Ca++, cAMP, and protein kinase C contribute to regulating the actin- and microtubule-dependent processes which produce retinomotor movements. The present application proposes the following specific approaches to further clarify the roles of these agents in regulation of photoreceptor and RPE motility: 1) To ascertain whether dopamine and light have similar effects on photoreceptor and RPE cyclic nucleotide levels by determining the effects of light, darkness, and dopamine on cAMP and cGMP levels in isolated photoreceptor fragments (rod inner-outer segments, RIS-ROS, and cone inner-outer segments, CIS-COS) and in isolated RPE cells; 2) To investigate the roles of Ca++, cAMP, and specific protein kinases in regulating retinomotor movements in rods, cones, and RPE cells by further characterization of phorbolester effects and by extending lysed cell motile model studies to rods and RPE; 3) To investigate changes in protein phosphorylation in motile RIS-ROS, CIS-COS, and RPE cells that occur in response to light, dark, dopamine, Ca++, cAMP, and protein kinase C; 4) To characterize the cytoskeletal components of RIS-ROS, CIS-COS, and RPE cells using SDS PAGE, Western Blots, and immunolocalization; 5) To use immunocytochemistry to identify changes in distribution of cytoskeletal components during morphogenesis of photoreceptors and RPE.
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