Everyday, the light-sensitive cells in our retinas, our photoreceptors, shed and renew a portion of their light-sensitive membrane. Why this process occurs is not completely understood, however; it is ubiquitous to all animals studied to date. It is essential for proper visual function. In fact, retinal degeneration occurs in mutant rats unable to shed their photosensitive membrane. In most animals, shedding of the photosensitive membrane is regulated by a biological clock (circadian rhythm) and triggered by that clock and/or by light. Relatively little is known, however, about how the biological clock and light interact to trigger and regulate daily shedding and renewal of photosensitive membrane. The lateral eye of the horseshoe crab (Limulus) has long served as an important system for the study of light-sensitive membrane shedding. Limulus provides the relatively unique advantage that, to date, there is no evidence of a biological clock in the eye; instead, circadian rhythms are communicated to the eye from a central clock in the brain along nerve fibers that run from the brain to the eye in the optic nerve. As a result, the effects of light and circadian rhythms on photoreceptor structure and function can be decoupled easily in this system by severing the optic nerve. In the currently funded proposal (EY13196-01), a model for the interaction of light and the biological clock in the regulation of daily transient photosensitive membrane shedding was developed and tested pharmacologically. This competing continuation application proposes to continue to evaluate the model through the following two specific aims. 1. Investigate whether protein synthesis is stimulated by long-term cAMP-dependent protein kinase activation during efferent priming of Limulus photoreceptors for transient rhabdom shedding. Protein synthesis inhibition, immunohistochemistry/immunoblotting (for CREB activity), and two-dimensional gel electrophoresis will be used to determine whether circadian priming of the lateral eye for transient shedding requires the synthesis of a new protein(s). If protein synthesis is required for the brain to prime the eye for transient shedding, the novel protein (or peptides from that protein) will be microsequenced (outsourced) for putative identification. 2. Identify the target substrate(s) for protein kinase C in the signaling cascade that triggers transient rhabdom shedding in response to light following circadian efferent priming. Immunohistochemical assays for serine and threonine phosphorylation will be used in conjunction with pharmacological activators and inhibitors of protein kinase C (PKC) and cAMP-dependent protein kinase to isolate the PKC substrate(s) that initiate transient shedding.
