Recent work from several laboratories has suggested that cytoskeletal components may be involved in several aspects of rod photoreceptor physiology. These include rod photoreceptor disc shedding and/or renewal,maintneance of rod outer segment (ROS) shape, as well as rapid light-induced disc spacing changes. At the present time, however, cytoskeletal elements within the light sensing organelle of the rod photoreceptor are virtually unstudied. Preliminary work by this applicant has demonstrated the existence and gross localization of actin, microtubules, fodrin, and calmodulin within the rod oter segment. In this application, projects are proposed: a) to continue the characterization and localization of conventional cytoskeletal elements in ROS: b) to undertake the biochemical characterization of ROS-unique filamentous elements previously observed only by electron microscopy, and c) to develop approaches for assessing the function of both classes of cytoskeletal components in rod photoreceptor physiology. The methods to be used in these studies are centered on the production of polyclonal and monoclonal specific antibodies to partially purified ROS-unique cytoskeletal elements. Appropriate antibodies will be selected on the basis of their staining pattern in isolated ROS. Because the ROS-unique filaments have such will-defined locations, e.g. at the ROS tips, or at the disc rims, each specific antibody can be easily identified by indirect immunofluorescent assay and its localization confirmed by electron microscopy. Once the antivodies to these ROS filaments are available, they may be used to further biochemically characterize the filaments and to determine the relationship of a particular element to other cytoskeletal elements (actin, microtubules, etc.). To investigate functions, simple light microscopic assays are to be developed to check for drug-and antibody-disruption of the filaments. The effect of filament-disrupting antibodies and drugs can be assessed by application of these agents to the living retina. These studies may be of relevance to the class of human retinal diseases which exhibit extensive photoreceptor involvement, including retinal degenerations such as retinitis pigmentosa or categories of night blindness in which there are sensitivity deficits. The often observed aberrations in normal photoreceptor shedding and renewal cycles could certainly implicate cytoskeletal elements in these disease processes.
