The location of three different cytoskeletal structures has recently been demonstrated within the rod photoreceptor outer segment (ROS). The molecular composition of one of these, the """"""""basal"""""""" or """"""""axonemal"""""""" domain, is also known. The remaining two cytoskeletal specializations, disc rim-associated filaments and clusters of tubules at the ROS tip, are as yet unidentified. Recent work from several laboratories has suggested that some or all of these filamentous and tubular elements may play an important role in photoreceptor physiology, particularly in membrane turnover. The work proposed here focuses on disc membrane renewal and explores the possible involvement of cytoskeletal elements in this process.
Specific aims are to: 1) use specific antibodies to probe the binding of ROS enzymes to known cytoskeletal structures; b) define the association of these cytoskeletal networks with ROS disc membranes using rapid freeze/deep etch microscopy; c) continue the molecular identification of the heretofore unidentified cytoskeletal elements in the ROS using monoclonal antibodies. The nature and specificity of the antibody effects on cytoskeletal interactions, combined with information about the molecular properties of ROS cytoskeletal assemblies, will help to define which elements are involved and by what mechanism they participate in photoreceptor turnover. These experiments represent a new approach to the study of membrane assembly in the ROS and could provide a novel set of probes for molecules related to disc membrane renewal. Such molecules in an aberrnat form, may be related to disease processes which result in the shortening of photoreceptor outer segments as observed in many forms of hereditary retinal degenerations. In addition, the simplified """"""""extracted cell"""""""" assays developed here might eventually be used to screen agents with potential to offset such difficulties in disc membrane assembly.
