MEMBRANE FUSION IN RETINAL ROD OUTER SEGMENTS

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DESCRIPTION (Adapted from applicant's abstract): The ability to maintain a constant length is the essence of the rod cell's ability to transduce light. These post-mitotic cells contain a stack of closed flattened membranous discs organized in a discontinuous lateral array within the rod outer segment (ROS). Whereas the program of outer segment renewal and disposal is defined as the coordinated process of disc morphogenesis (at base) and disc shedding (at tip), how the cell assembles and renews its architectural features remains obscure on a molecular level. The investigator proposes that a step during both of these processes, new disc closure and disc membrane packet formation prior to shedding, require the fusion of opposing membranes. The biochemical components mediating fusion processes in the ROS are largely unknown and their elucidation is the long-term focus of this research. To date, the best-characterized protein mediator of fusion within the ROS is peripherin/rds. To investigate the molecular mechanisms of these fusion processes with specific emphasis on the role of peripherin/rds, the investigator has developed a series of cell-free fusion assay systems, designed to increase the complexity of the target membrane during fusion. With the use of biophysical, molecular biology and cell biology techniques, the investigator has a complement of techniques that allows her to characterize the overall fusion process and to delineate the individual steps in the process and identify functional regions of the peripherin/rds and the structural requirements necessary for fusion competency of this protein. It is proposed to: (1) determine the role of peripherin/rds in ROS membrane fusion: (2) determine the role of phospho-peripherin/rds in ROS membrane fusion; (3) ascertain whether the protein components of the disc rim contribute to the fusion process; (4) address how the lipid and protein mediated fusion pathways converge to regulate the fusion proc3ess; and (5) determine what fusion machinery may be involved in disc morphogenesis. Using cell-free fusion assays, the investigator will investigate the overall fusion reaction involved in disc packet formation and disc morphogenesis. Using a COS cell expression system, the investigator will look at the effect of various mutations within peripherin/rds and rom-1 on the fusion process. To identify the functional regions of the proteins involved in the process, the investigator will investigate the effects of various peptides on the individual steps in the fusion reaction. Using FTIR and NMR, the investigator will determine the structural requirement of the peripherin/rds and membrane bilayer required for fusion to occur. An understanding of the molecular mechanism by which rod cells maintain a constant length is important to unraveling the mechanism of cell viability, degenerative disease processes and adaptation.