MEMBRANE BIOSYNTHESIS IN NORMAL AND DYSTROPHIC RETINA

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Specific Aims and Long Term Objectives: The biosynthesis of retinal cell membranes is being explored in frogs by subcellular fractionation and by evaluation of gene transcription and translation in mouse and rat retinas from animals bearing various forms of inherited retinal dystrophy. These studies are directed to understanding the cellular basis of sorting of membrane proteins from sites of synthesis to sites of function and the genetic controls that regulate these processes. They should contribute to understanding of retinal causes of blindness and to the organization of cells in tissues in normal and pathologic states. Experimental Design and Methods: Frog retinas will be incubated with [35S]-methionine and after homogenization, sucrose density gradient subcellular fractions bearing newly synthesized opsin and other outer segment proteins will be isolated. Preliminary studies have indicated the presence of a highly labeled low-density fraction with kinetics of labeling suggesting it contains the post-Golgi vesicles. This fraction will be used to determine the structure of the vesicles transporting opsin from the Golgi to the outer segment. Rabbit and mouse monoclonal antibodies will be generated to these partially purified fractions to characterize their composition. The vesicles will then be purified further by density-shift sucrose gradient fractionation by using gold- antibody conjugates bound to the membranes of the fraction. These studies are designed to determine if the vesicles carry unique molecules bearing an "address" to specifically sort newly synthesized opsin to the appropriate site in the photoreceptor in other cells tranfected with the opsin gene. Using renal tubular epithelial cell cultures transfected with the opsin gene, we are exploring polarity of expression. These cells provide a model system for studying photoreceptor cell polarity. Extensive EM studies of rats and mice bearing genes for inherited retinal dystrophies have demonstrated a uniform feature: the loss of the polarized distribution of opsin on the rod plasma membrane as the outer segment becomes damaged or fails to form. We are, therefore, examining the molecular control of gene expression of opsin and other proteins in these dystrophic rodents to evaluate alternative models to account for the altered localization of these molecules and the residual visual sensitivity in these retinas that lack outer the altered localization of these molecules and the residual visual sensitivity in these retinas that lack outer segments. Using cDNA radiolabelled probes, containing the opsin gene, we are evaluating the effects of retinal degeneration in C3H (rd) and O20/A (rds) mice and RCS rats on levels of gene transcription by quantitating mRNA expression throughout the light cycle and as the animals age. We have demonstrated at least five mRNA transcripts of the opsin gene exist in normal and dystrophic mice and four transcripts in rats in contrast to the single transcript in bovine and human retinas. These transcripts are differentially expressed as the animals age. We also have determined that expression of opsin genes in O20/A mice bearing the rds dystrophy is nearly normal and that translation rates of the mRNA are also nearly normal despite the presence of only 3% of the retinal opsin content when compared to age-matched control mice. Since outer segment structure is distorted in this dystrophy such that disks are not assembled, our data indicate that the gene defect interferes with disk morphogenesis rather than opsin synthesis.