Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors

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Project 001 (273): Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors, Robichaux, PL PROJECT SUMMARY/ABSTRACT In rod photoreceptor neurons of the vertebrate retina, rhodopsin (Rho) is the light-sensing, G protein- coupled receptor that is densely packaged into stacks of membrane discs within a specialized sensory cilium known as the outer segment. Rods continuously renew these discs, and so an enormous amount of Rho protein must be constantly synthesized and trafficked to the outer segment in each rod. To maintain this heavy biosynthesis of Rho, rods feature a highly regulated yet ill-characterized secretory and trafficking network. Rho trafficking in rod neurons is such an essential task that, when disrupted, it causes blinding retinal diseases such as retinitis pigmentosa. Recent evidence in mammalian rod photoreceptors suggest alternate, unconventional pathways for Rho trafficking; however, these pathways remain undiscovered. Another understudied component of Rho trafficking in rods is the essential structural waypoint known as the basal body, which is located at the critical boundary between the biosynthetic zone of rods and the thin connecting cilium bridge that leads to the outer segment. The basal body is composed of two centrioles that have associated sub-structures, including the centriolar satellites and distal appendages. These are each potential sites of Rho interaction and docking before transport into the outer segment; however, these structures have also not been rigorously studied in mammalian rods to date. Our preliminary data using advanced, super-resolution microscopy methods indicate that Rho molecules are densely accumulated in the basal body, and that endoplasmic reticulum (ER) membranes may also be located at the basal body. In the first aim of this proposal, we will first test if nascent Rho protein is being synthesized in local ER membranes within the basal body region of mammalian rods as part of a potential alternate Rho trafficking pathway. We will generate a novel knock-in ER fusion mouse to perform a comprehensive, super-resolution mapping of the entire local ER network in rods. We will quantitatively map onto this ER network the precise localization of Rho proteins, ribosomes (as rough ER), and Rho mRNA as sites for local Rho translation. Collectively, these experiments will define new Rho trafficking events and establish a new local ER network within the rod basal body. In our second aim, we will use complimentary approaches to test if Rho proteins dock onto the basal body at the centriolar satellites and distal appendages via specific protein- protein interactions with the component proteins of those two basal body sub-structures. We will perform a rigorous nanoscale mapping of these structures in mouse rods, followed by a quantitative test of Rho co- localization with the component proteins of the satellites and the distal appendages. We will corroborate these new interactions with biochemical co-precipitation assays. Finally, we will perform a novel, targeted inhibition of the satellites and distal appendages, by subretinal delivery of viral vectors carrying machinery to genetically disrupt these structures in mouse rods. With these experiments, we will establish the role of these basal body structures in Rho trafficking and rod photoreceptor homeostasis.