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A. F. Goldberg, L. M. Ritter, B. M. Tam, O. L. Moritz, N. Khattree, L. Dang; Identification and Localization of Outer Segment Scaffolding Interactions Using Bimolecular Fluorescence Complementation (BiFC) in Transgenic Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1112.
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Rod and cone photoreceptors are terminally differentiated post-mitotic neurons required for sight. Their membranous outer segment (OS) organelles respond to light via a G-protein mediated signaling cascade. OS architecture is elaborate, dynamic, and crucial for maintaining photoreceptor function and viability; however, technical limitations have hindered investigations of the molecular scaffolding that underlies this important structure. We have developed a novel approach to elucidate protein interactions that underlie OS scaffolding.
We are using BiFC to localize and analyze protein-protein interactions. Coding regions of several photoreceptor proteins, including rhodopsin, GARP2, and peripherin/rds have been genetically fused to fragments of green fluorescent protein (GFP), and the fusion proteins have been expressed in cultured cells and transgenic X. laevis photoreceptors. Western blot, immunocytochemical, FACS, confocal and electron microscopic analyses are being used to assess protein expression, localization, and interactions.
We find that both soluble and integral membrane photoreceptor proteins can be properly expressed as fusions with GFP fragments in cultured cells. Addition of GFP sequences to several photoreceptor proteins did not significantly alter their localization in transgenic X. laevis photoreceptors. Using several previously documented homotypic and heterotypic interactions as models, we observe that appropriate combinations of rhodopsin-, GARP2-, and peripherin/rds- GFP fusion proteins generate BiFC complexes. Subcellular localization of the BiFC complexes to discrete structures in transgenic X. laevis photoreceptors suggests mechanisms for their formation and function.
We have developed a novel approach to describe protein-protein interactions required for the distinctive structures of rod and cone OSs. Our results demonstrate the utility of using BiFC to define the role of these interactions for photoreceptor structure and retinal disease.
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