title: In situ visualization of protein interactions in sensory neurons: glutamic acid-rich proteins (GARPs) play differential roles for photoreceptor outer segment scaffolding.
abstract: Vertebrate photoreceptors initiate vision via a G-protein-mediated signaling cascade organized within a specialized cilium, the outer segment (OS). The membranous "stacked pancake" architecture of this organelle must be partially renewed daily to maintain cell function and viability; however, neither its static structure nor renewal process is well described in molecular terms. Glutamic acid-rich proteins (GARPs), including the cyclic nucleotide-gated cation channel (CNGB1) and GARP2 (a CNGB1 splice-variant), are proposed to contribute to OS organization in concert with peripherin/rds (P/rds), a retinal tetraspanin. We developed and applied an in situ fluorescence complementation approach that offers an unprecedented glimpse at the formation, trafficking, and localization of GARP-P/rds interactions in transgenic Xenopus laevis rod photoreceptors. Interactions for these (and other) proteins could be readily visualized using confocal microscopy. Nearly all associations, including CNGB1-P/rds interaction, were initiated within inner segments (ISs) before trafficking to OSs. In contrast, GARP2-P/rds interactions were only observed downstream, at or near sites of disk morphogenesis. These results suggest that GARP2-P/rds interaction participates directly in structuring disk stacks but CNGB1-P/rds interaction does not and instead serves mainly to localize plasma membrane ion channels. Altogether, the results lead us to propose that differential interaction of GARPs with P/rds may contribute to the broad phenotypic heterogeneity produced by inherited defects in P/rds. Analogous experiments applied to the synaptic protein RIBEYE suggest that monomers can oligomerize at the level of the IS before ribbon assembly and demonstrate the general applicability of this strategy for in situ analysis of protein interactions in sensory neurons.
authors.affiliation: Eye Research Institute, Oakland University, Rochester, Michigan 48309, USA.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Ritter
authors.forename: Nidhi
authors.initials: N
authors.lastname: Khattree
authors.forename: Beatrice
authors.initials: B
authors.lastname: Tam
authors.forename: Orson L
authors.initials: OL
authors.lastname: Moritz
authors.forename: Frank
authors.initials: F
authors.lastname: Schmitz
authors.forename: Andrew F X
authors.initials: AF
authors.lastname: Goldberg
mesh_terms: D000429:Alcohol Oxidoreductases; D000818:Animals; D030801:Animals, Genetically Modified; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism*; D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007814:Larva; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016253:Microscopy, Immunoelectron / Q000379:methods; D010750:Phosphoproteins / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000648:ultrastructure; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D055214:Retinal Photoreceptor Cell Outer Segment / Q000378:metabolism* / Q000648:ultrastructure; D012243:Rhodopsin / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000378:metabolism*; D014162:Transfection; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate / Q000378:metabolism; D014981:Xenopus
chemical_list: D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D018998:Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; D012243:Rhodopsin; D000429:Alcohol Oxidoreductases; C111341:Ctbp2 protein, mouse