Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes
J. Clin. Invest. Jen-Zen Chuang, et al. 114:131 doi:10.1172/JCI21136 [Go to this article.]

Figure 4
Endogenous v-arr was mislocalized in rodent rods that expressed R135L but not WT or Q344ter rhodopsin. (A) The schematic illustration shows the in vivo electroporation technique. Plasmid is injected into the subretinal space followed by electroporation. Tweezer-type electrodes are placed across the eye, with the anode facing the cornea. (B) A schematic illustration of the bicistronic expression vector pCAG-rhodopsin-IRES-GFP. These vectors permit both rhodopsin and GFP to be translated from a single mRNA and simultaneously expressed in the transfected cells. (C–H) Retinas were transfected with either pCAG-WT-IRES-GFP (C and D), pCAG-Q344ter-IRES-GFP (E and F), or pCAG-R135L-IRES-GFP (G and H). These retinas were immunolabeled with the antibodies indicated, followed by Alexa 594–conjugated secondary antibodies. Representative optical images of photoreceptor layer show transfected rhodopsin immunolabeling (C, E, and G). The GFP signals were directly visualized and are shown in the merge images in (D, F, and H). Note that mAb 3A6 effectively detected the WT but not the R135L mutant in transfected cells. (I–N) Retina sections prepared from eyes transfected with pCAG-WT-IRES-GFP (I and J), pCAG-Q344ter-IRES-GFP (K and L), and pCAG-R135L-GFP (M and N) were immunolabeled with anti–v-arr antibody (red). Representative confocal images of v-arr labeling (I, K, and M) and merge views with GFP⁺- transfected cells are shown (J, L, and N). The PM (arrow and arrowhead) and the intracellular vacuole accumulation of v-arr (open arrow) are marked. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 20 μm.