Frmpd1 Facilitates Trafficking of G-Protein Transducin and Modulates Synaptic Function in Rod Photoreceptors of Mammalian Retina

Outer plexiform layer (OPL) synaptic morphology is unaltered in Frmpd1^Δ1a mice. Morphologies of OPL synapses were examined by immunostaining using frozen retina sections from Post natal day 21 (P21) mice. Scale bar: 20 μm.

Frmpd1 localizes to rod inner segments and synapses. A, In vivo electroporation workflow. FLAG-tagged full-length Frmpd1 and GFP expression constructs under control of ubiquitin promoter (pUb) were coinjected subretinally in neonatal mouse pups at P1 and introduced to the retina via electroporation. At P21, eyes were harvested and processed for immunohistochemistry. B, Frmpd1 localization in dark-adapted retina. Mice were dark-adapted overnight before eyes were harvested and processed for immunohistochemistry to detect both GFP and FLAG-tagged Frmpd1 protein using anti-FLAG antibody (i). Immunofluorescence staining for the FLAG-Frmpd1 shows clear localization to both the rod inner segments (open arrowheads) and synapses (closed arrowheads; ii, iii). C, Frmpd1 localization in light-adapted retina. Mice were light-adapted for 1 h before eyes were harvested and processed for immunohistochemistry to detect both GFP and FLAG-tagged Frmpd1 protein using anti-FLAG antibody (i). As in dark-adapted retina, FLAG-Frmpd1 immunofluorescence staining localized to both rod inner segments (open arrowheads) and synapses (closed arrowheads; ii, iii). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Scale bar: 50 μm.

Frmpd1 interacts with Gpsm2 in the retina. A, Co-Immunoprecipitation (Co-IP) of Frmpd1 and Gpsm2. HEK293 cells were co-transfected with 3′ FLAG-Frmpd1 and mCherry-Gpsm2 expression constructs. Frmpd1-containing protein complexes were immunoprecipitated with anti-FLAG antibody and probed with anti-RFP (mCherry) to detect Gpsm2. Gpsm2-containing protein complexes were immunoprecipitated with anti-RFP antibody and probed with anti-FLAG to detect Gpsm2. B, Colocalization of Frmpd1 and Gpsm2 in HEK293 cells. HEK293 cells were co-transfected with 3′ FLAG-Frmpd1 and mCherry-Gpsm2 expression constructs and stained with anti-FLAG (green) antibody to visualize Frmpd1. Gpsm2 is visualized by mCherry protein fluorescence (red). C, Left panel, IP of Gpsm2 complexes from retina. Mice were light-adapted for 1 h, then dark-adapted for 1 h. Gpsm2-containing protein complexes were immunoprecipitated from retina lysate using anti-Gpsm2 antibody; 10% of input lysate and 50% of immunoprecipitated protein complexes were processed for immunoblotting with anti-Frmpd1 antibody. Asterisks (*) represent nonspecific band staining. Right panel, IP of Frmpd1 complexes from retina. Lysates were prepared as described, and Frmpd1-containing protein complexes were immunoprecipitated from retina lysate using anti-Frmpd1 antibody; 10% of input lysate and 50% of immunoprecipitated protein complexes were processed for immunoblotting with anti-Gpsm2 antibody. Asterisks (*) represent nonspecific band staining. D, Immunoblot of proteins pulled down from retina lysates with anti-Frmpd1 (left panel) and anti-Gpsm2 (right panel) antibodies and probed with Gαt antibodies.

Frmpd1 forms a ternary complex with Gpsm2 and Gαt. A, In vitro interaction of Frmpd1 with Gpsm2 and Gα_t*. Streptavidin resin beads with (+) and without (–) bound Avi-Gαt* were incubated with Gpsm2-FLAG and/or HA-thioredoxin tagged Frmpd1 fragment (residues 895–938 previously shown to contain Gpsm2 binding site). Immunoblots of pull down (PD) products from beads with (+) and without (–) bound Avi-Gαt* in presence of HA-thioredoxin-tagged Frmpd1 and/or Gpsm2-FLAG were probed with anti-HA and anti-FLAG antibodies. B, Steady state analysis of the Bio-Layer Interferometry (BLI) data (as shown in Fig. 5) for the binding of Gpsm2 to the Avi-Trx tagged Frmpd1 895–938 coupled to a streptavidin biosensor in the presence (+) or absence (–) of Gα_t*. Error bars show SEM. C, Kinetics of association and dissociation for Gpsm2 and Avi-thioredoxin tagged Frmpd1 895–938 coupled to a streptavidin biosensor as determined using Bio-Layer Interferometry (BLI). Representative curves are shown for black fitting in A. D, Kinetics of association and dissociation for Gpsm2 and Avi-thioredoxin-tagged Frmpd1 895–938 coupled to a streptavidin biosensor in the presence of Gαt* as determined using BLI. Representative curves are shown for orange fitting in A.

Rod bipolar cells (RBCs) light responses in Frmpd1^Δ1a and Gpsm2^−/− mice display decreased sensitivity compared with wild-type during Gα_t translocation. A, C, E, Representative dark-adapted response families of wild-type (A), Frmpd1^Δ1a (C), and Gpsm2^−/− (E) rods. B, D, F, Representative light response families of wild-type (B), Frmpd1^Δ1a (D), and Gpsm2^−/− (E) rods, following the Gα_t translocation protocol. G, Intensity-response relationships for dark adapted (DA) (black) and translocated (wild-type, green; Frmpd1^Δ1a, blue; Gpsm2^−/−, orange) rods. Solid and dashed arrows indicate half maximal flash strength (I_1/2) before and after translocation, respectively. H, J, L, Representative dark-adapted response families of wild-type (H), Frmpd1^Δ1a (J), and Gpsm2^−/− (L) RBCs. I, K, M, Representative response families of wild-type (I), Frmpd1^Δ1a (K), and Gpsm2^−/− (M) RBCs following the Gα_t translocation protocol. N, Intensity-response relationships for DA and translocated RBCs with color coding as in G. Solid and dashed arrows indicate half maximal flash strength (I_1/2) before and after translocation, respectively.

Frmpd1 facilitates dark-adapted (DA) return of Gα_t to rod outer segments. A, Wild-type, Frmpd1^Δ1a, and Gpsm2−/− mice were DA overnight, after which eyes were harvested and processed for immunohistochemistry to detect Gα_t in DA retina. B, DA mice were exposed to bright light (∼1000 lux) for 1.5 h to detect Gα_t translocation in light-adapted (LA) retina. C, Light-adapted mice were placed in darkness for 2 h to detect Gα_t in the retina during the course of dark adaptation (2-h DA). D, Example of transducin quantification for immunofluorescence preparations. Five equal-sized squares (regions of interest, ROIs) (yellow square) for outer segment (OS; A) (A), inner segment (IS; B) (B), synaptic terminal (ST; C) and background (bkg; D) were chosen for each image analyzed (see Materials and Methods, Transducin quantification assay). The total fluorescence (F_tot) was estimated by summing the fluorescence (F) values of the ROIs within the three relevant photoreceptor layers F_tot = (A – D) + (B – D) + (C – D). Relative intensity of each layer was then calculated as a ratio of the total: outer segments fluorescence (F_OS) = (A − D)/F_tot; inner segments fluorescence (F_IS) = (B − D)/F_tot; synaptic terminal fluorescence (F_ST) = (C − D)/F_tot. E, Quantification of relative intensity of the retinal layers at 2-h DA. Statistical significance is denoted with an asterisk where p < 0.05. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: 20 μm.