Fig 1: Co-expression of Gnaq, Gna11 and Gn14 in subtypes of pRGC. a Triple labelling of β-Gal (green), Gna14 (red) and Gnaq (yellow) antibodies confirms the co-expression of Gna14 and Gnaq in M1 type pRGCs. Levels and localisation of Gnaq labelling are similar between M1 cells and neighbouring non-melanopsin cells, whereas only M1 type cells show membrane-bound and intracellular labelling of Gna14 (white arrow). b, c Triple labelling of EYFP (green), Gna14 (red) and Gnaq (yellow) confirms the co-expression of Gna14 and Gnaq in M1-M5 type pRGCs. Expression of Gna14 is detected in all melanopsin cells, with distinctive membrane labelling of Gna14 evident for a subset of EYFP cells (white arrow), consistent with the percentage of M1 type pRGCs. The levels and distribution of Gnaq labelling show no obvious change within individual pRGCs regardless of their pattern of Gna14 labelling. d Triple labelling of β-Gal (green), Gnaq/11 (red) and Gnaq (yellow) antibodies confirms the co-expression of Gnaq and Gna11 Gα subunits in M1 type pRGCs. Levels of Gnaq labelling are similar between M1 cells and neighbouring non-melanopsin cells. By contrast, M1 cells show increased membrane bound Gnaq/11 labelling (white arrow), consistent with increased expression of Gna11 in these cells. e Triple labelling of EYFP (green), Gnaq/11 (red) and Gnaq (yellow) antibodies confirms the co-expression of Gnaq and Gna11 in M1-M5 type pRGCs. Both Gnaq/11 and Gnaq antibodies label all melanopsin cells, although increased levels of Gnaq/11 labelling are evident for only a subset of EYFP cells, with morphologies characteristic of M1 type pRGCs (white arrow)
Fig 2: Localisation of Gnaq/11 type Ga subunits in the mouse retina. a–c Immunolabelling of the mouse retina with antibodies recognising both Gnaq and Gna11 (termed Gnaq/11) (a) or antibodies specific for Gnaq (b) or Gna14 (c) confirms the widespread expression of Gnaq/11 type G proteins in the wildtype mouse retina (upper panels) and specifically in cells of the ganglion cell layer (lower panels). For all images DAPI nuclear counter stain is shown in blue. PR Photoreceptors, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. Additional images showing the detailed expression of Gnaq/11 type G proteins in the retina, and also data showing the specificity of these antibodies is shown in Supplementary Figs. 1 and 2
Fig 3: Expression of Gnaq, Gna11 and Gn14 in pRGC subtypes of the mouse retina. Double labelling of G protein antibodies and markers of specific pRGC subtypes shows the widespread expression of Gnaq, Gna11 and Gna14 in all pRGC subtypes. a, b Gnaq/11 immunoreactivity (red) was consistently detected for ß-Gal positive M1 cells (green) (a) and EYFP positive M1-M5 cells (green) (b), although levels of membrane bound Gnaq/11 labelling are increased for M1 type pRGCs. c, d Gnaq immunoreactivity (yellow) was consistently detected for ß-Gal positive M1 cells (green) (c) and EYFP positive M1-M5 cells (green) (d). The levels and pattern of Gnaq labelling were similar for ß-Gal positive M1 cells, EYFP positive M1-M5 cells and neighbouring non-melanopsin cells. e, f Gna14 immunoreactivity (red) was consistently detected for ß-Gal positive M1 cells (green) (e) and EYFP positive M1-M5 cells (green) (f). Membrane-bound Gna14 labelling was typically observed for M1 type pRGCs (e, white arrow), whereas only intracellular Gna14 labelling was detected for the majority of EYFP positive cells and neighbouring non-melanopsin cells. Membrane bound Gna14 labelling was observed for a subset of EYFP cells resembling M1 type pRGCs (f, white arrow). For all images DAPI nuclear counter stain is shown in blue. GCL is ganglion cell layer
Fig 4: Gnaq/11 and Gna14 G proteins perform overlapping roles in melanopsin phototransduction in vivo. a Summary of pupillary light responses observed from mice receiving intravitreal injections of siRNA targeting Opn4, Gna14, both Gnaq and Gna11, Gnaq and Gna11 and Gna14, or non-sequence control (NSC) siRNA. A significant attenuation of the pupil light response was observed following silencing of Opn4 and also following the simultaneous silencing of Gnaq, Gna11 and Gna14. A small, but significant reduction in pupil constriction was also observed following co-silencing of Gnaq and Gna11, but not following the silencing of Gna14 alone. Data are shown as mean ± SEM. * = t test p < 0.02, ** = t test p < 0.002. b Images showing pupil area immediately before the onset and termination of light stimulation for mice receiving the various siRNA. c Levels of Opn4 mRNA detected in the mouse retina following delivery of NSC siRNA and Opn4 siRNA. Data is shown as mean ± SEM. * = t test p = 0.035. d Images showing levels of melanopsin protein detected in whole retina flatmounts from mice 72 h post injection of NSC siRNA and Opn4 siRNA. e Images showing levels of Gna14 protein detected 72 h post injection of NSC siRNA and Gna14 siRNA. For both Opn4 and Gna14 siRNA notable but incomplete levels of protein silencing are observed
Fig 5: Melanopsin can couple to Gnaq, Gna11 and Gna14 in vitro. An in vitro cell line model of melanopsin signalling was used in combination with gene silencing and gene over expression techniques to show that melanopsin can couple to Gnaq, Gna11 and Gna14 Ga subunits. a Opn4L and Opn4S expressing Neuro-2A cells but not wildtype (WT) Neuro-2A cells show rapid and robust changes in intracellular calcium levels following the onset of image acquisition and resulting light stimulation (Rhod-2 images collected every 2 s using 100 ms exposures to 545 nm light at 7.9 × 1013 photons cm-2 s-1,10 nm bandwidth). Traces shown are the mean of all cells imaged in a single experiment (typically ~60–80 cells). Time lapse images (0–16 s) show rapid changes in intracellular calcium upon light exposure, with peak elevations typically observed within 10–20 s. b Graphs showing levels of mRNA expression observed following transfection of Neuro-2A cells with siRNA in vitro. Opn4 siRNA reduced expression of both isoforms of melanopsin, Opn4L and Opn4S, by 90–95 % (top left). Gnaq and Gna11 siRNA selectively silence Gnaq or Gna11 mRNA by 90 and 85 %, respectively (top right). Co-transfection of Gnaq and Gna11 siRNA resulted in 80–90 % silencing for both genes (top right). Co-silencing of Gnaq and Gna11 was also achieved following simultaneous transfection with plasmid DNA encoding Gna14 (lower left). Gna14 siRNA silenced Gna14 mRNA by 85–90 % (lower right). All gene expression data are normalised to the geometric mean expression of three housekeeping genes (Gapdh, Arbp, ß-actin) and shown as percentage expression compared to cells transfected with non-sequence control (NSC) siRNA. n = 5 replicate cultures for all groups. Data are shown as mean ± SEM. ** t test p < 0.01, *** t test p < 0.001. c, d Combining calcium imaging and siRNA transfection shows that both the long and short isoforms of melanopsin, Opn4L and Opn4S, can couple to Gnaq, Gna11 and Gna14 in vitro. c Graph showing the percentage of Opn4L and Opn4S expressing cells that show light induced changes in calcium levels following transfection with siRNA. Responses are eliminated following silencing of Opn4, and following co-silencing of Gnaq and Gna11. Robust responses are observed for cells expressing only Gnaq (receiving Gna11 siRNA), cells expressing only Gna11 (Gnaq siRNA) and cells expressing only Gna14 (Gnaq and Gna11 siRNA plus Gna14 plasmid). The number of replicate experiments (dishes) performed for each set of conditions is shown below the main graph. *** t test p < 0.001 following Bonferroni multiple test correction. d Traces showing responses recorded from melanopsin expressing cells receiving the different combinations of siRNA and plasmid DNA. Panels show responses from all individual cells within a single experiment (typically 50–60 cells)
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