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Figure 1. Changes in FRET efficiency between fluorescent α7 subunits upon co-transfection with RIC-3. Pixel based FRET was used to monitor assembly between α7-Venus and α7-Cerulean nAChR subunits. RIC-3 concentration was varied as molar ratio to total α7 plasmid: 0:1 (negative control), 0.02:1, 0.1:1, 1:1, and 5:1. Confocal images of transfected HEK293T cells showing α7-Cerulean (A, D, G), α7-Venus (B, E, H) and FRET efficiency (C, F, I) expression. FRET efficiency was higher in cells expressing RIC-3 at a 1:1 ratio to α7 (F) than cells not expressing RIC-3 (C) or cells expressing RIC-3 at a 5:1 ratio to α7 (I). (J) Summary plot showing that FRET efficiencies increased significantly (p < 0.0001, Kruskal-Wallis rank sum test) with increasing concentrations of RIC-3 relative to α7 but went to baseline at 5:1 ratio. There was a significant increase of FRET efficiency at 0.1:1 RIC-3 to α7 (p = 0.0004, Wilcoxon rank sum test) as compared to no RIC-3 cells and at 1:1 RIC-3 relative to α7 (p < 0.0001, Wilcoxon rank sum test). Numbers inside the plot represent the number of cells analyzed. (K) Control experiments validating the FRET measurements. α4YFP β2CFP shows significantly greater FRET efficiency than α4CFP β2YFP. This is expected because the transfection ratio favours an (α4)3(β2)2 stoichiometry [1] and the combination with the more acceptors (YFP) theoretically would have greater FRET efficiency. GYFP GCFP (GluClβ-YFP GluClα-CFP) are heteromeric glutamate-gated chloride channels found in invertebrates and show high FRET. Although they are members of the cys-loop family of receptors, they are not expected to assemble with any of the nicotinic receptors. Our negative control experiment, α4YFP GCFP (GluClα-CFP) shows very little FRET. A positive control experiment with RIC-3 and α7V α7C (α7-Venus α7-Cerulean) showing significant levels of FRET. Numbers inside the bars represent the number of cells analyzed.
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Figure 2. RIC-3 increases whole cell and cell surface expression of α-Bgt binding sites. Spectral confocal microscopy images of α-Bgt binding assays using Alexa 648 α-Bgt (Fl-Bgt) and performed under nonpermeabilizing conditions (A, B, C, D) without RIC-3, or (E, F, G, H) with RIC-3 (1:1 to α7), and permeabilizing conditions (I, J, K, L) without RIC-3 and (M, N, O, P) with RIC-3 (1:1 to α7). Emission signals from α7-Cerulean, α7-Venus, Fl-Bgt binding sites and the merged α7-Venus / Fl-Bgt images are shown for each corresponding cell. RIC-3 increases surface and intracellular α-Bgt binding sites.
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Figure 3. RIC-3 increases whole cell Fl-Bgt labelling and surface trafficking of α7 receptors but does not alter protein levels. Increasing concentrations of RIC-3 progressively augmented total Fl-Bgt binding under cell permeabilizing conditions (A, B). Fl-Bgt binding of surface α7 receptors also increased with RIC-3 but peaked at 1:1 RIC-3 to α7 and diminished at a 5:1 ratio (A, B). Total (A) and fold-change (B) of Fl-Bgt binding is shown. The surface and whole cell number of Fl-Bgt binding sites was quantified by measuring the integrated density of fluorescence intensities of Alexa 648-tagged α-bungarotoxin around the outer surface of each cell or within the cell, for non-permeabilizing and permeabilizing conditions, respectively. Significant difference levels comparing groups of RIC-3 coexpressing cells relative to no RIC-3 controls: * p < 0.05, ** p < 0.01, *** p < 0.001 Wilcoxon rank sum test post-hoc pair wise analysis. The number of cells analyzed for total receptor labelling in (A, B) are 0:1 (negative control, n = 14), 0.02:1 (n = 13), 0.1:1 (n = 14), 1:1 (n = 18), and 5:1 (n = 15). The number of cells analyzed for surface receptor labelling in (A, B) are 0:1 (n = 6), 0.02:1 (n = 8), 0.1:1 (n = 8), 1:1 (n = 11), and 5:1 (n = 11). (C, D) Mean emission intensity of the α7-Venus and α7-Cerulean fluorophores per HEK293T cell were determined at various concentrations of RIC-3. There was no significant change in either α7-Venus or α7-Cerulean fluorescent protein levels with various amounts of RIC-3 co-expressed inside the cells (p = 0.6, one-way ANOVAs; and p = 0.2, Kruskal-Wallis rank sum test, respectively).
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Figure 4. Effect of various concentrations of RIC-3 on FRET efficiencies between α4CFP and β2YFP subunits and protein expression levels of α4CFP and β2YFP subunits. Equimolar amounts of α4CFP and β2YFP subunits were coexpressed with various concentrations of RIC-3. The ratio of RIC-3 to α4CFP β2YFP nAChR cDNAs was varied to 0:1 , 0.02:1, 0.1:1, 1:1, and 5:1. (A) Quantification of FRET efficiencies between α4CFP and β2YFP subunits showed no significant change over various concentrations of RIC-3 up to 1:1 concentration (p = 0.08, one-way ANOVA). (B) Quantification of mean α4CFP fluorescence per cell showed a significant progressive increase with rising concentrations of RIC-3 (p < 0.0001, Kruskal-Wallis rank sum test). (C) The mean β2YFP fluorescence intensity per cell showed a significant increase with increasing concentrations of RIC-3 (p < 0.0001, Kruskal-Wallis rank sum test). (B, C) However, high RIC-3 concentrations at 5:1 reduced both α4CFP and β2YFP fluorescence intensities closer to baseline values. Significant difference levels comparing groups of RIC-3 coexpressing cells relative to no RIC-3 controls: * p = 0.02, ** p = 0.002, *** p < 0.0001, NS p > 0.05, Wilcoxon rank sum test post-hoc pair wise analyses.
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Figure 5. FRET efficiency measurements show that RIC-3 interacts with α7 and β2 nicotinic subunits. (A-I) Pixel based FRET was used to monitor assembly between CFP-RIC-3 and a fluorescently tagged nicotinic receptor subunit (either α7-Venus or β2YFP). (J) Summary data showing significantly higher levels of FRET efficiency between CFP-RIC-3 and α7-Venus at 1:1 ratio as compared to 5:1 ratio (p = 0.007, Welch two sample t-test). There was significantly greater FRET efficiency between CFP-RIC-3 and β2YFP at 0.2:1 (p = 0.003, Wilcoxon signed rank test post-hoc analysis) and 0.5:1 (p < 0.0001, Wilcoxon signed rank test post-hoc analysis) ratios as compared to 5:1 ratio. Also there was significantly greater FRET efficiency between CFP-RIC-3 and β2YFP at 0.2:1 (p = 0.009, Wilcoxon signed rank test post-hoc analysis) and 0.5:1 (p = 0.002, Wilcoxon signed rank test post-hoc analysis) ratios as compared to 1:1 ratio. No significant (NS) FRET efficiency could be detected between CFP-RIC-3 and α4YFP (p = 0.55, Kruskal-Wallis rank sum test) even though there was a similar trend as β2YFP. Therefore, equimolar RIC-3 interacts with α7 while RIC-3 optimally interacts with β2 at a 0.5:1 molar ratio.
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Figure 6. Effect of acute nicotine treatment on FRET efficiency and protein expression of α4CFP and β2YFP subunits in the presence or absence of RIC-3. Equimolar amounts of α4CFP and β2YFP cDNA were transfected either with (1:1 ratio of RIC-3 to nAChR subunit) or without RIC-3. Cells were incubated in various nicotine concentrations (0, 0.1, 1 and 10 μM) at 37°C 30 min before imaging. (A) In the absence of RIC-3 there was a significant progressive increase in FRET efficiency between α4CFP and β2YFP subunits, signifying receptor assembly, at all concentrations of nicotine (p = 0.002, Kruskal-Wallis rank sum test). With RIC-3 there was no change in FRET efficiency between α4CFP and β2YFP subunits with increasing concentrations of nicotine. Significant difference levels comparing groups of nicotine concentrations relative to no nicotine control: *, p = 0.03, **, p = 0.002 (Wilcoxon rank sum tests). Quantification of mean α4CFP (B) and β2YFP (C) fluorescence intensities per cell showed no change with increasing nicotine concentrations whether RIC-3 (at 1:1 ratio) was present or absent. However, the mean α4CFP and β2YFP fluorescence intensities with RIC-3 was significantly greater than without RIC-3 at all nicotine concentrations (p < 0.0001, RIC-3 factor, two-way ANOVA; for both α4CFP and β2YFP subunits). For pairwise comparison of α4CFP and β2YFP fluorescence between no RIC-3 and RIC-3 coexpression at each nicotine concentration significance levels are reported as p values (post-hoc pairwise Tukey’s HSD tests).
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Figure 7. Anti-HA epitope antibody binding confirms no change in nAChR protein with 30 min nicotine and no change in nAChR conformation within 3 min of nicotine. (A) Equimolar amounts of α4CFP and β2YFP cDNA were transfected in HEK293T cells and time lapse imaging of FRET was performed at 30°C. Cells were imaged for FRET at 1 and 3 min before and 1 and 3 min during 10 μM nicotine incubation. There was no significant change (p = 0.88, one-way repeated measures ANOVA, n = 12) in FRET efficiency between nicotinic receptor subunits with bath applied nicotine during the 3 min nicotine incubation. (B) 30 min application of 10 μM nicotine did not alter anti-HA epitope antibody binding of HA epitope tagged α4 (α4-HA) and β2 (β2-HA) subunits expressed in HEK293T cells. (C) RIC-3 coexpression with α4-HA and β2-HA subunits resulted in significantly enhanced levels of anti-HA antibody labelling of α4-HA (p < 0.0001, Wilcoxon rank sum test) and β2-HA (p < 0.0001, Wilcoxon rank sum test) subunits as compared to cells not coexpressing RIC-3.
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Figure 8. Effect of acute nicotine treatment on FRET efficiency of α7-Cerulean and α7-Venus subunits in the presence or absence of RIC-3. Equimolar amounts of α7-Venus and α7-Cerulean were transfected either without RIC-3 or with RIC-3 at a 1:1 ratio to the nAChR subunit. Cells were incubated at various concentrations of nicotine (0, 0.1, 1 and 10 μM) at 37°C for 30 min prior to imaging. Whether RIC-3 was present or absent, there was no change in FRET efficiency between α7-Venus and α7-Cerulean subunits over increasing concentrations of nicotine (30 min) (p = 0.3, and p = 0.12, Kruskal-Wallis rank sum tests, respectively). However, the FRET efficiency between α7-Venus and α7-Cerulean was signficantly greater in the presence than the absence of RIC-3 at each of the nicotine concentrations (*, p < 0.05, **, p < 0.01, Wilcoxon rank sum test or t-test).
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