Fig. 1
From: Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling
FZD6 forms dimers. a HEK293 cells expressing V5-FZD6-mCherry and FZD6-GFP were used for dcFRAP. Arrows depict a ROI of 1.80 × 1.80 µm that was photobleached. Size bar, 10 µm. Lower panels provide fluorescence prior (left), directly after (bleach), and 100 s after (post-bleach) photobleaching. Size bar–2 µm. b, c Curves provide fluorescence intensity data from dcFRAP in the absence (gray) and presence (red) of biotinylated anti-V5/avidin crosslinking (CL) for V5-FZD6-mCherry and FZD6-GFP. The CL-induced decrease in the mobile fraction of FZD6-GFP indicates receptor–receptor interaction. d Bar graph shows fluorescence intensity averages of the mobile fraction across different conditions within the time frame of 85–101 s (including 15 s pre-bleach measurements). White bars—V5-FZD6-mCherry; gray bars—FZD6-GFP; hatched—CL. N = 21 ROIs before CL; N = 31 after CL from three independent experiments. P = 0.0045; t = 2.975; df = 50. e–g Corresponding negative control dcFRAP experiments for FZD6 interaction employing V5-FZD6-mCherry and CB1-GFP. P = 0.7030; t = 0.3831; df = 61. N = 29 ROIs before CL; N = 34 ROIs after CL from three independent experiments. Error bars provide standard error of the mean (s.e.m.). **P < 0.01; ns non significant (two-tailed t-test). For a statistical comparison of the FZD6/FZD6 and FZD6/CB1 data sets presented in d and g, see Supplementary Fig. 1a. Only ROIs wherein the crosslinked receptor population was in excess to the uncrosslinked were included in the data analysis (see Supplementary Fig. 1b). h HEK293 cells expressing V5-FZD6-mCherry/FZD6-GFP used for FCCS. i FCCS amplitudes from a representative experiment are shown. Autocorrelations in green—FZD6-GFP and red—V5-FZD6-mCherry; cross-correlation amplitude in black. G(τ) provides the fluorescence correlation amplitude. τ/s gives time shift per s. j The scatter plot summarizes FCCS values obtained in cells co-expressing FZD6-GFP/V5-FZD6-mCherry (filled circles) or cannabinoid CB1 receptor (CB1-GFP)/V5-FZD6-mCherry (open circles). Data presentation shows the percentage of dimerization over varying numbers of green over red particles. The crosses/dotted line represent a theoretical model assuming 100% FZD6 dimerization. The crosstalk correction in the model assumed that CB1-mCherry was monomeric. N = 10 cells expressing FZD6-GFP/V5-FZD6-mCherry; N = 25 cells expressing CB1-GFP/V5-FZD6-mCherry from 6 independent experiments. Data are based on two to eight 10 s acquisition periods per cell (see also Supplementary Fig. 1b)
