Fig. 2

vbSPT analysis of the diffusion of rhodopsin and Gα_t in disc membrane. a Effect of light and GTP on the optimal HMM of rhodopsin diffusion. Coloured bars, shown in D_100 ms-histograms (grey bars), indicate D_state and occupancies in a representative optimal three-state HMM of rhodopsin calculated from single-molecule tracking data of ~400 trajectories with ~35-frames length on average. Top and upper-middle panels are from dark-adapted discs without or with GTP, respectively. Lower-middle and bottom panels are from light-exposed discs (20% of rhodopsin isomerized) without or with GTP, respectively. The HMM data are means of three experiments. Grey arrowheads indicate medians of D_100 ms. Significant differences (P) within histograms, vs. the top panel, by Mann−Whitney U test are indicated. b Bar graph of D_state (left) and stacked bar graph of state occupancies (right) indicating mean ± SD (N = 3). Statistically significant differences by two-way ANOVA are labelled. *P < 0.05, ***P < 0.001 vs. corresponding state in the dark in the absence of GTP, by Tukey’s multiple-comparison test. c Light-dose-dependence of rhodopsin’s D_100 ms in the presence or absence of GTP. d Light-dose-dependence of rhodopsin’s D_state (upper) and occupancies (lower). e Effect of 20 mM MCD on D_state and occupancies in HMM of rhodopsin in dark-adapted or 20%-photoisomerized discs in the absence of GTP. f Schematic picture of IgG-crosslinked rhodopsin. Fluorescent Fab′-1D4 was rendered bivalent with anti-mouse-κ-light-chain antibody IgG. g D_state and occupancies in HMM of IgG-crosslinked rhodopsin in dark-adapted discs (upper) and 20 mM methyl-β-cyclodextrin-treated discs (lower). h Bar graph of D_state (left) and stacked bar graph of occupancies (right) of IgG-crosslinked rhodopsin, indicating mean ± SD (N = 2). i Schematic diagram of rhodopsin dimer light-dependently stabilized by binding with fluorescently labelled transducin (G_t). j D_state and occupancies in three-state HMM of fluorescent Gα_t in dark-adapted discs (upper) and 20%-photoisomerized discs (lower) in the presence (hatched bar) or absence (solid bar) of 0.5 mM GTP. Coloured bars are indicated in corresponding D_100 ms-histograms. No optimal HMM was determined for activated-Gα_t. k Bar graph of D_state (left) and stacked bar graph of occupancies (right) of Gα_t in various conditions