Fig. 1: ORs in the PM form metastable homodimers with lifetimes of 120 ~ 150 ms, without involving the N-terminal extracellular domain.
a Typical (among 20 experiments) image sequence of simultaneous two-color single fluorescent molecule observations, showing transient homo-colocalization and co-diffusion of single molecules of SNAPf-KOR tagged with SNAP-CF 660 R (magenta) and SNAP-Surface 549 (green), with their trajectories. b PCCFs for the SNAPf-KOR spots (mean ± SEM after area normalization; 18 movies). Green and grey bars indicate the results of the correct and 180°-rotated overlays of the simultaneously observed magenta and green images. The colocalization index is defined as the mean of PCCF values for 0—100 nm divided by the mean of PCCF values for 400–500 nm (see Supplementary Fig. 2c–e). c Colocalization indexes showing the homo-colocalizations of three ORs. In the box plots, horizontal bars, crosses, boxes, and whiskers indicate median values, mean values, interquartile ranges (25–75%), and 10–90% ranges, respectively. * and ns represent significant (p < 0.05) and non-significant (p ≥ 0.05) differences, respectively (Tukey’s multiple comparison test). The data set used for multiple comparison is indicated by the group of lines in the figure. All of the statistical parameters and analysis results including sample size n and p values are provided in Supplementary Data 2. d Histograms showing the distributions of colocalization durations for correct and rotated overlays. Control histograms for rotated overlays (grey) were fitted by single exponential functions (black), providing the lifetime of the incidental overlap events (τinci). Histograms for correct overlays (colors) were fitted by the sum of two exponential functions: The faster decay time (τ1) was close to τinci, and the slower decay time provided the true homodimer lifetime (τ2; see Supplementary Note 1 for the theory). The homodimer lifetimes, after correction for the trackable duration lifetimes of the two fluorescent probes, are shown in the boxes. e Method to determine the true dissociation rate constant (koff), considering the multiple binding events during an observed colocalization duration. See Supplementary Note 1. Source data are provided as a Source Data file.
