Extended Data Fig. 9: Single-molecule behaviors of V2R, βarr and PLCδ-PH.

(a) Images of cells expressing LgBiT-V2R (SF650-FiBiT labeling) and, Halo-PLCδ-PH (labeled with SF650-FiBiT and Halo-JF549 labeling), respectively, and EGFP-βarr2 measured across three channels. (b) The intensity profile along the red line for each channel. Scale bar: 3 μm (c) The mean local density (red, left axis) and its first-order difference (blue, right axis) were plotted versus log(r), where r is the radius of the circle around each particle to calculate the local density. (d, e) Comparison of the mean densities estimated by hand (d) and by the local density analysis algorithm (e). Yellow lines in the upper panels were drawn by hand. The densities in the upper panels (yellow characters) were calculated as spot number/area of a frame. The densities in the lower panels were estimated by the mean local density analysis of the same frame. (f) Representative TIRF microscopic image of EGFP-βarr2 (βarr2-WT, C-3Q, or NC-4Q) at the before AVP stimulation (basal). The bright spots of tracked βarr2 are surrounded by white lines. Scale bar: 3 µm. (g) Numbers of HMM states and its selection. Single-molecule-tracking data for βarr2 (black), V2R (blue) and PLCδ-PH (red) molecules were classified based on the one- to five-state HMMs. Note that the four-state model, although the differences among the three to five states were modest, gave the top score for the lower bound values. (h) MSD-Δt plots of βarr2, V2R and PLCδ-PH after 15 min AVP stimulations under the βarr2 WT (black)-, C-3Q (blue)-, or NC-4Q (red)-expressing conditions. WT (n = 36), C-3Q (n = 32), NC-4Q (n = 23); n represents individual cells analyzed across two independent biological replicates. In Extended Data Fig. 9g, h, bars and error bars represent mean and SEM, respectively.

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