Fig. 8: Two-step process for βarr2-PTH₁R association.

A Principle of the analysis. Dual-color single-molecule was performed under TIRF illumination with βarr2^mNG and PTH₁R^iRFP (B) or PTH^TMR in cells expressing PTH₁R^CFP(C). Under the assumption that all molecules of βarr2^mNG detected on the TIRF plane are on the plasma membrane. The duration of βarr2^mNG molecules before their coincidental detection (<80 nm) with a receptor was quantified; this “pre-colocalization time” reflects the time βarr2^mNG spent free at the PM before collision with PTH₁R^iRFP. Scheme created in BioRender. Pacheco Romero, J. (2025) https://BioRender.com/t93c242. B, C Pre-colocalization time-frequency distributions for βarr2^mNG interacting with PTH₁R^iRFP (B) or PTH^TMR (C) (left panels). The data was fit with a single exponential decay. Histograms were produced from pooled data derived from n = 7/61(βarr2^PAmCherry/PTH₁R^iRFP) and n = 7/568 (βarr2^PAmCherry/PTH^TMR) cells/average events per cell in 3 experiments. 3D plot of trajectories showing fast association of βarr2^mNG (green) with PTH₁R^iRFP (red) (B, right panel) or PTH^TMR (red) (C, right panel). Dots in trajectories indicate the start point. D, E Distribution of PTH₁R^iRFP, PTH^TMR, and βarr2^PAmCherry trajectories on endogenous clathrin clusters (clathrin light-chain A, CLTA^mNG). Examples showing the fluorescent distribution of CLTA^mNG and the resulting binary mask (white line contour). Magenta, green, and red trajectories represent single molecule tracking of PTH₁R^iRFP, PTH^TMR, and βarr2^PAmCherry, respectively. In panel (E), cells were treated with PTH or PTH + TGX221. F–H The ratio of single-molecule detected inside over outside clathrin-domains normalized by the area of the cell. Data are the mean ± 95% confidence intervals (C.I.). For PTH₁R^iRFP, n = 16/26738 (basal), n = 20/26637 (PTH), n = 13/39071 (PTH + TGX221) cells/average detections per cell of N = 3 experiments. ns, not significant by one-way ANOVA with a Tukey’s multiple comparison (G). For PTH^TMR, n = 9/436960 from N = 3 independent experiments and for βarr2^PAmCherry n = 20/14050 (PTH) and n = 15/10929 (PTH + TGX221) of N = 4 and 5 experiments, respectively. **P = 0.0074 by unpaired t-test (H).