Fig. 3: Diffusion modes of PTH and PTH1R.
A Scheme illustrating both labeled PTH1R and peptide ligand used to record dual-color single molecule imaging (upper), and representative trajectories of PTH1RmNG before (green-left) and after (green-center) stimulation with PTHTMR (magenta-center). The merge of trajectories depicts the coincidental detection of PTH1RmNG and PTHTMR (right). Scale bar: 2 µm. Scheme created in BioRender. Pacheco Romero, J. (2025) https://BioRender.com/q57m113. B, C MSD vs time intervals plots of PTH1RmNG (B) and PTHTMR (C). Time points indicate the cohorts of trajectories after adding PTHTMR over which mean squared displacement was calculated for each particle. Data are mean ± S.E.M. of n = 12 cells for both, PTH1RmNG and PTHTMR. D, E Diffusion (D) and motion (E) time-lapses of PTH1RmNG alone (black), in the presence of PTHTMR (magenta) or the inverse agonist, IA-PTHTMR (teal). Individual trajectories were analyzed at intervals of 10 s. Ligands were added at T = 30 s (vertical dotted line). Data are the mean ± S.E.M. of n = 11 cells derived from the same experiment used to calculate panels (B, C). F, G Averaged diffusion coefficient (F) and motion (G) of PTH1RmNG before and after PTHTMR, inverse agonist, or unlabeled PTH. Data are the mean ± S.E.M. of n = 10/1081 (PTH1RmNG), n = 12/1481 (PTH1RmNG + PTHTMR), n = 11/3027 (PTH1RmNG + IA-PTHTMR) and n = 18/143 (PTH1RmNG + PTH), cells/average trajectories per cell of N = 3 independent experiments. ****P < 0.0001 and ns, not significant (P = 0.8633 in F and P = 0.39 in G) by one-way ANOVA with a Tukey’s multiple comparison. H Example of interacting PTH1RmNG–PTHTMR or PTH1RmNG– IA-PTHTMR trajectories. Scale bar: 0.5 µM. I Diffusion coefficients of agonists PTHTMR (n = 12/777), PTH1-14TMR (n = 11/1562), LAPTHTMR (n = 9/824), and the inverse agonist IA-PTHTMR (n = 11/619). Only the trajectories that showed coincidental detection with PTH1RmNG were considered for this analysis. Data are mean ± S.E.M. n = cells/average trajectories per cell. ****P < 0.0001, ***P < 0.001, **P < 0.01 by one-way ANOVA with a Dunnett multiple comparison.
