Fig. 2: Single molecule measurements of PTHTMR. | Nature Communications

Fig. 2: Single molecule measurements of PTHTMR.

From: Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid hormone–GPCR–arrestin complex

Fig. 2

A, B 3D-printed device for imaging the top cell surface by TIRF. The black lid with a screw-type plunger fits in the glass-bottom dishes. The plunger holds the sample coverslip down and ensures its correct adjustment at the bottom of the dish while preventing compression of cells (A). Representative trajectories of receptor and ligand at the top membrane (B). Scale bar, 2 µm. Scheme was created in BioRender. Pacheco Romero, J. (2025) https://BioRender.com/p78d742. C Comparison of diffusion of PTHTMR in the bottom and top membranes. Data are the mean ± S.E.M. of Bottom plasma membrane (PM) (black), n = 11/5751, and top PM (teal), n = 18/156 cells/average trajectories per cell. ns, not significant with P = 0.92 by unpaired t-test. D Comparison of diffusion of PTHTMR in HEK293 cells expressing PTH1RmNG (black), for comparison, the same data as panel C is shown (bottom PM), MC3T3 (gray), n = 18/686 and RPTEC cells, n = 19/604 cells/average trajectories per cell. Data are the mean ± S.E.M. ns, not significant by one-way ANOVA with a Tukey’s multiple comparison. E Diffusion coefficient of trajectories of PTH labeled with either TMR or fluorescein in cells expressing PTH1RmNG or PTH1RiRFP, respectively. Only trajectories that showed coincidental detection with the receptor were considered for the analysis. Mean ± S.E.M. of n = 9/75 (PTHfluo) and n = 12/777(PTHTMR) cells/average trajectories per cell. ns, not significant by unpaired t-test. F Diffusion coefficient of PTH1RiRFP in presence or absence of PTHfluo. Mean ± S.E.M. of n = 9/3674 cells/average trajectories per cell. **P = 0.0086 by unpaired t-test. G, H FRAP recordings in confocal and TIRF microscopy for PTHTMR. A representative cell before, after, and recovering from photobleaching. The scheme depicts the optical section that was selected for photobleaching. Scale bar, 10 µm. Time courses of fluorescence recovery and quantification of mobile fraction from FRAP experiments (H). Mean ± 95% C.I. of n = 12 cells of N = 3 experiments. ns, not significant by unpaired t-test. Scheme created in BioRender. Pacheco Romero, J. (2025) https://BioRender.com/i51c035.

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