Fig. 1: SPT analysis reveals that ligand binding is gated and restricted to a subset of EGFR.

A Diagram showing labeling strategies for detection of ligand-bound receptors (via EGF-Cy3B, top panels) or all receptors, regardless of ligand binding status (via Fab-Cy3). Shown (left panels) are representative single-frames of time-lapse image series obtained by TIRF-M using each labeling strategy, as used for SPT analysis. Scale 3 µm. Also shown (right panels) are the results of the tracking showing traces of the EGFR particles over 25 frames (0.5 s). Scale 10 µm B–D Results of SPT analysis. B Shown are the mean ± SE of the fraction of all EGFR tracks, as labelled by Fab-Cy3B (left panels, “total receptor”), or the fraction of only ligand-bound EGFR, as labeling EGF-Cy3B (right panels, “ligand-bound receptor”) that exhibit mobile, confined or immobile behaviour. Shown are mean ± SE of diffusion coefficient (C) or the confinement radius (D). EGF-Cy3B (ligand-bound) data is from 5 independent experiments, and Fab-Cy3B (total EGFR) data is from 4 independent experiments. Each experiment involved detection and tracking of >500 EGFR objects. ^*p < 0.05. E Diagram showing SNAP-reagent EGFR labeling strategy that detects all receptors, regardless of ligand binding status. Also shown (right panels) are representative single-frames of time-lapse image series obtained by TIRF-M using each labeling strategy, as used for SPT analysis. Scale 3 µm. F–H Results of SPT analysis with SNAP-A488 (no EGF) and Fab-Cy3B performed immediately one after the other in the same cells, or SNAP-A488 (in the presence of 10 ng/mL EGF) and EGF-Cy3B (at 10 ng/mL) similarly performed sequentially in the same cells. F Shown are the mean ± SE of the fraction of all EGFR tracks that exhibit mobile, confined or immobile behaviour. Also shown are mean ± SE of diffusion coefficient (G) or the confinement radius (H). For E–H, the data is from 3 independent experiments. Each experiment involved detection and tracking of >500 EGFR objects. ^*p < 0.05. Statistical analysis and p-values are indicated in Supplementary Table 1. Source data are provided as a Source Data file.