Figure 3. Receptor Clustering Increases the Probability of ZAP70 Recruitment.
Ligand and ZAP70-GFP binding to DNA-CARζ by TIRF imaging with a 16-mer ligand at 0.1 (A–C) and 1 ligand per μm2 (D and E). Dose-response curves (A and D) are based on data in Figure 2B. The blue line overlaid on the fluorescence intensity represents detected step changes marking new ligand binding or ZAP70 recruitment events. The dashed red lines mark the quantal ligand fluorescence intensities determined using a hidden Markov model analysis (see Figure S3 and STAR Methods). Single or multiple ligand-binding events that could be followed for >30 s were scored for ZAP70-GFP recruitment. The initial ZAP70-GFP recruitment was referenced to the number of bound ligands (as in A and D).
(A) TIRF images of Atto647N-labeled 16-mer DNA ligands. Left panel, single bound ligands are marked by yellow circles. Bar, 5 μm. Region of interest overlaid with tracked single-molecule trajectories. ROI bar, 1 μm. Right panels, fluorescence-intensity time series for the 16-mer ligand and the corresponding ZAP70-GFP fluorescence intensity. Ligand 1, example of a ligand-receptor pair that does not recruit a ZAP70-GFP. Ligand 2, a less common example of ZAP70-GFP recruitment (often transient as shown here) to a single bound 16-mer ligand.
(B) Quantification of ZAP70-GFP recruitment at 0.1 ligands/μm2 of 16-mer. Bar plot shows the percentage of single ligated receptors and clusters (black bars) and percentage of ZAP70 recruitment (gray bars) for single bound ligands that can be tracked for >30 s. Mean ± SD from n = 9 cells.
(C) Quantification of ZAP70 dwell times at single bound 16-mer ligands (n = 15).
(D) TIRF images of 16-mer DNA ligands at 1 ligands/μm2. Bar, 5 μm. Region of interest (red box) shows three ligand-receptor clusters (labeled 1–3). ROI bar, 1 μm. Fluorescence-intensity time series from the DNA ligand and ZAP70-GFP of the three microclusters shown on the right. For additional traces, see Figure S4 and Movies S3, S4, S5, and S6.
(E) Quantification of ZAP70 recruitment at 1 ligands/μm2 of 16-mer. Organization of bar plot same as shown in (B). Results are mean ± SD from n = 6 cells.
(F) Distribution of ZAP70 dwell times at single bound receptors (n = 16) and receptor-ligand clusters (n = 70). For 32 clusters with ZAP70 dwells time of > 100 s, the measurement was truncated by the end of image acquisition.
See also Figure S4.