Figure 6. Formation of Microclusters from Single Ligand-Receptor-Binding Event.

(A) A TIRF image of receptor-bound 16-mer DNA ligand; receptor-ligand clusters grow by adjacent ligand-binding events (red arrows numbered 1–3) and by merging and fusion (red box numbered 4). Bar, 2.5 μm. See Movie S7. Clusters grow by sequential addition of newly bound ligand; the blue lines overlaid on the fluorescence intensities are detected step changes (see STAR Methods; Figure S3). Time series (red box, numbered 4) below follows fusion of two clusters (Bar, 1 μm).

(B) The rate of new ligand-binding events near to an existing receptor-bound ligand (quantal intensity increase in an existing diffraction-limited spot) or outside of these zones (sudden appearance of a new bound ligand in the contact area between the cell and SLB) for DNA-CARζ (red) and DNA-CAR_TCR (blue). The ligand-receptor on-rate is expressed as events per second per μm² membrane surface area (using 0.126 μm² for a diffraction-limited spot). Mean ± SEM from n = 125 binding events from 5 cells and n = 60 binding events from 4 cells for the DNA-CARζ and DNA-CAR_TCR, respectively.

See also Figure S6D.