Figure 3.

Average receptor diffusion displays two different phases of dependence on the number of proteins in the average cluster. (A) Average D_S (as in Fig. 2C) is shown as a function of average N from the same live-cell experiments (as in Fig. 1C). Each point corresponds to values of D_s and N at a given time before (diamonds) and after (circles) stimulation averaged over the 11 cells imaged, and data from individual cells are binned every 15 s to facilitate averaging. Time after the addition of antigen is indicated by the color bar. Antigen (1 μg/ml) was added after the cells were imaged for 5 min. The solid black lines represent linear fits of points between 0 and 1 min and between 1:45 and 15 min after antigen stimulation, weighted by the inverse of the mean ± SE in D_S and N for each point. Points spanning these two regimes are indicated with arrows and labeled with the time after antigen addition. (B) Average intensity of the cytoplasmic Ca²⁺ indicator Fluo-4 over a population of cells imaged as described in Materials and Methods in the Supporting Material. The increase in Fluo-4 intensity after antigen stimulation indicates the onset of Ca²⁺ mobilization. The time period coinciding with the timing of the transition from the first regime to the second in A is highlighted by the shaded region. (C) The cumulative distribution of cells exhibiting an initial Ca²⁺ response indicates that the majority (>90%) of cells have initial Ca²⁺ responses between 1 and 4 min after antigen stimulation. The time point when 50% of the responding cells have exhibited a Ca²⁺ response is indicated by the open circle. To see this figure in color, go online.