Figure 1. FcγRIIB-T232 exhibits... | Rockefeller University Press

Figure 1.

$Figure 1. FcγRIIB-T232 exhibits significantly slower lateral mobility than FcγRIIB-I232. (A) Representative FRAP images at the indicated time points showing the recovery of FcγRIIB-YFP fluorescence intensity in A20II1.6 B cells. The red circle shows the bleaching area. Bars, 1.5 µm. (B, left) Representative FRAP curve at 25°C experimental condition. (Right) Statistical comparison of the FRAP recovery kinetics from A20II1.6 B cells expressing either FcγRIIB-I232 or FcγRIIB-T232. Error bars represent mean ± SEM from at least 20 cells in three independent experiments. AU, arbitrary units. (C) Representative 2D FRAP images at the indicated time points showing the recovery of FcγRIIB-YFP fluorescence intensity in ST486 B cells. The red circle shows the bleaching area. Bars, 1.5 µm. (D, left) Statistical comparison of FRAP recovery kinetics at 37°C from ST486 B cells expressing either FcγRIIB-I232 or FcγRIIB-T232. Error bars represent mean ± SEM from at least 18 cells in three independent experiments. (Right) Comparison of the diffusion coefficient with each dot representing one measurement for the diffusion coefficient from one cell. Bars represent mean ± SD from at least 18 cells in three independent experiments. Two-tailed Student's t tests were performed with the p-value indicated. **, P < 0.01. (E) Representative trajectories in the whole TIRF imaging time course of either FcγRIIB-I232 or FcγRIIB-T232 on the plasma membrane of ST486 B cells. (F) A series of mathematical comparisons of the Brownian diffusion of FcγRIIB-I232 or FcγRIIB-T232 molecules from ST486 cells in CPD plots (left), MSD plots (middle), or scatter plots (right). Bars represent median value. The p-value in CDP plots is <0.0001 in Kolmogorov-Smirnov tests. (G) PICS analysis of single-molecule TIRF images from either FcγRIIB-I232 or FcγRIIB-T232. (H and I) Two exponential PICS analysis for both the fast (H) and slow (I) fractions of FcγRIIB molecules. (E–I) The results shown are representative of one of at least three independent experiments. See also Videos 1–6.$

FcγRIIB-T232 exhibits significantly slower lateral mobility than FcγRIIB-I232. (A) Representative FRAP images at the indicated time points showing the recovery of FcγRIIB-YFP fluorescence intensity in A20II1.6 B cells. The red circle shows the bleaching area. Bars, 1.5 µm. (B, left) Representative FRAP curve at 25°C experimental condition. (Right) Statistical comparison of the FRAP recovery kinetics from A20II1.6 B cells expressing either FcγRIIB-I232 or FcγRIIB-T232. Error bars represent mean ± SEM from at least 20 cells in three independent experiments. AU, arbitrary units. (C) Representative 2D FRAP images at the indicated time points showing the recovery of FcγRIIB-YFP fluorescence intensity in ST486 B cells. The red circle shows the bleaching area. Bars, 1.5 µm. (D, left) Statistical comparison of FRAP recovery kinetics at 37°C from ST486 B cells expressing either FcγRIIB-I232 or FcγRIIB-T232. Error bars represent mean ± SEM from at least 18 cells in three independent experiments. (Right) Comparison of the diffusion coefficient with each dot representing one measurement for the diffusion coefficient from one cell. Bars represent mean ± SD from at least 18 cells in three independent experiments. Two-tailed Student's t tests were performed with the p-value indicated. **, P < 0.01. (E) Representative trajectories in the whole TIRF imaging time course of either FcγRIIB-I232 or FcγRIIB-T232 on the plasma membrane of ST486 B cells. (F) A series of mathematical comparisons of the Brownian diffusion of FcγRIIB-I232 or FcγRIIB-T232 molecules from ST486 cells in CPD plots (left), MSD plots (middle), or scatter plots (right). Bars represent median value. The p-value in CDP plots is <0.0001 in Kolmogorov-Smirnov tests. (G) PICS analysis of single-molecule TIRF images from either FcγRIIB-I232 or FcγRIIB-T232. (H and I) Two exponential PICS analysis for both the fast (H) and slow (I) fractions of FcγRIIB molecules. (E–I) The results shown are representative of one of at least three independent experiments. See also Videos 1–6.