Figure 6

FRAP analysis of the lateral mobility of flotillin-2-EGFP, expressed without or with flotillin-1-mCherry in T-lymphocytes. Freshly isolated human T-lymphocytes were transfected with either flotillin-2-EGFP alone (A,B,E,F) or with flotillin-2-EGFP and flotillin-1-mCherry together (C,D,G,H), followed by a 4 hour incubation prior to FRAP analysis. Just before the FRAP analysis, cells were incubated for 15 minutes in the absence (left panels) or presence (right panels) of 40 ng/ml SDF-1, followed by bleaching of EGFP using excitation at 473 nm for all samples. Images of representative cells 2 seconds before (pre) and 7- 133 seconds after (post) completion of bleaching are shown, as indicated. Bleached regions of interest (ROI) are shown as red circles. Bar, 10 μm. (E-H) Fluorescence recovery time course after photobleaching of EGFP (mean ± s.e.m.) averaged from 11-16 cells derived from 3-4 independent experiments, as indicated in the panels. The prebleaching intensity was set to 100 in panels E-H. The values were corrected for bleaching due to acquisition of frames assessed in control ROIs corresponding to non-bleached regions analyzed in the same cells where localized bleaching was performed (not shown in A-D). Note that these FRAP experiments were carried out at 20°C, where cells are not any more polarized but still feature flotillin caps. At 37°C rapid shape changes and migration precluded accurate bleaching, acquisition and evaluation of FRAP data.