Figure 3.

FLIP analysis of free and clustered YFP-FasL and Fas-YFP. HeLa cells transfected with YFP-FasL and Fas-YFP, respectively, were cultured individually or together with HeLa cells expressing CFP fusion proteins of the corresponding receptor or ligand. Images of a representative cell from each experimental group are shown. Bleaching areas covering most of the cell except the analyzed FasL-Fas cluster and/or a control region in the plasma membrane were defined (dotted lines). Cells were initially bleached for 5 min to bleach the majority of the highly diffusible intracellular fraction of the YFP fusion proteins. After this, bleaching was continued, and images were recorded in 1-min intervals. Using these images, loss of fluorescence in nonbleached areas, which contained clusters or control membrane, was determined, as described in Materials and methods. These areas were defined as ROI, and are indicated (dotted line, bleach area; solid line, ROI of clustered or free YFP fusion protein; dashed line, control ROI). Measurements of clustered YFP fusion proteins (Fas-YFP, closed squares top diagram; YFP-FasL, closed squares bottom diagram) were corrected for each time point for the residual fluorescence of the corresponding “free” protein outside the bleach region. Fluorescence intensities of nonclustered Fas-YFP (open squares top diagram) and YFP-FasL (open squares bottom diagram) determined outside the bleach regions were corrected for the rest fluorescence within the bleach region. Corrected fluorescence intensities were normalized against the relative fluorescence intensity obtained after the initial five bleach cycles. This time point of each experiment was defined as t = 0 min. Averaged (n = 12–15) normalized relative fluorescence intensities were shown in the diagrams. The fluorescence loss indicating dissociation was linearized by plotting the natural logarithm of the relative fluorescence intensities over time. The third row in the top part shows a control experiment where besides Fas-YFP membrane CFP were coexpressed to demonstrate that the overall morphology of bleached cells was not affected.