Figure 6: Increased association of LFA-1 with the cytoskeleton upon Cer formation.

(A) Dual colour images of lifeact-GFP transfected monocytes and SPT of LFA-1 nanoclusters (red trajectories) in control (left) and SMase (right) treated cells. After transfection with lifeact-GFP and, if applicable treatment with SMase, monocytes were labeled with TS2/4-Atto647N and seeded on fibronectin. Movies were recorded at 100ms/frame in TIRF mode. Custom-written software was employed to quantify and normalize the actin signal in each cell by assigning the lowest actin intensity to 0 (blue) and the highest to 1 (yellow), as indicated in the color code (bottom). White dotted lines represent the cell boundaries. Within the control cell, selected area i) represents an example of a low actin signal, while selected area ii) is an example of a high actin signal in control cells, superimposed with the corresponding LFA-1 trajectories (red). Both selected areas are shown as enlarged images next to the control cell. (B) Distribution of the normalized actin intensity associated with each LFA-1 trajectory in unperturbed (blue) or SMase treated cells (green). Each histogram contains at least 140 trajectories taken from 15 cells in multiple experiments. The data represents the mean ± SD obtained after bootstrapping of actin intensities measured in all experiments. The inset shows the difference between the normalized frequency of localizations of LFA-1 trajectories in unperturbed cells and upon SMase treatment as function of the normalized actin signal. P-values were compared to unperturbed cells by 2way ANOVA with Bonferroni post-test, ***<0.001. (C) Total percentages of LFA-1 trajectory localizations in unperturbed (blue) and SMase treated cells (green) associated with the normalized actin-GFP fluorescent signal for values <0.4, 0.4–0.7 and >0.7 as extracted from B.