Figure 4: Metabolic depletion of SM reduces LFA-1 adhesion without affecting its lateral mobility.

(A) Monocyte adhesion to ICAM-1-Fc-coated fluorescent beads at 37 °C in unperturbed and myriocin-treated cells. The % of adhesion represents the amount of cells that have bound beads as determined by flow cytometry. NKI-L15 mAb was used to block LFA-1. The data show one representative experiment of 4 ± SD. Differences were assessed by 1way ANOVA test, ***<0.001. (B) Relative expression of αL– and β2- specific epitopes were assessed by flow cytometry. Mean fluorescent intensity changes are displayed as relative to control sample (expression levels in unperturbed cells set as = 1, dotted line). The data represent the mean ± SEM of 5–7 independent experiments. (C) Total SM content and PM cholesterol levels were determined by lysenin and filipin III labelling, respectively, while PM GM levels were detected by Ctx-AF647 and PM GPI-APs were detected by FLAER. The relative expression was assessed by flow cytometry and mean fluorescent intensity changes are relative to the respective unperturbed sample (expression levels in untreated cells set as = 1, dotted line). The data represent the mean ± SEM of 3–5 independent experiments. Differences were assessed by 1way ANOVA test, *<0.05, ***<0.001. (D) Normalized semilog distribution of D_1–4 values for LFA-1 nanoclusters in unperturbed control (grey) or myriocin-treated cells (red). Vertical dotted lines represent the average D_1–4 values in control (black) and myriocin-treated cells (red). Each histogram contains at least 406 trajectories taken from 30 cells in 3–5 experiments. (E) Percentage of LFA-1 nanocluster mobility classified as immobile, slow and fast mobile in unperturbed and myriocin treated monocytes. The error bars represent the mean ± SEM of 3–5 independent experiments. 2way ANOVA and Bonferroni post-test were applied to the full distribution of sub-population values calculated from at least 30 cells in 3–5 experiments. (F,G) Square displacement plots of slow and fast moving LFA-1 populations by fitting the CPD at different time lags (461–658 trajectories). Slow and fast diffusing components were fitted to a model of i) free (dashed lines) and ii) anomalous (continuous lines) diffusion.