Fig. 4.

Membrane cholesterol regulates p75^NTR diffusivity and response to NGF. (A) Box-plot for D values from trajectories of wt p75^NTR or mut p75^NTR in SK-N-BE(2) cells in resting conditions (black) and up to 15 min after NGF administration (gray); the distribution for dim p75^NTR (blue) is also shown. Boxes represent 25th to 75th percentiles, lines represent medians, and dashed lines represent means. ***P < 0.001, Kruskal–Wallis test (with Dunn’s means comparison). (B) Plot of membrane cholesterol (CHO) content (mean intensity ± SEM of filipin III-stained cells) versus D of p75^NTR single molecules (peak ± full width at half maximum of its distribution as in SI Appendix, Fig. S8C) in 6 different cell models. Cholesterol content is normalized to SK-N-BE(2) cell results. The black line indicates linear fit. HEK, human embryonic kidney 293 cells. (C) TIRF images of filipin III-stained SK-N-BE(2) cells exhibiting modulation of cholesterol levels (quantified as mean intensity ± SEM) with mevastatin (meva., green) or soluble cholesterol (chol., red). *P < 0.05 and ***P < 0.001, 1-way ANOVA. (Scale bar, 10 μm.) (D) Same graph as in A, obtained for NGF-stimulated wt and mut S6-p75^NTR trajectories in SK-N-BE(2) cells (gray), treated with mevastatin (green) and cholesterol (red). ***P < 0.001, Kruskal–Wallis test (with Dunn’s means comparison). (E) Outline of the colocalization experiment with TIRF images of cholera toxin B subunit (CT-B)-stained GM1 (green) and p75^NTR single molecules (red). (Scale bar, 5 μm; Inset, 1 μm.) (F) Quantification of wt/mut p75^NTR and CT-B colocalization in the absence or presence of NGF. Box-plots show median and 25th to 75th percentiles of average p75^NTR intensity inside CT-B–stained domains (Ī_{raft p75}) over average p75^NTR intensity within the whole cell (Ī_{total p75}). Whiskers indicate Tukey intervals, and red circles represent individual data. *P = 0.016, 1-way ANOVA (with Bonferroni comparison of means). ns, not significant at the 0.05 level.