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. Author manuscript; available in PMC: 2022 Aug 9.
Published in final edited form as: Cell Rep. 2022 Jul 12;40(2):111063. doi: 10.1016/j.celrep.2022.111063

Figure 2. C14* motif dictates differential sorting between the lysosomal and recycling pathways.

Figure 2.

(A) PM concentrations of GM1 C18:0 (turquoise, with C14* motif) and GM1 C18:1Δ9 (magenta, without C14* motif) as assessed by FACS using AlexaFluor-labeled streptavidin immediately after loading and after a 3-h chase. Quantification (right panel) with means ± SEMs, each dot equals 2 biological replicates measuring 20,000 cells each. ****p ≤ 0.0001 by unpaired t test.

(B) PM recycling as in (A) for the GM1 structural library (GM1 species with C14* motif in turquoise, GM1 species without C14* motif in magenta). Means ± SEMs. One-way ANOVA and Tukey’s multiple comparison.

(C) Venn diagram for ceramide acyl chain structure-dependent GM1 sorting in the endosomal system. Recycling GM1 species without C14* motif are in magenta and GM1 species entering the lysosomal pathway containing the C14* motif are in turquoise.

(D) Change in recycling rates under mildly cholesterol-depleting conditions (simvastatin treatment, 24 h, 10 μM). Recycling assay as in (A). ****p ≤ 0.0001 by 1-way ANOVA and Tukey’s multiple comparison.

(E) Fluid phase uptake by fluorescent dextran (left) and lysosomal trafficking of EGF (right) measured by FACS in A431 cells depleted or not of cholesterol as indicated (growth to over-confluency, growth in high-density lipoprotein (HDL)-depleted serum, 10 μM simvastatin, 100 nM T0901317 for 24 h, 1 μg/mL U18666A for 12 h, or using the respective CRISPR-Cas9 KO line). No differences were observed as measured by 1-way ANOVA and Tukey’s multiple comparison.

(F) Images of Tfn recycling tubules (red) in A431 cells treated or not with 2.5 mMβCD and GM1 C18:0 (green). Scale bars, 5 and 0.5 μm, respectively.

(G) Entry of GM1 C16:0 or C16:1Δ9 into recycling tubules in cholesterol-depleted A431 cells as indicated. Mean gray values of fluorescent GM1 in both endosomal vesicle and tubule were recorded, and the ratio of GM1 fluorescence in tubule over vesicle was calculated for each condition. Each dot represents one tubule versus vesicle ratio. Means ± SEMs. ****p ≤ 0.0001 by 1-way ANOVA and Tukey’s multiple comparison.