Fig 2: CERT inhibitor HPA-12 reduces production and sphingolipid content of EVs. (a) Fluorescent HPA-12 (HPA-12-NBD) chemical structure. Single plane photomicrograph of fluorescence live microscopy of HPA-12-NBD-treated (green) and tdTomato-CD9 (red) transfected cells showing partial colocalization. Scale bar 10 µm. b) 100k EV numbers measured by NTA and normalized to cells after 24 h treatment with syn-HPA-12 or the less potent stereoisomer anti-HPA-12. Bar graph shows average ± SEM of N = 3/group. One-way ANOVA, Tukey posthoc testing (*p < 0.05). (c) Quantification of CD81 and Tsg101 by Western blots. Bar graphs show average ± SEM of N = 4/group. One-way ANOVA, Tukey posthoc testing *p < 0.05. (d) Cer d18:1/16:0 and SM d18:1/16:0 levels measured in the 100k EVs fraction. Bar graphs show average ± SEM with N = 3/group. Unpaired t-test (*p < 0.05). (e) 100k EVs numbers measured by NTA and normalized to cells after 24 h treatment with syn-HPA-12 and/or Cer d18:1/16:0. Bar graph shows average ± SEM with N = 3/group. One-way ANOVA, Tukey's posthoc testing (*p < 0.05). (f) Quantification of CD81, Tsg101 and Alix by Western blots. Bar graphs show average ± SEM of two independent experiments with each N = 4/group. One-way ANOVA, Tukey's posthoc testing *p < 0.05. (g) Representative photomicrographs of Tsg101 and CERT PLA signals after treatment with vehicle or synHPA-12 (8 µM). Scale bar 10 µm. (h). Box and Whiskers plot of PLA signals normalized to the number of nuclei of three independent experiments including a total of 12–20 pictures/condition. Unpaired t-test (***p < 0.001).

Fig 3: PI4P produced by PI4KIIa controls a direct ceramide transport from the ER to the MVE and CERT-mediated EV generation. (a) Confocal photomicrographs displaying immunolabeled CERT (red) partially colocalizing with the ER immunolabeled with Calnexin (green) and MVE immunolabeled with LBPA (magenta) at contact sites. (b) Linescan analyses with fluorescence intensities of the ER (Calnexin), MVE (LPBA) and CERT along the arrow shown in A. (c) TEM image of Immunogold labelling of CERT in the limiting membrane of MVE and in proximity to ER. Arrow shows labelling on limiting membrane of MVE. (d) Confocal photomicrographs showing BODIPY FL C5-Cer redistribution to the Golgi region without or with PI4KIIa inhibitor (NC03) 10 µM or 5 µM. HeLa CERT-deficient cell line was used as negative control. (e) Quantification of the relative MFI measured at the Golgi region after BODIPY FL C5-Cer incubation in absence or presence of 10 µM or 5 µM of PI4KIIa inhibitor. Bar graph represents average ± SEM of N = 8–18/group. One-way ANOVA, Tukey's posthoc testing (*p < 0.05; **p < 0.01). (f) Number of EVs in the 100k fraction measured by NTA after 24 h incubation with vehicle, 50 nM of PI4KIIIß inhibitor or with 5 µM of PI4KIIa inhibitor (NC03). Bar graph represents average ± SEM of N = 3/group. One-way ANOVA, Sidak's posthoc testing (*p < 0.05; **p < 0.01). (g) Quantification of Flotillin-1 by Western blots. Bar graphs represent average ± SEM of N = 3/group. (h) Cer d18:1/16:0 and SM d18:1/16:0 levels measured in the 100k × g EV fraction or cell lysate after 24 h incubation with vehicle, 50 nM of PI4KIIIß inhibitor or with 5 µM of PI4KIIa inhibitor (NC03). Bar graph shows average ± SEM with N = 3/group. One-way ANOVA, Sidak's posthoc testing (*p < 0.05).

Fig 4: Schematic representation of CERT function in EV biogenesis. There are two routes for CERT-mediated EV biogenesis. The indirect pathway via the trans-Golgi network which is regulated by PI4P generated by PI4PIIIß and involves SM production. This pathway is related to SMS1, SMS2 and N-Smase-2 dependent EV formation. The direct pathway is located in ER-endosomes/MVE contact sites. The enzyme PI4PIIa regulates CERT transfer of ceramide to the MVE. At the MVE, a complex is formed by Tsg101 and CERT, which requires ceramide.

Fig 5: CERT is associated with the MVE and interacts with Tsg101. (a) Confocal photomicrograph of fluorescent labelling of CERT (green) showing partial colocalization/juxtaposition (white) with EEA1 or LBPA (magenta) in N2a cells. Scale bar 5 µm. (b) CERT detected by immunoblot in the same fractions of Alix after EV purification by sucrose gradient ultracentrifugation 0.29–1.75M. (c) Confocal photomicrograph of PLA assay (green) in tdTomato-CD9 (red) transfected N2a cells indicating complex formation between Tsg101 and CERT (arrow). DAPI (blue) was used to stain the nuclei. Scale bar 5 µm. (d) Binding kinetics of rTsg101 to immobilized rCERTL measured by ELISA. The Michaelis-Menten constant (Km) equalled 3.59nM with Vmax equal to 22.7nM. (e) Representative photomicrographs of Tsg101 and CERT PLA signals (red), after treatment with vehicle and 10 µM FB1. Scale bar 5 µm. (f) Box and Whiskers plot of 14–23 pictures per condition, of n = 3 independent experiments, showing PLA signals normalized to the number of nuclei (blue). Unpaired t-test (*p < 0.05). (g) AlphaFold2 highest-ranked structure prediction of the complex between (a) the human START domain of CERT (green A) and the human UEV domain of Tsg101 (chain coloured in light blue B). In (b) the coloured polymers based on pLDDT (score = 93.3) and in (c) PAE plot (pTMscore = 0.742). In (d) the 3D structure of the complex (START domain in green A and UEV domain in magenta B) and in (e) the colour surface electrostatic potential of the complex.