Figure 4.
ER-LE MCSs impact fusion activity—ORP1L. (A) Confocal analysis of ORP1L -wt, -∆ORDPHDPHD or -∆ORD (red) expression in CD63-pHluorin (green) HeLa cells. (B) Correlative Light-Electron Microscopy (CLEM) on section on CD63-pHluorin cells transfected with ORP1L -wt or -∆ORD and post-stained with DAPI. Top panel: large overview, with squares indicating zoom areas depicted right below. Subsequent panels below: zoom of boxes indicated in top panel and Fig. S1, B and C, respectively, with below same pictures including transparent orange and blue masks indicate ER and MVB, respectively. (C) Immunofluorescent labeling for LAMP1 (blue) in CD63-pHluorin (green) HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. (D) Quantification of CD63 overlaps with LAMP1 in cells as in C. Measured over >4 cells in n = 3 experiments using Mander’s Overlap Coefficient. (E) Fusion activity of CD63-pHluorin HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Measured over >60 cells from n > 3 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (Fand G) Western blotting analysis of cell lysates and small EVs isolated from CD63-pHluorin HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Multiple comparisons using ordinary one-way ANOVA. (H) Still of Video 4 of CD63-eGFP HeLa cells transfected with ORP1L-∆ORD-RFP. (I) Motility of CD63 compartments in CD63-eGFP HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Student’s two-tailed two-sample t test, red line indicates median. n = 40–70 organelles. (J) Fusion activity of CD63-pHluorin HeLa cells transfected with HA-ORP1L-wt, -FFAT or control (ctrl). Measured over >100 cells from n > 4 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (K and L) Fusion activity of CD63-pHluorin HeLa cells overexpressing RILP (K) or SKIP (L). Measured over >37 cells from n > 2 independent experiments using Student’s two-tailed two-sample t test. Red line indicates median. Bars: 5 µm, unless stated otherwise. Source data are available for this figure: SourceData F4.

ER-LE MCSs impact fusion activity—ORP1L. (A) Confocal analysis of ORP1L -wt, -∆ORDPHDPHD or -∆ORD (red) expression in CD63-pHluorin (green) HeLa cells. (B) Correlative Light-Electron Microscopy (CLEM) on section on CD63-pHluorin cells transfected with ORP1L -wt or -∆ORD and post-stained with DAPI. Top panel: large overview, with squares indicating zoom areas depicted right below. Subsequent panels below: zoom of boxes indicated in top panel and Fig. S1, B and C, respectively, with below same pictures including transparent orange and blue masks indicate ER and MVB, respectively. (C) Immunofluorescent labeling for LAMP1 (blue) in CD63-pHluorin (green) HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. (D) Quantification of CD63 overlaps with LAMP1 in cells as in C. Measured over >4 cells in n = 3 experiments using Mander’s Overlap Coefficient. (E) Fusion activity of CD63-pHluorin HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Measured over >60 cells from n > 3 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (Fand G) Western blotting analysis of cell lysates and small EVs isolated from CD63-pHluorin HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Multiple comparisons using ordinary one-way ANOVA. (H) Still of Video 4 of CD63-eGFP HeLa cells transfected with ORP1L-∆ORD-RFP. (I) Motility of CD63 compartments in CD63-eGFP HeLa cells transfected with ORP1L -wt, -∆ORDPHDPHD or -∆ORD. Student’s two-tailed two-sample t test, red line indicates median. n = 40–70 organelles. (J) Fusion activity of CD63-pHluorin HeLa cells transfected with HA-ORP1L-wt, -FFAT or control (ctrl). Measured over >100 cells from n > 4 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (K and L) Fusion activity of CD63-pHluorin HeLa cells overexpressing RILP (K) or SKIP (L). Measured over >37 cells from n > 2 independent experiments using Student’s two-tailed two-sample t test. Red line indicates median. Bars: 5 µm, unless stated otherwise. Source data are available for this figure: SourceData F4.

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