Fig. 3. The Rab7 interactor ORP1L regulates the formation of ER-lysosome-mitochondria three-way contacts in Hela cells.

a Representative image of a cell expressing GFP-ORP1L and mApple-TOM20 acquired by SIM microscopy. White arrows show ORP1L positive lysosomes-mitochondria contacts. Scale bars: 10 µm and 1 µm (inset). b Representative image of two ORP1L positive lysosome-mitochondria contacts, indicated by white arrows, marked by the ER. Line-scan analysis of relative fluorescence intensities from the yellow line in merge is shown. Scale bar: 1 µm. c Representative time lapse image of ORP1L positive lysosomes forming stable contact with mitochondria indicated by white arrows on the first frame. Scale bar: 1 µm. d, e Percentage of Lamp1-mCherry and mCherry-ORP1L positive lysosomes in contact with mitochondria (d) and e minimum duration of these contacts in cells co-expressing mito-GFP. Cells from three independent experiments. Two-sided unpaired t-test. f, g Percentage of Lamp1-mCherry positive lysosomes in contact with mitochondria (mito-BFP) (f) and g minimum duration of these contacts in cells expressing GFP, GFP-ORP1L, or the D478A mutant. Cells from two independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. h–j Percentage of lysosome-mitochondria contacts that were marked by the ER: h when ORP1 expression was downregulated. Cells from three independent experiments, one-way ANOVA with Dunnett’s Multiple Comparison Test; i in ORP1L KO cells and control cells. Cells from three independent experiments, two-sided unpaired t-test; and j when VAPA and VAPB expression levels were downregulated. Data from three independent experiments, Two-sided unpaired t-test. k Cytosolic GAI-ΔANKORP1L can be recruited to GID1-Rab7 lysosomes upon GA₃-AM treatment. l Representative image of a cell expressing mTq2-GAI-ΔANKORP1L and iRFP-GID1-Rab7 before and after GA₃-AM treatment (10 µM). Scale bar: 10 µm. m Normalized fluorescence intensity of mTq2-GAI-ΔANKORP1L WT and D478A colocalizing with iRFP-GID1-Rab7 lysosomes at the indicated times before and after GA₃-AM treatment. Cells from four independent experiments. n Relative ER fluorescence intensity at lysosome-mitochondria contacts at the indicated times before and after GA₃-AM treatment (shaded area represent the area within one SEM). Cells from four independent experiments. Two-way ANOVA, Sidak’s multiple comparisons test. d–j, m, n All graphs show the mean ± SEM.