Fig. 2: Hypoxia promotes mitochondria-lysosome contacts and induces lysosome entering into megamitochondria.

a HeLa cells expressing LAMP1–mCherry, TOMM20-GFP and mito-BFP were treated with normoxia or hypoxia for 24 h, and analyzed by confocal microscopy. Representative images were shown, white arrowhead: lysosome. b, c HeLa cells were treated with normoxia or hypoxia for 24 h, and then immunostained with anti-LAMP1 and anti-TOMM20 antibodies, and analyzed by 3D imaging with confocal microscopy with Airyscan (b). The bottom images are mitochondria (green) and lysosome (red) which were displayed using 3D surface reconstructions of the middle images. The events of mitochondria-lysosome contacts (M-L contacts) and lysosome within megamitochondria (L within M) from 10 cells were then quantified (c) according to the criteria detailed in “Methods”, and the number of “M-L contacts” and “L within M” per 100 lysosomes was displayed. d, e HeLa cells treated with hypoxia (24 h) were analyzed by Focused ion beam/scanning electron microscopy (FIB-SEM), and representative images showing lysosome within mitochondria were displayed (d). 3D reconstruction and segmentation FIB-SEM images was shown (e). “M”: mitochondrion, “L”: lysosome. White, mitochondrial outer membrane; Cyan, mitochondrial inner boundary membrane; Purple Red, lysosome. f, g HeLa cells expressing LAMP1-GFP cells were treated with normoxia or hypoxia for 24 h, and then were fixed for GFP immunogold staining and immunoelectron microscopy analysis. red arrowhead: immuno-gold particle (f). The number of mitochondria and lysosomes containing immunogold particles was quantified, and 100 mitochondria or 100 lysosomes were analyzed for each experiment (g). h, i HeLa cells expressing LAMP1–mCherry (red) and mito-GFP (green) were treated with normoxia (h) or hypoxia (i) for 24 h, and then were tracked by time-lapse imaging with a confocal microscopy. Representative time-lapse confocal 3D images in living HeLa cells were displayed (n = 30 examples from 21 cells). j HeLa cells were exposed to normoxia or hypoxia for 24 h, then were fixed by high pressure freezing and analyzed by TEM. The red “L”: lysosome, the purple “M”: mitochondrion. All bars represent mean ± SEM, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA. P values are indicated in the figure. Source data are provided as a Source Data file.