Figure 2.
Mitochondrial contacts promote inter-lysosomal untethering events. (A) Super-resolution SIM of lysosomes (LAMP1-mGFP) and mitochondria (mApple-TOM20) in live HeLa cells showing inset corresponding to C (t = 0 s). Scale bar, 5 μm. (B) Confocal microscopy of lysosomes (LAMP1-mGFP) and mitochondria (mApple-TOM20) in live HeLa cells showing inset corresponding to D (t = 0 s). Scale bar, 5 μm. (C) SIM imaging of inter-lysosomal (L-L) untethering (yellow arrow; bottom) event marked by mitochondria–lysosome (M-L) untethering event (yellow arrow; top) in live HeLa cell (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bar, 0.5 μm. Video 4 corresponds to C. (D) Confocal time-lapse microscopy showing lysosomal cluster of L-L tethers subsequently undergoing untethering events (yellow arrows; bottom) marked by mitochondria–lysosome (M-L) untethering (yellow arrows; top) in live HeLa cells (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bar, 0.5 μm. Video 5 corresponds to D. (E and F) Linescans showing inter-lysosomal tethering (L-L) and mitochondria–lysosome tethering (M-L) from SIM imaging corresponding to C (t = 84 s) before untethering events. (G) The majority of inter-lysosomal untethering events are marked by mitochondria (Observed), compared with mitochondrial localization by random chance (Expected) in live HeLa cells (n = 97 events from 24 cells). (H and I) Quantification of fate of L-L tethering (remain tethered or undergo untethering event) after 10 s of no mitochondrial tether (H; n = 18 total events from 11 cells (– mitochondria), or a mitochondria–lysosome (M-L) untethering event (I; n = 86 total events from 24 cells (M-L contact untether). (J and K) Histogram of L-L untethering event compared with mitochondria–lysosome (M-L) formation (J) or untethering (K; n >80 events from 24 cells). (L and M) Histogram of L-L initial formation compared with mitochondria–lysosome (M-L) formation (L) or untethering (M; n >80 events from 24 cells). Mean ± SEM; Fisher’s exact test (G), unpaired two-tailed t test (H and I); ***, P < 0.001 (G and H); *, P = 0.0171 (I).

Mitochondrial contacts promote inter-lysosomal untethering events. (A) Super-resolution SIM of lysosomes (LAMP1-mGFP) and mitochondria (mApple-TOM20) in live HeLa cells showing inset corresponding to C (t = 0 s). Scale bar, 5 μm. (B) Confocal microscopy of lysosomes (LAMP1-mGFP) and mitochondria (mApple-TOM20) in live HeLa cells showing inset corresponding to D (t = 0 s). Scale bar, 5 μm. (C) SIM imaging of inter-lysosomal (L-L) untethering (yellow arrow; bottom) event marked by mitochondria–lysosome (M-L) untethering event (yellow arrow; top) in live HeLa cell (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bar, 0.5 μm. Video 4 corresponds to C. (D) Confocal time-lapse microscopy showing lysosomal cluster of L-L tethers subsequently undergoing untethering events (yellow arrows; bottom) marked by mitochondria–lysosome (M-L) untethering (yellow arrows; top) in live HeLa cells (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bar, 0.5 μm. Video 5 corresponds to D. (E and F) Linescans showing inter-lysosomal tethering (L-L) and mitochondria–lysosome tethering (M-L) from SIM imaging corresponding to C (t = 84 s) before untethering events. (G) The majority of inter-lysosomal untethering events are marked by mitochondria (Observed), compared with mitochondrial localization by random chance (Expected) in live HeLa cells (n = 97 events from 24 cells). (H and I) Quantification of fate of L-L tethering (remain tethered or undergo untethering event) after 10 s of no mitochondrial tether (H; n = 18 total events from 11 cells (– mitochondria), or a mitochondria–lysosome (M-L) untethering event (I; n = 86 total events from 24 cells (M-L contact untether). (J and K) Histogram of L-L untethering event compared with mitochondria–lysosome (M-L) formation (J) or untethering (K; n >80 events from 24 cells). (L and M) Histogram of L-L initial formation compared with mitochondria–lysosome (M-L) formation (L) or untethering (M; n >80 events from 24 cells). Mean ± SEM; Fisher’s exact test (G), unpaired two-tailed t test (H and I); ***, P < 0.001 (G and H); *, P = 0.0171 (I).

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