Figure S1.
EM and super-resolution imaging of inter-lysosomal tethering dynamics. (A) TEM of inter-lysosomal tethering (L) near mitochondria (M) in untreated HeLa cells. Scale bar, 50 nm. (B) Quantification of percentage of lysosomes in an inter-lysosomal tether (distance between lysosomal membranes <30 nm) from EM images (n = 38 cells). Mean ± SEM. (C and D) Super-resolution live-cell SIM of inter-lysosomal tethering (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bars, 0.5 μm. (E) Confocal time-lapse imaging of inter-lysosomal (L-L) tethering over time (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (F and G) Confocal time-lapse microscopy of lysosomal clusters composed of multiple inter-lysosomal tethers (white arrows, inset in G) which subsequently untether (yellow arrows, inset in G). Scale bars, 5 μm (F); 0.5 μm (G). Video 3 corresponds to G. (H–J) Quantification of state of inter-lysosomal tether (remain tethered or undergo untethering event) after 30 s (H), 60 s (I) or 120 s (J) of initial tether formation (n = 88 events from 25 cells). (K and L) Super-resolution live-cell SIM showing L-L untethering event marked by mitochondria–lysosome (M-L) untethering event (yellow arrows, inset in L) in live HeLa cells (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bars, 5 μm (K); 0.5 μm (L). (M–P) Linescans corresponding to L showing inter-lysosomal tethering (L-L) and mitochondria–lysosome tethering (M-L) at t = 0 s (M and N) followed by inter-lysosomal untethering and mitochondria–lysosome untethering events at t = 7 s (O and P; mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Mean ± SEM; unpaired two-tailed t test (H–J); N.S. not significant (I); *, P = 0.02 (H); *, P = 0.029 (J).

EM and super-resolution imaging of inter-lysosomal tethering dynamics. (A) TEM of inter-lysosomal tethering (L) near mitochondria (M) in untreated HeLa cells. Scale bar, 50 nm. (B) Quantification of percentage of lysosomes in an inter-lysosomal tether (distance between lysosomal membranes <30 nm) from EM images (n = 38 cells). Mean ± SEM. (C and D) Super-resolution live-cell SIM of inter-lysosomal tethering (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bars, 0.5 μm. (E) Confocal time-lapse imaging of inter-lysosomal (L-L) tethering over time (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (F and G) Confocal time-lapse microscopy of lysosomal clusters composed of multiple inter-lysosomal tethers (white arrows, inset in G) which subsequently untether (yellow arrows, inset in G). Scale bars, 5 μm (F); 0.5 μm (G). Video 3 corresponds to G. (H–J) Quantification of state of inter-lysosomal tether (remain tethered or undergo untethering event) after 30 s (H), 60 s (I) or 120 s (J) of initial tether formation (n = 88 events from 25 cells). (K and L) Super-resolution live-cell SIM showing L-L untethering event marked by mitochondria–lysosome (M-L) untethering event (yellow arrows, inset in L) in live HeLa cells (mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Scale bars, 5 μm (K); 0.5 μm (L). (M–P) Linescans corresponding to L showing inter-lysosomal tethering (L-L) and mitochondria–lysosome tethering (M-L) at t = 0 s (M and N) followed by inter-lysosomal untethering and mitochondria–lysosome untethering events at t = 7 s (O and P; mitochondria mApple-TOM20; lysosome LAMP1-mGFP). Mean ± SEM; unpaired two-tailed t test (H–J); N.S. not significant (I); *, P = 0.02 (H); *, P = 0.029 (J).

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