Figure 1.
Inter-lysosomal tethering modulates lysosomal network dynamics. (A and B) TEM of two lysosomes tethered together (L, arrows) in untreated HeLa cells. Inset (B) shows inter-lysosomal tether. Scale bars, 1 μm (A); 50 nm (B). (C–F) Super-resolution SIM of inter-lysosomal (L-L) tethering (white arrows, D) in live HeLa cells (LAMP1-mGFP). Inset (D) shows inter-lysosomal tether formation. Corresponding linescans before tether formation (precontact; t = 0 s) and subsequent tethering (contact; t = 7 s) are shown in E and F. Scale bars, 1 μm (C); 0.5 μm (D). Video 1 corresponds to D. (G) Quantification of percentage of lysosomes in an inter-lysosomal tether (duration >10 s) from confocal live-cell microscopy videos (n = 25 cells). (H) Quantification of minimum duration of inter-lysosomal tethering (n = 88 events from 25 cells). (I) Examples of SIM imaging of L-L tethers (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (J) SIM imaging of L-L tethering (white arrows) and subsequent L-L contact untethering (yellow arrow) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (K) Confocal microscopy image of lysosomes in live HeLa cells (LAMP1-mGFP) showing inset corresponding to O (t = 0 s). Scale bar, 5 μm. (L) The majority of inter-lysosomal tethers undergo untethering events rather than fusion within 120 s of initial contact formation (n = 64 events from 25 cells). (M and N) Rate of lysosomal untethering events vs. fusion events in live HeLa cells in events/min (M) and frequency of events over time of lysosomal untethering events vs. fusion events (%; N; n = 149 total events from 14 cells). (O–S) Confocal time-lapse microscopy of L-L tethering (white arrows) and subsequent untethering (yellow arrows) in live HeLa cells (LAMP1-mGFP). Scale bars, 0.5 μm. Video 2 corresponds to S. Mean ± SEM; unpaired two-tailed t test (L, M, and N); ***, P < 0.001 (L, M, and N).

Inter-lysosomal tethering modulates lysosomal network dynamics. (A and B) TEM of two lysosomes tethered together (L, arrows) in untreated HeLa cells. Inset (B) shows inter-lysosomal tether. Scale bars, 1 μm (A); 50 nm (B). (C–F) Super-resolution SIM of inter-lysosomal (L-L) tethering (white arrows, D) in live HeLa cells (LAMP1-mGFP). Inset (D) shows inter-lysosomal tether formation. Corresponding linescans before tether formation (precontact; t = 0 s) and subsequent tethering (contact; t = 7 s) are shown in E and F. Scale bars, 1 μm (C); 0.5 μm (D). Video 1 corresponds to D. (G) Quantification of percentage of lysosomes in an inter-lysosomal tether (duration >10 s) from confocal live-cell microscopy videos (n = 25 cells). (H) Quantification of minimum duration of inter-lysosomal tethering (n = 88 events from 25 cells). (I) Examples of SIM imaging of L-L tethers (white arrows) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (J) SIM imaging of L-L tethering (white arrows) and subsequent L-L contact untethering (yellow arrow) in live HeLa cells (LAMP1-mGFP). Scale bar, 0.5 μm. (K) Confocal microscopy image of lysosomes in live HeLa cells (LAMP1-mGFP) showing inset corresponding to O (t = 0 s). Scale bar, 5 μm. (L) The majority of inter-lysosomal tethers undergo untethering events rather than fusion within 120 s of initial contact formation (n = 64 events from 25 cells). (M and N) Rate of lysosomal untethering events vs. fusion events in live HeLa cells in events/min (M) and frequency of events over time of lysosomal untethering events vs. fusion events (%; N; n = 149 total events from 14 cells). (O–S) Confocal time-lapse microscopy of L-L tethering (white arrows) and subsequent untethering (yellow arrows) in live HeLa cells (LAMP1-mGFP). Scale bars, 0.5 μm. Video 2 corresponds to S. Mean ± SEM; unpaired two-tailed t test (L, M, and N); ***, P < 0.001 (L, M, and N).

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