Figure 5.
Regulation of lysosomal tethering dynamics by Mid51 on mitochondria. (A–F) Confocal time-lapse microscopy showing increased mitochondria-lysosomal (M-L) tether formation (white arrows; A and B) and prolonged M-L tethering (white arrows; insets in C and D) in live HeLa cells expressing Mid51 (mitochondria mCherry-Mid51; lysosome LAMP1-mGFP) compared with Mff (mitochondria mCherry-Mff). Corresponding linescans show M-L untethering for Mff (E) vs. prolonged tethering for Mid51 (F). Scale bars, 5 μm (A and B); 0.5 μm (C and D). Video 6 corresponds to D, and Video 7 corresponds to C. (G) Quantification of increased percentage of lysosomes in M-L tethers for Mid51; n = 13 cells (Mff); n = 25 cells (Mid51). (H) Quantification of prolonged M-L tethering duration for Mid51; n = 68 events from 13 cells (Mff); n = 151 events from 25 cells (Mid51). (I–N) Confocal time-lapse microscopy showing lysosomes (I and J) and prolonged inter-lysosomal (L-L) tethering (white arrows; insets in K and L) in live HeLa cells expressing Mid51 (mCherry-Mid51; lysosome LAMP1-mGFP) compared with Mff (mCherry-Mff). Corresponding linescans show L-L untethering for Mff (M) vs. prolonged tethering for Mid51 (N). Scale bars, 5 μm (I and J); 0.5 μm (K and L). (O) Quantification of percentage of lysosomes in L-L tethers; n = 13 cells (Mff); n = 25 cells (Mid51). (P) Quantification of prolonged L-L tethering duration for Mid51; n = 40 events from 13 cells (Mff); n = 68 events from 21 cells (Mid51). Mean ± SEM; unpaired two-tailed t test (G, H, O, and P); N.S., not significant (O); *, P = 0.029 (G); **, P = 0.002 (H); *, P = 0.035 (P).

Regulation of lysosomal tethering dynamics by Mid51 on mitochondria. (A–F) Confocal time-lapse microscopy showing increased mitochondria-lysosomal (M-L) tether formation (white arrows; A and B) and prolonged M-L tethering (white arrows; insets in C and D) in live HeLa cells expressing Mid51 (mitochondria mCherry-Mid51; lysosome LAMP1-mGFP) compared with Mff (mitochondria mCherry-Mff). Corresponding linescans show M-L untethering for Mff (E) vs. prolonged tethering for Mid51 (F). Scale bars, 5 μm (A and B); 0.5 μm (C and D). Video 6 corresponds to D, and Video 7 corresponds to C. (G) Quantification of increased percentage of lysosomes in M-L tethers for Mid51; n = 13 cells (Mff); n = 25 cells (Mid51). (H) Quantification of prolonged M-L tethering duration for Mid51; n = 68 events from 13 cells (Mff); n = 151 events from 25 cells (Mid51). (I–N) Confocal time-lapse microscopy showing lysosomes (I and J) and prolonged inter-lysosomal (L-L) tethering (white arrows; insets in K and L) in live HeLa cells expressing Mid51 (mCherry-Mid51; lysosome LAMP1-mGFP) compared with Mff (mCherry-Mff). Corresponding linescans show L-L untethering for Mff (M) vs. prolonged tethering for Mid51 (N). Scale bars, 5 μm (I and J); 0.5 μm (K and L). (O) Quantification of percentage of lysosomes in L-L tethers; n = 13 cells (Mff); n = 25 cells (Mid51). (P) Quantification of prolonged L-L tethering duration for Mid51; n = 40 events from 13 cells (Mff); n = 68 events from 21 cells (Mid51). Mean ± SEM; unpaired two-tailed t test (G, H, O, and P); N.S., not significant (O); *, P = 0.029 (G); **, P = 0.002 (H); *, P = 0.035 (P).

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