Figure 8.
Distinct Mid51 mutants differentially regulate lysosomal tethering and network dynamics. (A–F) Confocal time-lapse microscopy showing prolonged mitochondria-lysosomal (M-L) tether formation (white arrows; insets in C and D) with oligomerization domain mutant Mid51(R169W) in live HeLa cells compared with Drp1-binding domain mutant Mid51(Y240N) (mitochondria mCherry-Mid51(Y240N) or mCherry-Mid51(R169W), lysosome LAMP1-mGFP). Corresponding linescans show M-L untethering for Mid51(Y240N) (E) vs. prolonged tethering for Mid51(R169W) (F). Scale bars, 5 μm (A and B); 0.5 μm (C and D). Video 9 corresponds to D. (G and H) Quantification and histogram of prolonged mitochondria–lysosome (M-L) tethering duration for Mid51(R169W); n = 101 events from 18 cells (Mid51(Y240N)); n = 99 events from 15 cells (Mid51(R169W)). (I–N) Confocal time-lapse microscopy showing prolonged inter-lysosomal (L-L) tether formation (white arrows; insets in K and L) with oligomerization domain mutant Mid51(R169W) in live HeLa cells compared with dominant optic atrophy mutant Mid51(Y240N) (lysosome LAMP1-mGFP). Corresponding linescans show M-L untethering for Mid51(Y240N) (M) vs. prolonged tethering for Mid51(R169W) (N). Scale bars, 5 μm (I and J); 0.5 μm (K and L). Video 10 corresponds to L. (O and P) Quantification and histogram of prolonged L-L tethering duration for Mid51(R169W); n = 51 events from 19 cells (Mid51(Y240N)); n = 55 events from 13 cells (Mid51(R169W)). (Q and R) Quantification and corresponding histogram showing the motility of individual lysosomes is increased after an untethering event (Free lyso) compared with while in an inter-lysosomal tether (In L-L contact; n = 18 events from 7 cells). (S–U) Quantification showing Fis1 (LA) oligomerization mutant disrupts lysosomal motility (S), lysosomal distribution (T), and lysosomal cargo trafficking dynamics (U). S, n = 82 events from 16 cells (Fis1(WT)); n = 79 events from 17 cells (Fis1(LA)); T, n = 2,994 lysosomes from 20 cells (Fis1(WT)); n = 4,141 lysosomes from 20 cells (Fis1(LA)); U, n = 20 cells (30 min), 20 cells (1.5 h), 19 cells (4 h; Fis1(WT)); n = 18 cells (30 min), 18 cells (1.5 h), 18 cells (4 h; Fis1(LA)). (V–X) Quantification showing Mid51(R169W) oligomerization mutant disrupts lysosomal motility (V), lysosomal distribution (W), and lysosomal cargo trafficking dynamics (X). V, n = 79 events from 17 cells (Mid51(Y240N); n = 74 events from 16 cells (Mid51(R169W)); W, n = 1,846 lysosomes from 15 cells (Mid51(Y240N)); n = 2,191 lysosomes from 15 cells (Mid51(R169W)); X, n = 12 cells (30 min), 15 cells (1.5 h), 13 cells (4 h; Mid51(Y240N)); n = 25 cells (30 min), 25 cells (1.5 h), 21 cells (4 h; Mid51(R169W)). Mean ± SEM; unpaired two-tailed t test (G, O, and Q–X); ***, P < 0.001 (G,T,W, and X); **, P = 0.002 (O); *, P = 0.0383 (Q); *, P = 0.0281 (S); *, P = 0.0396 (U); **, P = 0.0027 (V); **, P = 0.0021 (X).