Figure 6.
Drp1 GTP hydrolysis modulates lysosomal tethering dynamics. (A–F) Confocal time-lapse microscopy showing increased lysosomes in mitochondria–lysosome (M-L) tether formation (white arrows; A and B) and insets (white arrows; C and D) in live HeLa cells upon inhibition of Drp1 GTP hydrolysis by mutant Drp1 (K38A) (mitochondria mito-BFP [pseudocolored red]; lysosome LAMP1-mGFP). Corresponding linescans show M-L not in contact for Drp1(WT) (E) vs. M-L tethering for Drp1(K38A) (F). Scale bars, 5 μm (A and B); 0.5 μm (C and D). (G) Quantification of increased percentage of lysosomes in mitochondria–lysosome (M-L) tethers for Drp1(K38A); n = 21 cells (Drp1(WT)); n = 20 cells (Drp1(K38A)). (H) Quantification of increased percentage of lysosomes in inter-lysosomal (L-L) tethers for Drp1(K38A); n = 21 cells (Drp1(WT)); n = 20 cells (Drp1(K38A)). (I–P) Confocal time-lapse microscopy showing increased L-L tether formation (white arrows; I and J) and prolonged L-L tethering (white arrows; insets in K–N) in live HeLa cells expressing Drp1(WT) and Drp1(K38A) (lysosome LAMP1-mGFP). Corresponding linescans show prolonged L-L tethering for Drp1(WT) (O) and Drp1(K38A) (P). Scale bars, 5 μm (I and J); 0.5 μm (K–N). (Q) Quantification of mitochondria–lysosome (M-L) tethering duration for Drp1(WT) and Drp1(K38A); n = 38 events from 19 cells (control); n = 82 events from 21 cells (Drp1(WT)); n = 104 events from 19 cells (Drp1(K38A)). (R) Quantification of prolonged L-L tethering duration for Drp1(WT) and Drp1(K38A); n = 88 events from 25 cells (control); n = 36 events from 15 cells (Drp1(WT)); n = 58 events from 15 cells (Drp1(K38A)). Mean ± SEM; unpaired two-tailed t test (G and H), ANOVA with Tukey’s post hoc test (Q and R); N.S., not significant (Q); **, P = 0.0097 (G); *, P = 0.044 (H); ***, P < 0.001 (R).