Figure 3.
Inter-lysosomal untethering is mediated by Rab7 GTP hydrolysis via mitochondrial TBC1D15 recruitment by Fis1 oligomers. (A–F) Confocal time-lapse microscopy showing increased inter-lysosomal (L-L) tether formation (white arrows; A and B) and prolonged tethering (white arrows; insets in C and D) upon inhibition of Rab7 GTP hydrolysis by constitutively active GTP-bound mutant Rab7(Q67L) in live HeLa cells (Rab7(WT)-GFP or Rab7(Q67L)-GFP). Corresponding linescans show L-L untethering for Rab7(WT) (E) vs. prolonged tethering for Rab7(Q67L) (F). Scale bars, 5 μm (A, B); 0.5 μm (C, D). (G and H) Quantification and histogram of prolonged L-L tethering duration for Rab7(Q67L) (n = 39 events from 13 cells (Rab7(WT)); n = 42 events from 14 cells (Rab7(Q67L)). (I–N) Confocal time-lapse microscopy showing increased L-L tether formation (white arrows; I and J) and prolonged tethering (white arrows; insets in K and L) by the Rab7-GAP mutant TBC1D15(D397A) that has defective GAP activity in live HeLa cells (lysosome LAMP1-mGFP). Corresponding linescans show L-L untethering for TBC1D15(WT) (M) vs. prolonged tethering for TBC1D15(D397A) (N). Scale bars, 5 μm (I and J); 0.5 μm (K and L). (O and P) Quantification and histogram of prolonged L-L tethering duration for TBC1D15(D397A) (n = 51 events from 17 cells (TBC1D15(WT)); n = 48 events from 16 cells (TBC1D15(D397A)). (Q–V) Confocal time-lapse microscopy showing increased L-L tether formation (white arrows; Q and R) and prolonged tethering (white arrows; insets in S and T) by mutant Fis1 (LA) that has defective oligomerization and is unable to recruit TBC1D15 to mitochondria in live HeLa cells (lysosome LAMP1-mGFP). Corresponding linescans show L-L untethering for Fis1(WT) (U) vs. prolonged tethering for Fis1(LA) (V). Scale bars, 5 μm (Q and R); 0.5 μm (S and T). (W and X) Quantification and histogram of prolonged L-L tethering duration for Fis1(LA) (n = 54 events from 18 cells (Fis1(WT)); n = 36 events from 12 cells (Fis1(LA)). Mean ± SEM; unpaired two-tailed t test (G, O, and W); ***, P < 0.001 (G and W); *, P = 0.0327 (O).