Figure S2.
Rab7 GTP hydrolysis and Drp1 GTP hydrolysis machinery regulate inter-lysosomal tethers. (A) Percentage of inter-lysosomal (L-L) untethering events marked by no mitochondria (− Mito) or by mitochondria in contact with at least one of the two lysosomes (+ Mito; n = 97 total events from 24 cells). (B) Mitochondria–lysosome (M-L) untethering is more closely coupled to L-L untethering vs. formation (n = 86 total events from 24 cells). (C) Mitochondria–lysosome (M-L) formation is more closely coupled to L-L formation vs. untethering (n = 80 total events from 24 cells). (D) Quantification of increased percentage of lysosomes in inter-lysosomal tether upon inhibition of Rab7 GTP hydrolysis by constitutively active GTP-bound mutant Rab7(Q67L) in live HeLa cells; n = 25 cells (Control); n = 13 cells (Rab7(WT)); n = 15 cells (Rab7(Q67L)). (E) Quantification of increased percentage of lysosomes in inter-lysosomal tether by the Rab7-GAP mutant TBC1D15(D397A) that has defective GAP activity in live HeLa cells; n = 18 cells (TBC1D15(WT)); n = 24 cells (TBC1D15(D397A)). (F) Quantification of increased percentage of lysosomes in inter-lysosomal tether by mutant Fis1 (LA) that has defective oligomerization and is unable to recruit TBC1D15 to mitochondria in live HeLa cells; n = 26 cells (Fis1(WT)); n = 16 cells (Fis1(LA)). (G and H) Quantification of percentage of lysosomes in mitochondria–lysosome (M-L) tethers (G) and mitochondria–lysosome (M-L) tethering duration (H) for SKIP(WT) and mutants SKIP(AAA) and SKIP(ΔRUN); n = 75 events from 15 cells (SKIP(WT)); n = 80 events from 16 cells (SKIP(AAA)); n = 75 events from 14 cells (SKIP(ΔRUN)). (I and J) Quantification of percentage of lysosomes in L-L tethers (I) and L-L tethering duration (J) for SKIP(WT) and mutants SKIP(AAA) and SKIP(ΔRUN); n = 75 events from 15 cells (SKIP(WT)); n = 71 events from 16 cells (SKIP(AAA)); n = 75 events from 14 cells (SKIP(ΔRUN)). (K and L) Loss of Fis1 disrupts inter-lysosomal contact formation (K) and tethering duration (L); n = 31 events from 10 cells (WT HCT116); n = 75 events from 15 cells (Fis1−/− HCT116). (M and N) Loss of Mid51 disrupts mitochondria–lysosome contact formation (M) and tethering duration (N); n = 67 events from 15 cells (WT HeLa); n = 65 events from 15 cells (Mid51−/− HeLa). (O and P) Loss of Mid51 disrupts inter-lysosomal contact formation (O) and tethering duration (P); n = 55 events from 15 cells (WT HeLa); n = 65 events from 15 cells (Mid51−/− HeLa). (Q and R) Loss of Drp1 and Mff disrupt mitochondria–lysosome contact tethering duration (R) but not contact formation (Q); n = 47 events from 10 cells (WT HCT116); n = 55 events from 11 cells (Drp1−/− HCT116); n = 75 events from 15 cells (Mff−/− HCT116). (S and T) Loss of Drp1 and Mff disrupt inter-lysosomal contact formation (S) and tethering duration (T); n = 31 events from 10 cells (WT HCT116); n = 55 events from 11 cells (Drp1−/− HCT116); n = 72 events from 15 cells (Mff−/− HCT116). Mean ± SEM; unpaired two-tailed t test (A–C, E, F, and K–P); ANOVA with Tukey’s post hoc test (D, G–J, and Q–T); N.S. not significant (G–J); ***, P < 0.001 (A–C and E); *, P = 0.02 (D); *, P = 0.02 (F); ***, P = 0.0005 (K); ***, P = 0.0006 (L); *, P = 0.0102 (M); **, P = 0.0058 (N); ***, P = 0.0004 (O); ***, P = 0.0003 (P); *, P = 0.031 (R, WT vs. Drp1−/−); **, P = 0.0061 (R, WT vs. Mff−/−); *, P = 0.0105 (S, WT vs. Drp1−/−); *, P = 0.0305 (S, WT vs. Mff−/−); **, P = 0.0054 (T, WT vs. Drp1−/−); **, P = 0.0064 (T, WT vs. Mff−/−).

Rab7 GTP hydrolysis and Drp1 GTP hydrolysis machinery regulate inter-lysosomal tethers. (A) Percentage of inter-lysosomal (L-L) untethering events marked by no mitochondria (− Mito) or by mitochondria in contact with at least one of the two lysosomes (+ Mito; n = 97 total events from 24 cells). (B) Mitochondria–lysosome (M-L) untethering is more closely coupled to L-L untethering vs. formation (n = 86 total events from 24 cells). (C) Mitochondria–lysosome (M-L) formation is more closely coupled to L-L formation vs. untethering (n = 80 total events from 24 cells). (D) Quantification of increased percentage of lysosomes in inter-lysosomal tether upon inhibition of Rab7 GTP hydrolysis by constitutively active GTP-bound mutant Rab7(Q67L) in live HeLa cells; n = 25 cells (Control); n = 13 cells (Rab7(WT)); n = 15 cells (Rab7(Q67L)). (E) Quantification of increased percentage of lysosomes in inter-lysosomal tether by the Rab7-GAP mutant TBC1D15(D397A) that has defective GAP activity in live HeLa cells; n = 18 cells (TBC1D15(WT)); n = 24 cells (TBC1D15(D397A)). (F) Quantification of increased percentage of lysosomes in inter-lysosomal tether by mutant Fis1 (LA) that has defective oligomerization and is unable to recruit TBC1D15 to mitochondria in live HeLa cells; n = 26 cells (Fis1(WT)); n = 16 cells (Fis1(LA)). (G and H) Quantification of percentage of lysosomes in mitochondria–lysosome (M-L) tethers (G) and mitochondria–lysosome (M-L) tethering duration (H) for SKIP(WT) and mutants SKIP(AAA) and SKIP(ΔRUN); n = 75 events from 15 cells (SKIP(WT)); n = 80 events from 16 cells (SKIP(AAA)); n = 75 events from 14 cells (SKIP(ΔRUN)). (I and J) Quantification of percentage of lysosomes in L-L tethers (I) and L-L tethering duration (J) for SKIP(WT) and mutants SKIP(AAA) and SKIP(ΔRUN); n = 75 events from 15 cells (SKIP(WT)); n = 71 events from 16 cells (SKIP(AAA)); n = 75 events from 14 cells (SKIP(ΔRUN)). (K and L) Loss of Fis1 disrupts inter-lysosomal contact formation (K) and tethering duration (L); n = 31 events from 10 cells (WT HCT116); n = 75 events from 15 cells (Fis1−/− HCT116). (M and N) Loss of Mid51 disrupts mitochondria–lysosome contact formation (M) and tethering duration (N); n = 67 events from 15 cells (WT HeLa); n = 65 events from 15 cells (Mid51−/− HeLa). (O and P) Loss of Mid51 disrupts inter-lysosomal contact formation (O) and tethering duration (P); n = 55 events from 15 cells (WT HeLa); n = 65 events from 15 cells (Mid51−/− HeLa). (Q and R) Loss of Drp1 and Mff disrupt mitochondria–lysosome contact tethering duration (R) but not contact formation (Q); n = 47 events from 10 cells (WT HCT116); n = 55 events from 11 cells (Drp1−/− HCT116); n = 75 events from 15 cells (Mff−/− HCT116). (S and T) Loss of Drp1 and Mff disrupt inter-lysosomal contact formation (S) and tethering duration (T); n = 31 events from 10 cells (WT HCT116); n = 55 events from 11 cells (Drp1−/− HCT116); n = 72 events from 15 cells (Mff−/− HCT116). Mean ± SEM; unpaired two-tailed t test (A–C, E, F, and K–P); ANOVA with Tukey’s post hoc test (D, G–J, and Q–T); N.S. not significant (G–J); ***, P < 0.001 (A–C and E); *, P = 0.02 (D); *, P = 0.02 (F); ***, P = 0.0005 (K); ***, P = 0.0006 (L); *, P = 0.0102 (M); **, P = 0.0058 (N); ***, P = 0.0004 (O); ***, P = 0.0003 (P); *, P = 0.031 (R, WT vs. Drp1−/−); **, P = 0.0061 (R, WT vs. Mff−/−); *, P = 0.0105 (S, WT vs. Drp1−/−); *, P = 0.0305 (S, WT vs. Mff−/−); **, P = 0.0054 (T, WT vs. Drp1−/−); **, P = 0.0064 (T, WT vs. Mff−/−).

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