Figure S7.
Rab7 and TBC1D15 localization and regulation of lysosomal networks by Fis1 and Mid51. (A) Quantification of lysosomes in mitochondria–lysosome (M-L) tether (left) or not in M-L tether (right) for the percentage of lysosomes positive for Rab7 in Fis1(WT) and Fis1(LA) conditions in live HeLa cells; n = 181 total lysosomes from 19 cells (Fis1(WT)); n = 170 total lysosomes from 13 cells (Fis1(LA)). (B) Quantification of lysosomes in mitochondria–lysosome (M-L) tether (left) or not in M-L tether (right) for the percentage of lysosomes positive for Rab7 in Mid51(Y240N) and Mid51(R169W) conditions in live CRISPR-Cas9 genetically edited HCT116 mutant Mid51 cells; n = 55 total lysosomes from 10 cells Mid51(Y240N)); n = 71 total lysosomes from 13 cells (Mid51(R169W)). (C–E) Localization of mitochondrial-targeted TBC1D15 in live HeLa cells (mCherry-tagged mitoTBC1D15 artificially targeted to the outer mitochondrial membrane via the TOM20 transmembrane domain) showing localization around the mitochondrial matrix (mEmerald-mito) with corresponding linescan (D), and linescan showing colocalization with the outer mitochondrial membrane (mEmerald-TOM20; E). (F and G) Quantification of percentage of lysosomes in inter-lysosomal (L-L) tether (F) and L-L tether duration (G) in Fis1(LA) live HeLa cells with or without mitoTBC1D15 (n = 75 events from 15 cells [− mitoTBC1D15]; n = 75 events from 15 cells [+ mitoTBC1D15]). (H and I) Quantification of percentage of lysosomes in L-L tether (H) and L-L tether duration (I) in live CRISPR-Cas9 genetically edited HCT116 mutant Mid51(R169W) cells with or without mitoTBC1D15 (n = 75 events from 15 cells [− mitoTBC1D15]; n = 75 events from 15 cells [+ mitoTBC1D15]). (J and K) Fis1(LA) oligomerization mutant does not regulate lysosomal acidification (percentage of lysosomes positive for LysoTracker; J) or lysosomal density (normalized to Fis1(WT); K). J, n = 20 cells (Fis1(WT)), n = 18 cells (Fis1(LA)); K, n = 15 cells (Fis1(WT)), n = 15 cells (Fis1(LA)). (L and M) Mid51(R169W) oligomerization mutant does not regulate lysosomal acidification (percentage of lysosomes positive for LysoTracker; L) or lysosomal density (normalized to Mid51(Y240N); M). L, n = 21 cells (Mid51(Y240N)); n = 25 cells (Mid51(R169W)); M, n = 15 cells (Mid51(Y240N)), n = 15 cells (Mid51(R169W)). (N–P) Quantification showing Drp1(K38A) mutant does not regulate lysosomal acidification (N) but disrupts lysosomal motility (O) and lysosomal cargo trafficking dynamics (P). N, n = 26 cells (Drp1(WT)); n = 19 cells (Drp1(K38A)); O, n = 73 events from 16 cells (Drp1(WT)); n = 74 events from 16 cells (Drp1(K38A)); P, n = 21 cells (30 min), 17 cells (1.5 h), 15 cells (4 h; Drp1(WT)); n = 17 cells (30 min), 23 cells (1.5 h), 16 cells (4 h; Drp1(K38A)). Mean ± SEM; unpaired two-tailed t test (A, B, and F–P); N.S., not significant (A, B, and F–N); *, P = 0.037 (O); ***, P < 0.001 (P).

Rab7 and TBC1D15 localization and regulation of lysosomal networks by Fis1 and Mid51. (A) Quantification of lysosomes in mitochondria–lysosome (M-L) tether (left) or not in M-L tether (right) for the percentage of lysosomes positive for Rab7 in Fis1(WT) and Fis1(LA) conditions in live HeLa cells; n = 181 total lysosomes from 19 cells (Fis1(WT)); n = 170 total lysosomes from 13 cells (Fis1(LA)). (B) Quantification of lysosomes in mitochondria–lysosome (M-L) tether (left) or not in M-L tether (right) for the percentage of lysosomes positive for Rab7 in Mid51(Y240N) and Mid51(R169W) conditions in live CRISPR-Cas9 genetically edited HCT116 mutant Mid51 cells; n = 55 total lysosomes from 10 cells Mid51(Y240N)); n = 71 total lysosomes from 13 cells (Mid51(R169W)). (C–E) Localization of mitochondrial-targeted TBC1D15 in live HeLa cells (mCherry-tagged mitoTBC1D15 artificially targeted to the outer mitochondrial membrane via the TOM20 transmembrane domain) showing localization around the mitochondrial matrix (mEmerald-mito) with corresponding linescan (D), and linescan showing colocalization with the outer mitochondrial membrane (mEmerald-TOM20; E). (F and G) Quantification of percentage of lysosomes in inter-lysosomal (L-L) tether (F) and L-L tether duration (G) in Fis1(LA) live HeLa cells with or without mitoTBC1D15 (n = 75 events from 15 cells [− mitoTBC1D15]; n = 75 events from 15 cells [+ mitoTBC1D15]). (H and I) Quantification of percentage of lysosomes in L-L tether (H) and L-L tether duration (I) in live CRISPR-Cas9 genetically edited HCT116 mutant Mid51(R169W) cells with or without mitoTBC1D15 (n = 75 events from 15 cells [− mitoTBC1D15]; n = 75 events from 15 cells [+ mitoTBC1D15]). (J and K) Fis1(LA) oligomerization mutant does not regulate lysosomal acidification (percentage of lysosomes positive for LysoTracker; J) or lysosomal density (normalized to Fis1(WT); K). J, n = 20 cells (Fis1(WT)), n = 18 cells (Fis1(LA)); K, n = 15 cells (Fis1(WT)), n = 15 cells (Fis1(LA)). (L and M) Mid51(R169W) oligomerization mutant does not regulate lysosomal acidification (percentage of lysosomes positive for LysoTracker; L) or lysosomal density (normalized to Mid51(Y240N); M). L, n = 21 cells (Mid51(Y240N)); n = 25 cells (Mid51(R169W)); M, n = 15 cells (Mid51(Y240N)), n = 15 cells (Mid51(R169W)). (N–P) Quantification showing Drp1(K38A) mutant does not regulate lysosomal acidification (N) but disrupts lysosomal motility (O) and lysosomal cargo trafficking dynamics (P). N, n = 26 cells (Drp1(WT)); n = 19 cells (Drp1(K38A)); O, n = 73 events from 16 cells (Drp1(WT)); n = 74 events from 16 cells (Drp1(K38A)); P, n = 21 cells (30 min), 17 cells (1.5 h), 15 cells (4 h; Drp1(WT)); n = 17 cells (30 min), 23 cells (1.5 h), 16 cells (4 h; Drp1(K38A)). Mean ± SEM; unpaired two-tailed t test (A, B, and F–P); N.S., not significant (A, B, and F–N); *, P = 0.037 (O); ***, P < 0.001 (P).

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