Mid51 oligomerization domain mutant preferentially disrupts mitochondrial and lysosomal untethering dynamics. (A–C) Confocal time-lapse microscopy of mitochondria–lysosome (M-L) tethering (white arrows; inset in B) with corresponding linescan (C) showing prolonged M-L tethering duration by oligomerization domain mutant Mid51(R169W) in live HeLa cells (mitochondria mCherry-Mid51(R169W), lysosome LAMP1-mGFP). Scale bars, 5 μm (A); 0.5 μm (B). (D–F) Confocal time-lapse microscopy of inter-lysosomal (L-L) tethering (white arrows; inset in E) with corresponding linescan (F) showing prolonged L-L tethering duration by Mid51(R169W) in live HeLa cells (lysosome LAMP1-mGFP). Scale bars, 5 μm (D); 0.5 μm (E). (G and H) Quantification of percentage of lysosomes in mitochondria–lysosome (M-L) tethers (G) and L-L tethers (H) in Mid51(Y240N) and Mid51(R169W) conditions in live HeLa cells; n = 19 cells (Mid51(Y240N)); n = 15 cells (Mid51(R169W)). (I and J) Quantification in CRISPR-Cas9 genetically edited HCT116 mutant Mid51 cells of percentage of lysosomes in mitochondria–lysosome (M-L) tethers; I, n = 21 cells (Mid51(Y240N)); n = 18 cells (Mid51(R169W)); prolonged mitochondria–lysosome (M-L) tether duration by Mid51(R169W)); J; n = 38 events from 16 cells (Mid51(Y240N)); n = 36 events from 17 cells (Mid51(R169W)). Mean ± SEM; unpaired two-tailed t test (G–J); N.S., not significant (G–I); *, P = 0.019 (J).