Fig. 5.

Mitophagy trafficking dynamics: mitophagy occurs piecemeal. (A) Isosurface object projections of confocal time-lapse imaging of fasted skeletal muscle in 5 dpf ubi:mito-Keima larva. Scale bar: 4 µm. (A′) Magnified view of region indicated in A; mitophagy occurs piecemeal. An EGFP–Lc3⁺ surface is interacting with a Keima440^high filamentous mitochondrion (86 min; *). Then, a small portion of the longer filamentous mitochondrion becomes Keima561^high, indicating pH change and mitophagy (89 min; *). Scale bars: 1 µm. (B) Maximum intensity projection (MIP) and isosurface object representations displaying healthy mitochondria (Keima488/561), mitolysosomes (Keima561/488), EGFP–Lc3⁺ objects, and LysoTracker Deep Red⁺ objects. Red dashed lines outline highlights an autolysosome containing degraded mitochondria. Yellow dashed lines outline highlights an autophagosome containing no mitochondria. Scale bars: 2 µm. (C) Classification of EGFP–Lc3⁺ objects as autophagosomes or autolysosomes. Those in contact with LysoTracker⁺ objects are autolysosomes, and the remaining are autophagosomes. (D) Volume quantification of autophagosomes and autolysosomes. (E) Colocalization analysis between mitolysosomes (Keima561^high objects) and autophagosomes or autolysosomes. (F) Colocalization analysis between healthy mitochondria and autophagosomes or autolysosomes. In D–F and I data are shown as mean± s.e.m. P-values were calculated with an unpaired, two-tailed Student's t-test. Δ, percentage change between autophagosomes and autolysosomes. n=4 individual 6 dpf larvae. (G,H) MIP of 7 dpf ubi:mito-Keima larvae injected with an inducible DN-Drp1 construct. Compare the piecemeal mitophagy pattern in the control to the wholesale mitophagy pattern in the injected (white dotted outlines). Mitochondrial morphology is also altered in DN-Drp1-injected larvae (arrows). Graph (H) depicts mitophagy index calculations from EGFP⁺;DN-Drp1 cells and paired, neighboring EGFP⁻;WT cells. Each dot is an individual cell for a total of 9 pairs of cells from three different animals. P, paired, two-tailed Student's t-test. Scale bars: 4 µm. (I) Colocalization analysis between mitolysosomes and Lc3⁺ objects.