Figure 1. Characterization of
aco-2:gfp C elegans strain:(A) Confocal imaging of five (aligned) Day 3 adults, showing ACO-2::GFP fluorescence throughout the worms and their developing germline (scale bar 100 µm).(B) ACO-2::GFP (green) and MitoTracker Red (red) staining in Day 5 (top panels) and Day 11 (bottom panels) worms. In Day 5 worms, colocalization (yellow) is pronounced, but by Day 11 there are large portions of the worm devoid of MitoTracker Red staining (arrow). Top panel scale bar 20 µm and bottom panel scale bar 50 µm.(C) Fragmentation and rounding of the mitochondrial network upon aging. (i.) Confocal images (max projections) of the tail region of
aco-2::gfp worms (green top panels) across representative ages: Day 1 (left panels); Day 7 (middle panels); and Day 17 (right panels). Binary images help visualize the mitochondrial network (black and white middle panels), and individual mitochondrial 'objects' were identified using an automated image analysis pipeline (red outlines, bottom panels), and image insets in the top right corner show a zoom-in of corresponding 'example objects' indicated with colored arrows (Scale bar = 20 µm.) (ii.) A density-plot of object eccentricity across age. For each mitochondrial object 'eccentricity' was measured, where eccentricity values approaching 1 correspond to shapes which are more 'elongated' (vs. more 'rounded'-where a perfectly circular object has a value of 0). Dashed vertical lines identify the mean value for object eccentricity at Day 1 (green line); Day 7 (orange line); and Day 17 (purple line). Example objects from Day 1 (green object), Day 7 (orange object), and Day 17 (purple object) are displayed on the upper left side of the graph. (iii.) Bar graph of the percent of 'elongated objects' (those with an eccentricity value between 0.9-1.0). This demonstrates that there is a significant and progressive drop in the proportion of mitochondria that have an elongated/tubular morphology from Day 1 (green bar) to Day 7 (orange bar) to Day 11 (purple bar), consistent with the mitochondrial network becoming more fragmented and rounded as the worms age. Data is plotted as the mean ± SEM, each point is a worm, n = 3 - 20 worms, p values calculated using Kruskal-Wallis test, p = 0.0009.(D) Fragmentation and rounding of the mitochondrial network upon paraquat (PQ) exposure. (i.) Confocal images (max projections) of the tail region of
aco-2::gfp worms (green top panels) after exposure to PQ for 48 hours starting at Day 1: 0 mM (left panels); 5 mM (middle panels); and 10 mM (right panels). As in panel 'C', binary images are shown (middle panels), as are identified mitochondrial 'objects' (bottom panels). (Scale bar = 20 µm.) (ii.) A density plot of object eccentricity upon PQ exposure. For each mitochondrial object 'eccentricity' was measured as described in panel 'C'. Dashed vertical lines identify the mean value for object eccentricity with 0 mM (green line); 5 mM (orange line); and 10 mM (purple line). Example objects from 0 mM (green object), 5 mM (orange object), and 10 mM (purple object) are displayed on the upper left side of the graph. (iii.) Bar graph showing the percent of 'elongated objects', demonstrating that PQ causes a significant reduction in the proportion of mitochondria that have an elongated/tubular morphology: 0 mM (green bar); 5 mM (orange bar); 10 mM (purple bar). Data is plotted as the mean ± SEM, each point is a worm, n = 3 - 4 worms, p values calculated using Kruskal-Wallis test, p = 0.036.