Fig. 3. Overexpression of drp1 suppresses clu null mutant phenotypes in Drosophila.

a Survival of wild-type (black line) and clu^f04554 null mutant (red line) flies, as well as those with overexpression of clu (blue line) or drp (green line) in the clu^f04554 background. Expression of UAS-clu and UAS-drp1 were driven by Mef2-GAL4. Days with 50%, 25%, and 5% survival for these flies are shown in Supplementary Table 1. b Percentage of live flies on representative days. Statistical analysis of day-to-day survival rate is shown in Supplementary Table 2: overexpression of drp1 significantly increases the survival rates of clu^f04554 mutants throughout Day 4 to Day 16. As controls, the introduction of Mef2-GAL4 or UAS-drp1 alone into the clu^f04554 background does not result in significant differences in survival rates as compared to those of the clu^f04554 mutants (one-way ANOVA with post hoc Tukey’s HSD test, with P-values displayed in the graph. P < 0.05: significantly different from clu^f04554 mutants). c–e Confocal microscopy images of the thoracic muscle double-labeled with mitoGFP (green) and TUNEL (red). c’–e’ Toluidine Blue staining of plastic sections of embedded thoraces. c”–e” TEM ultrastructural images of thoracic muscle. Red dashed lines mark the boundaries of representative mitochondria. drp1 overexpression suppresses TUNEL-positive signals indicative of cell death in clu^f04554 mutants (c–e, quantified in f), largely restores tissue integrity (c’–e’, quantified in g) and mitochondrial cristae structure (c”–e”, quantified in h) in clu^f04554 mutants, and results in small mitochondrial size (e”). Scale bars: 5 μM in (c–e, c’–e’), 1 μM in (c”–e”). Expression of UAS-mitoGFP (c–e) and UAS-drp1 (e, e’, e”) were driven by Mef2-GAL4. f–h Quantification of muscle death (f), tissue disintegration (g), and damaged mitochondria with broken cristae (h). For each genotype, 3 different male flies were analyzed (mean ± SEM, n = 3, one-way ANOVA with post hoc Tukey’s HSD test). f, g For each male fly, 30–50 individual muscle pieces were analyzed, and the percentage of TUNEL-positive muscle (f) or that of disintegrated muscle (g) was calculated. h For each male fly, 30–50 individual mitochondria were analyzed, and the percentage of vacuolated mitochondria with broken cristae was calculated.