Extended Data Fig. 6: Mitochondrial fragmentation is sufficient for germline mitochondrial DNA selection.
From: Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline
a, b, Stills of live images illustrating the effect that reducing the expression of Mitofusin in the germline (b) has on the morphology of mitochondria compared to controls (a). When mitofusin is knocked down (nos-GAL4 driving UAS-mfn shRNA2 TRiP.HMC0388332) the mitochondria in the stem cells are fragmented. The mitochondria (white) were labelled with a mitochondrially targeted eYFP and cell membranes (blue) were labelled with CellMask Deep Red Plasma membrane Stain. Stem cells and cysts are outlined in red. c, c′, c″, c‴, The knockdown of mitofusin in the germline by expressing mitofusin RNAi (nos-GAL4 driving UAS-mfn shRNA2 TRiP.HMC03883) results in selection for wild-type mtDNA (green) occurring in stem cells. The germarium was also reacted with anti-Vasa antiserum (c″) to mark the germline and delineate the stem cells and cysts. Wild-type D. yakuba mtDNA, greyscale in c, green in c‴; mutant D. melanogaster mtDNA, greyscale in c′, magenta in c‴. Mutant mtDNA is readily detected in the soma but not in the germline. d, Scatter plot comparing the percentage of mutant mtDNA, as assayed by qPCR, of ovaries in which mitofusin was weakly knocked down in the germline (Mfn KD; nos-GAL4 driving UAS-mfn long hairpin RNA1 TRiP.JF0165049; Ctrl, n = 23; Mfn KD, n = 24) and of ovaries in which Drp1 was overexpressed in the germline (Drp1 OE; nos-GAL4 driving UAS-Drp1.E; Ctrl, n = 11; Drp1 OE, n = 11). The percentage of mutant mtDNA in each case was normalized to the percentage of mutant mtDNA in control ovaries to illustrate that the overexpression of Drp1 enhances selection to a similar extent as does a weak reduction in the expression of Mitofusin. e, Scatter plot of the amount of mutant D. melanogaster (purple) and wild-type D. yakuba (green) mtDNA, as assayed by qPCR, in ovaries in which the expression of Mitofusin was weakly reduced (Ctrl, n = 20; Mfn OE, n = 21) or in which Drp1 was overexpressed in the germline (Ctrl, n = 10; Drp1 OE, n = 10), normalized to the amount of mutant and wild-type mtDNA in control ovaries (Ctrl) expressing mCherry RNAi in the germline. Reducing the expression of Mitofusin or overexpressing Drp1 results in a decrease in mutant mtDNA. f–h, The effect of germline overexpression of Mitofusin (Mfn) and Drp1 on copy number (f), ATP levels (g), and mitochondrial motility (h) in homoplasmic wild-type D. melanogaster ovaries (see Supplementary Note 1). f, Scatter plot of the amount of mtDNA, as assayed by qPCR, in homoplasmic ovaries in which Mfn (n = 24) or Drp1 (n = 24) was overexpressed in the germline, normalized to the amount of mtDNA in control ovaries (Ctrl; nos-GAL4 driving UAS-mCherry RNAi, n = 23). g, Scatter plot of the amount of ATP in homoplasmic ovaries overexpressing Mfn (n = 5) or Drp1 (n = 5) in the germline under control of Maternal α-Tubulin Gal4, normalized to the amount of ATP in control ovaries (Ctrl; Maternal α-Tubulin Gal4 driving UAS-mCherry RNAi, n = 5). h, Scatter plot of mitochondrial motility in homoplasmic ovaries overexpressing Mfn (n = 5) or Drp1 (n = 5) in the germline. Motility was assessed by measuring mean mitochondrial displacement using live confocal microscopy and Imaris analysis software.
