Figure 2. Mitochondrial fragmentation... | Rockefeller University Press

Figure 2.

$Mitochondrial fragmentation and nucleoid segregation in germarium region 2A are essential for mtDNA selective inheritance. (A) Mitochondria labeled by ATPs staining and mtDNA labeled by TFAM-GFP in Dm germarium. The images are representative z-stack projections (1.5 µm) of a germarium acquired with STED microscopy. The developmental regions of germarium are indicated. Images are oriented with the germarium anterior toward the left. Scale bar, 10 µm. (B) Mitochondrial nucleoids labeled by TFAM-GFP and mitochondria labeled by ATPs staining in a germarium are displayed in magnified views. Both control and Fis1 knockdown ovaries driven by bam-gal4 are shown. The representative images of region 1 are from the anterior end, where GSCs or cystoblasts reside. The white-dashed outlines are elongated mitochondria containing multiple nucleoids. Arrowheads point to mitochondria without nucleoids. The arrow points to clustered mitochondria, which are excluded from data analyses. The ATPs staining was uneven, with less intensity in the regions where nucleoids are located. We defined two adjacent but distinct ATPs puncta as a single mitochondrion if they appeared in the same contour and were connected to one nucleoid (magenta dashed outlines). Scale bar, 2 µm. (C) The number of nucleoids per mitochondrion was determined using TFAM-GFP and ATPs staining shown in B in regions 1 (GSCs or cystoblasts) and 2A from control and Fis1 knockdown ovaries (n = 5 germaria for each genotype). The fraction of each group is shown. Note that the fraction of mitochondria containing multiple nucleoids was increased in region 2A of Fis1 knockdown driven by bam-gal4. (D) Quantification of mitochondrial nucleoid number (n = 5) and mtDNA copy number (n = 6) in each germ stem cell (GSC) in germarium. CV, coefficient of variance. (E) Knockdown of Fis1 in germarium region 2A, using a bam-Gal4 driver, compromised the selection against the mutant mtDNA in heteroplasmic mt:CoIT300I Dm. The proportion of mutant ts mtDNA in progeny was decreased by 15% compared with their mothers on average. In Fis1 knockdown fly, this negative selection was diminished. Data presented are means ± SD (n = 10 for each genotype). P < 0.005.$

Mitochondrial fragmentation and nucleoid segregation in germarium region 2A are essential for mtDNA selective inheritance. (A) Mitochondria labeled by ATPs staining and mtDNA labeled by TFAM-GFP in Dm germarium. The images are representative z-stack projections (1.5 µm) of a germarium acquired with STED microscopy. The developmental regions of germarium are indicated. Images are oriented with the germarium anterior toward the left. Scale bar, 10 µm. (B) Mitochondrial nucleoids labeled by TFAM-GFP and mitochondria labeled by ATPs staining in a germarium are displayed in magnified views. Both control and Fis1 knockdown ovaries driven by bam-gal4 are shown. The representative images of region 1 are from the anterior end, where GSCs or cystoblasts reside. The white-dashed outlines are elongated mitochondria containing multiple nucleoids. Arrowheads point to mitochondria without nucleoids. The arrow points to clustered mitochondria, which are excluded from data analyses. The ATPs staining was uneven, with less intensity in the regions where nucleoids are located. We defined two adjacent but distinct ATPs puncta as a single mitochondrion if they appeared in the same contour and were connected to one nucleoid (magenta dashed outlines). Scale bar, 2 µm. (C) The number of nucleoids per mitochondrion was determined using TFAM-GFP and ATPs staining shown in B in regions 1 (GSCs or cystoblasts) and 2A from control and Fis1 knockdown ovaries (n = 5 germaria for each genotype). The fraction of each group is shown. Note that the fraction of mitochondria containing multiple nucleoids was increased in region 2A of Fis1 knockdown driven by bam-gal4. (D) Quantification of mitochondrial nucleoid number (n = 5) and mtDNA copy number (n = 6) in each germ stem cell (GSC) in germarium. CV, coefficient of variance. (E) Knockdown of Fis1 in germarium region 2A, using a bam-Gal4 driver, compromised the selection against the mutant mtDNA in heteroplasmic mt:CoI^T300I Dm. The proportion of mutant ts mtDNA in progeny was decreased by 15% compared with their mothers on average. In Fis1 knockdown fly, this negative selection was diminished. Data presented are means ± SD (n = 10 for each genotype). P < 0.005.