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Figures
- Fig. 1.
Genotype and phenotype of sesB1 flies. (A) Aligned partial sequences of sesB mRNA, isoform A (sesB+), commencing at nucleotide 962 of NCBI database entry NM_078554, and human ANT1 mRNA (NCBI database entry NM_001151 from nucleotide 863). The corresponding amino acid sequences and the base substitutions (red) and corresponding amino acid changes in the sesB1 allele, verified in the strain analyzed here, are also shown. The ANT1 gene mutation shown here is that reported in an Italian family with adPEO (Kaukonen et al., 2000). Each mutation results in the substitution of a hydrophobic residue in transmembrane segment VI of the protein (Pebray-Peyroula et al., 2003) by another hydrophobic residue. (B) The day of eclosion of flies of the genotypes indicated; means±s.e.m. are shown from five replicate vials. FM7 is the X-chromosomal balancer chromosome used to maintain heterozygosity for sesB1. (C) The square-root of the time taken to recover from mechanical shock (‘bang sensitivity’) for batches of ≥25 flies of each sex and genotype that were individually analyzed; means±s.e.m. are shown. The day of eclosion and bang sensitivity of sesB1 flies were significantly different from those of wild-type (wt) or heterozygous flies of the same sex (P<0.01). (D) Response of adult males of the indicated strains to sound. The mean number of responders±s.d. amongst groups of six males from three replicate groups of each genotype are shown. Bang-sensitive strains are: tko25t, point mutant of technical knockout, which encodes mitoribosomal protein S12 (Shah et al., 1997); bss, bang senseless, a point mutant of paralytic, which encodes a voltage-gated Na+ channel (Parker et al., 2011); sesB1; and sda, slamdance, which encodes aminopeptidase N (Zhang et al., 2002). tko25t, sesB1 and sda are non-responsive, whereas bss has normal hearing. (E) Lifespan curves for sesB1 and wild-type control flies of the sexes indicated (groups of 100 flies of each sex and genotype) when maintained at 25°C. Three replicate experiments gave similar results – i.e. mean lifespans of 11–13 days for sesB1 males and 13–18 days for sesB1 females. (F) GFP that was targeted to the mitochondria was expressed in indirect flight muscles of wild-type and sesB1 flies (in both genotypes in the presence of a transgene encoding UAS-mito-HA-GFP and the Mef2-GAL4 driver). The mitochondrial network appears normal in sesB1. (G) Dark-field micrograph of the dissected ovaries from 2-day-old wild-type and sesB1 females. (H) Phase-contrast micrograph of developing embryos from eggs laid by sesB1 females. The vast majority of embryos failed to hatch, and only a tiny number (~0.1%) completed development.
- Fig. 2.
Mitochondrial metabolism in sesB1 flies. (A) Apparent respiratory control ratio (RCR; the relative rate of oxygen consumption before and after the addition of ADP) of isolated mitochondria from flies of the strains and sexes indicated, using different substrate mixes, based on the data shown in Table 1. Note that the value obtained with the pyruvate and proline substrate mix is not a true RCR, because of the secondary activation of isocitrate dehydrogenase by ADP (Miwa et al., 2003). However, a similar trend is observed with both substrate mixes. (B) Steady-state ATP levels in the extracts from flies of the strains and sexes indicated, normalized to the value in wild-type (wt) flies in the given sex. The means of results from five or six biological replicates for each group are shown. (C) Steady-state lactate levels in extracts from 10-day-old flies of strains and sexes indicated. n=6 for males, n=3 for females. (A–C) Means±s.d., *P<0.05, **P<0.01 by using Student’s t-test. (D) Representative blue native electrophoresis gels, which were stained histochemically for complex I and IV, of extracts from flies of the strains and sexes indicated. Arrows indicate the major complexes that were stained; their apparent molecular weights were extrapolated from the migration of markers.
- Fig. 3.
Summary of transcriptomic analysis of sesB1 flies. (A–D) Diagrammatic representation of degree of overlap between probesets that had altered expression from wild type; fly strains and sexes are indicated. For details, see supplementary material Tables S1–S7. (E) GO classification by molecular function of the genes that showed altered expression in sesB1 flies of both sexes (panel A; supplementary material Table S1).
- Fig. 4.
Testing rescue of sesB1 by altered mtDNA background or spargel overexpression. (A,B) The day of eclosion of flies of the genotypes and sexes indicated is shown; means±s.d. from three replicate experiments. FM7 is the X-chromosomal balancer chromosome used to maintain heterozygosity for sesB1, which gives a wild-type phenotype. BER-1;sesB1 denotes cybrid flies (BER-1 mtDNA, sesB1 mutation and nuclear background). srlGR (an additional genomic copy of spargel) was homozygous, as indicated. (C) The square-root of the recovery time from mechanical shock (bang sensitivity) of flies of the genotypes and sexes indicated; means±s.e.m. are shown. srlGR flies in this experiment were hemizygous. The number of individually analyzed flies of each genotype was between 19 and 24 for the different classes. In B and C, #P<0.05, *P<0.01 between sesB1 and other data classes. Note that t-test or ANOVA cannot be meaningfully implemented for A owing to the skewed distribution of the day of eclosion in the BER-1 mtDNA background; hence the data are only indicative. The partial rescue of bang sensitivity by overexpression of spargel is statistically significant, but because an entire chromosome is replaced in this experiment, we cannot exclude a contribution of the genetic background. (D) Relative spargel mRNA levels, in flies of the sex and genotype indicated, normalized to the value in the same sex for wild-type flies. Only the lines that overexpressed spargel showed a significant increase in the mRNA, denoted by *, as above. (E) The mean proportion of eggs that developed into adults for the offspring of females, of the genotypes indicated, mated to wild-type males. Means±s.d. from three replicate experiments are shown.
- Fig. 5.
Partial rescue of sesB1 by AOX expression. (A) The day of eclosion of flies of the genotypes and sexes indicated, reared at 22°C. Where present, da-GAL4 and UAS-AOX transgenes were hemizygous. ** significant differences between sesB1 and other data classes (P<0.01). The means±s.d. from eight to 14 replicate experiments are shown. (B) Bang sensitivity – the square-root of recovery time from mechanical shock of 3-day-old flies of the genotypes and sexes indicated, reared at 22°C; means±s.e.m. are shown. 79–215 flies of each class were analyzed individually. **P<0.01 between data classes for each sex (calculated by Student’s t-test with Bonferroni correction). (C) Mean numbers of offspring per vial as a result of mating females of the indicated genotypes (reared at 22°C) with wild-type (Oregon R) males. +, hemizygous; ++, homozygous; −, absence of a given marker, balancer or transgene. *P<0.05, **P<0.01 between sesB1;AOX and other data classes for each sex (Student’s t-test). (D) Lifespan curves for male flies of the indicated genotypes. Canton S (CS) and Oregon R (OR) are different strain backgrounds, which show curtailed lifespans similar to that of sesB1. Where present together, da-GAL4 and UAS-AOX transgenes were hemizygous. When UAS-AOX alone was present, it was homozygous in order to control for the number of copies of the mini-white selectable marker. The survival curve for AOX-expressing males is significantly different from all of the control lines, including sesB1 in either genetic background (log-rank test, P<0.001). However, given that the increase in mean lifespan was quantitatively minor, and was not seen in females, a contribution from the genetic background cannot be excluded.
- Fig. 6.
Lack of rescue of sesB1 by Ndi1 expression. (A) The day of eclosion of flies of the genotypes and sexes indicated; means±s.e.m. from three biological replicates are shown. Where present, da-GAL4 and UAS-Ndi1 transgenes were hemizygous. **P<0.01 between sesB1;Ndi1 and other data classes for each sex (Student’s t-test with Bonferroni correction where appropriate). (B) The mean numbers of offspring per vial resulting from the mating of females of the genotypes indicated with wild-type (Oregon R) males. +, hemizygous; ++, homozygous; −, absence of a given marker, balancer or transgene.
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