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Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.

Chen Z, Qi Y, French S, Zhang G, Covian Garcia R, Balaban R, Xu H - Mol. Biol. Cell (2014)

Bottom Line: In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme.Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoI(T300I) homoplasmy in the eye caused severe neurodegeneration at 29°C.Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.

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mt:CoIT300I disrupts cytochrome c oxidase activity. (A) Western blot analysis of total tissue extracts of mt:CoIT300I and wt flies cultured at 25 or 29°C after eclosion at 25°C, with antibodies against CoI, CoIV, ATP synthase α-subunit (ATPs-α), and tubulin. (B) Spectrophoto­metric measurements of cytochrome a (cyt. a) and cytochrome c (cyt. c) amounts in isolated mitochondria from 2-d-old mt:CoIT300I and wt flies raised at 25°C (mean ± SD, n = 3). (C) Difference absorbance spectrum of wt and mt:CoIT300I mitochondria at 25°C or after incubation at 29°C for 30 min. Peaks for cytochrome c (cyt. c) and cytochrome a (cyt. a) are labeled. (D) Ubiquitous AOX expression rescues the mt:CoIT300I adult lethality at 29°C. Flies carrying a UAS-AOX transgene were crossed to flies carrying both the homoplasmic mt:CoIT300I mtDNA and a ubiquitous driver actin-Gal4 (ac-Gal4) on the nuclear genome. Because of maternal transmission of mtDNA, only the progeny of mutant females inherited the mt:CoIT300I mutation (right). These flies died as pupae unless they inherited the ac-Gal4 driver, that is, Cy+ (CyO balancer chromosome carries a dominant Cy marker; Cy+ means non-Cy). Overexpression of UAS-AOX under control of ac-Gal4 had no effect on the viability of flies inheriting wide-type (wt) mtDNA from their mothers (left). Asterisk, the CyO escapers died quickly after eclosion.
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Figure 1: mt:CoIT300I disrupts cytochrome c oxidase activity. (A) Western blot analysis of total tissue extracts of mt:CoIT300I and wt flies cultured at 25 or 29°C after eclosion at 25°C, with antibodies against CoI, CoIV, ATP synthase α-subunit (ATPs-α), and tubulin. (B) Spectrophoto­metric measurements of cytochrome a (cyt. a) and cytochrome c (cyt. c) amounts in isolated mitochondria from 2-d-old mt:CoIT300I and wt flies raised at 25°C (mean ± SD, n = 3). (C) Difference absorbance spectrum of wt and mt:CoIT300I mitochondria at 25°C or after incubation at 29°C for 30 min. Peaks for cytochrome c (cyt. c) and cytochrome a (cyt. a) are labeled. (D) Ubiquitous AOX expression rescues the mt:CoIT300I adult lethality at 29°C. Flies carrying a UAS-AOX transgene were crossed to flies carrying both the homoplasmic mt:CoIT300I mtDNA and a ubiquitous driver actin-Gal4 (ac-Gal4) on the nuclear genome. Because of maternal transmission of mtDNA, only the progeny of mutant females inherited the mt:CoIT300I mutation (right). These flies died as pupae unless they inherited the ac-Gal4 driver, that is, Cy+ (CyO balancer chromosome carries a dominant Cy marker; Cy+ means non-Cy). Overexpression of UAS-AOX under control of ac-Gal4 had no effect on the viability of flies inheriting wide-type (wt) mtDNA from their mothers (left). Asterisk, the CyO escapers died quickly after eclosion.

Mentions: To understand the biochemical basis of the temperature sensitivity of mt:CoIT300I, we assayed the composition and activity of the respiratory chain complexes of mutant flies at both 25 and 29°C. Although CoI protein levels were comparable in mt:CoIT300I and wt flies (Figure 1A), cytochrome c oxidase (COX) activity in the mutant was decreased to ∼30% of wt activity at 25°C (Supplemental Figure S1A). The mutant COX appears unstable at restrictive condition, as the COX activity of mt:CoIT300I extract quickly diminished to <5% of wild type after incubating at 29°C for 40 min (Hill, Chen, et al., 2014).


Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.

Chen Z, Qi Y, French S, Zhang G, Covian Garcia R, Balaban R, Xu H - Mol. Biol. Cell (2014)

mt:CoIT300I disrupts cytochrome c oxidase activity. (A) Western blot analysis of total tissue extracts of mt:CoIT300I and wt flies cultured at 25 or 29°C after eclosion at 25°C, with antibodies against CoI, CoIV, ATP synthase α-subunit (ATPs-α), and tubulin. (B) Spectrophoto­metric measurements of cytochrome a (cyt. a) and cytochrome c (cyt. c) amounts in isolated mitochondria from 2-d-old mt:CoIT300I and wt flies raised at 25°C (mean ± SD, n = 3). (C) Difference absorbance spectrum of wt and mt:CoIT300I mitochondria at 25°C or after incubation at 29°C for 30 min. Peaks for cytochrome c (cyt. c) and cytochrome a (cyt. a) are labeled. (D) Ubiquitous AOX expression rescues the mt:CoIT300I adult lethality at 29°C. Flies carrying a UAS-AOX transgene were crossed to flies carrying both the homoplasmic mt:CoIT300I mtDNA and a ubiquitous driver actin-Gal4 (ac-Gal4) on the nuclear genome. Because of maternal transmission of mtDNA, only the progeny of mutant females inherited the mt:CoIT300I mutation (right). These flies died as pupae unless they inherited the ac-Gal4 driver, that is, Cy+ (CyO balancer chromosome carries a dominant Cy marker; Cy+ means non-Cy). Overexpression of UAS-AOX under control of ac-Gal4 had no effect on the viability of flies inheriting wide-type (wt) mtDNA from their mothers (left). Asterisk, the CyO escapers died quickly after eclosion.
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Related In: Results  -  Collection

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Figure 1: mt:CoIT300I disrupts cytochrome c oxidase activity. (A) Western blot analysis of total tissue extracts of mt:CoIT300I and wt flies cultured at 25 or 29°C after eclosion at 25°C, with antibodies against CoI, CoIV, ATP synthase α-subunit (ATPs-α), and tubulin. (B) Spectrophoto­metric measurements of cytochrome a (cyt. a) and cytochrome c (cyt. c) amounts in isolated mitochondria from 2-d-old mt:CoIT300I and wt flies raised at 25°C (mean ± SD, n = 3). (C) Difference absorbance spectrum of wt and mt:CoIT300I mitochondria at 25°C or after incubation at 29°C for 30 min. Peaks for cytochrome c (cyt. c) and cytochrome a (cyt. a) are labeled. (D) Ubiquitous AOX expression rescues the mt:CoIT300I adult lethality at 29°C. Flies carrying a UAS-AOX transgene were crossed to flies carrying both the homoplasmic mt:CoIT300I mtDNA and a ubiquitous driver actin-Gal4 (ac-Gal4) on the nuclear genome. Because of maternal transmission of mtDNA, only the progeny of mutant females inherited the mt:CoIT300I mutation (right). These flies died as pupae unless they inherited the ac-Gal4 driver, that is, Cy+ (CyO balancer chromosome carries a dominant Cy marker; Cy+ means non-Cy). Overexpression of UAS-AOX under control of ac-Gal4 had no effect on the viability of flies inheriting wide-type (wt) mtDNA from their mothers (left). Asterisk, the CyO escapers died quickly after eclosion.
Mentions: To understand the biochemical basis of the temperature sensitivity of mt:CoIT300I, we assayed the composition and activity of the respiratory chain complexes of mutant flies at both 25 and 29°C. Although CoI protein levels were comparable in mt:CoIT300I and wt flies (Figure 1A), cytochrome c oxidase (COX) activity in the mutant was decreased to ∼30% of wt activity at 25°C (Supplemental Figure S1A). The mutant COX appears unstable at restrictive condition, as the COX activity of mt:CoIT300I extract quickly diminished to <5% of wild type after incubating at 29°C for 40 min (Hill, Chen, et al., 2014).

Bottom Line: In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme.Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoI(T300I) homoplasmy in the eye caused severe neurodegeneration at 29°C.Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.

Show MeSH
Related in: MedlinePlus