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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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Tissue-specific mt:CoIT300I homoplasmy. (A) Tissue-specific expression of MitoXhoI shifted heteroplasmic mt:CoIT300I flies to homoplasmy in fat body and eye disk. A 4.0-kb mtDNA fragment flanking the XhoI site was amplified by PCR from flies with the indicated genotypes (1–6) and further digested by XhoI enzyme. The wt mtDNA carrying the XhoI recognition site could be digested into two fragments (1.6 and 2.4 kb), whereas mutant mtDNA was resistant to XhoI digestion. Here 1–6 denote the genotypes and tissues of different Drosophila lines. Note that tissue-specific expression of MitoXhoI under control of Cg-Gal4 (Cg>mitoXhoI) or eyeless-Gal4 (ey>mitoXhoI) completely eliminated the wt mtDNA and resulted in 100% of mt:CoIT300I in fat body or eye disk, respectively, whereas the whole body remains heteroplasmic. (B–D) mt:CoIT300I homoplasmy in the eye leads to photoreceptor degeneration. (B) Rhabdomeres observed by optical neutralization from 2- or 8-d-old heteroplasmic flies with homoplasmic eyes. Eye tissues were made homoplasmic by eye-specific expression of MitoXhoI under control of eyeless-Gal4 driver (ey>mitoXhoI). Adult flies were kept at 29°C under a 12-h light/12-h dark cycle. (C) Mean number of rhabdomeres per ommatidium, as determined by optical neutralization as a function of age. The numbers of rhabdomeres decreased rapidly in homoplasmic eyes (-o-, ey>mitoXhoI [wt+ mt:CoIT300I]) but were rescued by coexpression of Letm1 (-Δ-, ey >mitoXhoI +letm1 [wt+ mt:CoIT300I]). Expression of MitoXhoI in flies carrying a mtDNA synonymous mutation (mt:CoIsyn) showed no rhabdomere loss (-×-, ey>mitoXhoI [mt:CoIsyn]), demonstrating that overexpression of MitoXhoI per se was not detrimental to photoreceptor cells. Data are presented as means ± SD; n ≥7 flies. (D) Retinal morphology of 2-d or 2-wk-old heteroplasmic flies with or without homoplasmic eyes, as examined by transmission electron microscopy. In addition to the flies used in C, heteroplasmic flies without any nuclear transgene (w1118 [wt+ mt:CoIT300I]) were examined as an additional control. C, cell body; R, rhabdomere. Note the disorganized rhabdomeres (arrowheads) and vesicular structures (arrow) indicating degeneration. Nuclear genotypes are Cg-Gal4/UAS-mitoXhoI (Cg>mitoXhoI), ey-Gal4/UAS-mitoXhoI (ey>mitoXhoI),and ey-Gal4/UAS-mitoXhoI; UAS-letm1/+ (ey >mitoXhoI +letm1).
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Figure 4: Tissue-specific mt:CoIT300I homoplasmy. (A) Tissue-specific expression of MitoXhoI shifted heteroplasmic mt:CoIT300I flies to homoplasmy in fat body and eye disk. A 4.0-kb mtDNA fragment flanking the XhoI site was amplified by PCR from flies with the indicated genotypes (1–6) and further digested by XhoI enzyme. The wt mtDNA carrying the XhoI recognition site could be digested into two fragments (1.6 and 2.4 kb), whereas mutant mtDNA was resistant to XhoI digestion. Here 1–6 denote the genotypes and tissues of different Drosophila lines. Note that tissue-specific expression of MitoXhoI under control of Cg-Gal4 (Cg>mitoXhoI) or eyeless-Gal4 (ey>mitoXhoI) completely eliminated the wt mtDNA and resulted in 100% of mt:CoIT300I in fat body or eye disk, respectively, whereas the whole body remains heteroplasmic. (B–D) mt:CoIT300I homoplasmy in the eye leads to photoreceptor degeneration. (B) Rhabdomeres observed by optical neutralization from 2- or 8-d-old heteroplasmic flies with homoplasmic eyes. Eye tissues were made homoplasmic by eye-specific expression of MitoXhoI under control of eyeless-Gal4 driver (ey>mitoXhoI). Adult flies were kept at 29°C under a 12-h light/12-h dark cycle. (C) Mean number of rhabdomeres per ommatidium, as determined by optical neutralization as a function of age. The numbers of rhabdomeres decreased rapidly in homoplasmic eyes (-o-, ey>mitoXhoI [wt+ mt:CoIT300I]) but were rescued by coexpression of Letm1 (-Δ-, ey >mitoXhoI +letm1 [wt+ mt:CoIT300I]). Expression of MitoXhoI in flies carrying a mtDNA synonymous mutation (mt:CoIsyn) showed no rhabdomere loss (-×-, ey>mitoXhoI [mt:CoIsyn]), demonstrating that overexpression of MitoXhoI per se was not detrimental to photoreceptor cells. Data are presented as means ± SD; n ≥7 flies. (D) Retinal morphology of 2-d or 2-wk-old heteroplasmic flies with or without homoplasmic eyes, as examined by transmission electron microscopy. In addition to the flies used in C, heteroplasmic flies without any nuclear transgene (w1118 [wt+ mt:CoIT300I]) were examined as an additional control. C, cell body; R, rhabdomere. Note the disorganized rhabdomeres (arrowheads) and vesicular structures (arrow) indicating degeneration. Nuclear genotypes are Cg-Gal4/UAS-mitoXhoI (Cg>mitoXhoI), ey-Gal4/UAS-mitoXhoI (ey>mitoXhoI),and ey-Gal4/UAS-mitoXhoI; UAS-letm1/+ (ey >mitoXhoI +letm1).

Mentions: Wild-type mtDNA has an XhoI site, whereas the mt:CoIT300I mitochondrial genome does not and is therefore resistant to XhoI digestion. We reasoned that expression of a mitochondrially targeted form of XhoI, MitoXhoI (Xu et al., 2008), in a heteroplasmic background would destroy wt mtDNA specifically and shift mt:CoIT300I heteroplasmy toward homoplasmy (Supplemental Figure S4A). As a test of principle, we ubiquitously expressed MitoXhoI under control of a tubulin-Gal4 driver (tub-Gal4) in heteroplasmic larvae and quantified mtDNAs based on their sensitivity to XhoI digestion (Supplemental Figure S4B). Expression of MitoXhoI efficiently eliminated wt mtDNA, resulting in nearly 100% mt:CoIT300I. The absolute amount of mtDNA in the heteroplasmic flies expressing MitoXhoI was similar to that in wt flies (Supplemental Figure S4C), suggesting that the copy number of mtDNA remains constant after removal of wt mtDNA. We further tested the efficacy of this approach by generating homoplasmy in specific tissues—larval fat body and eye disk—because of the ease of dissecting homogeneous tissues. Expression of MitoXhoI in fat body driven by Cg-Gal4 or in eye disk by eyeless-Gal4 (ey-Gal4) in the heteroplasmic background completely removed wt mtDNA in the fat body or eye disk, respectively, whereas other tissues remained heteroplasmic (Figure 4A).


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)

Tissue-specific mt:CoIT300I homoplasmy. (A) Tissue-specific expression of MitoXhoI shifted heteroplasmic mt:CoIT300I flies to homoplasmy in fat body and eye disk. A 4.0-kb mtDNA fragment flanking the XhoI site was amplified by PCR from flies with the indicated genotypes (1–6) and further digested by XhoI enzyme. The wt mtDNA carrying the XhoI recognition site could be digested into two fragments (1.6 and 2.4 kb), whereas mutant mtDNA was resistant to XhoI digestion. Here 1–6 denote the genotypes and tissues of different Drosophila lines. Note that tissue-specific expression of MitoXhoI under control of Cg-Gal4 (Cg>mitoXhoI) or eyeless-Gal4 (ey>mitoXhoI) completely eliminated the wt mtDNA and resulted in 100% of mt:CoIT300I in fat body or eye disk, respectively, whereas the whole body remains heteroplasmic. (B–D) mt:CoIT300I homoplasmy in the eye leads to photoreceptor degeneration. (B) Rhabdomeres observed by optical neutralization from 2- or 8-d-old heteroplasmic flies with homoplasmic eyes. Eye tissues were made homoplasmic by eye-specific expression of MitoXhoI under control of eyeless-Gal4 driver (ey>mitoXhoI). Adult flies were kept at 29°C under a 12-h light/12-h dark cycle. (C) Mean number of rhabdomeres per ommatidium, as determined by optical neutralization as a function of age. The numbers of rhabdomeres decreased rapidly in homoplasmic eyes (-o-, ey>mitoXhoI [wt+ mt:CoIT300I]) but were rescued by coexpression of Letm1 (-Δ-, ey >mitoXhoI +letm1 [wt+ mt:CoIT300I]). Expression of MitoXhoI in flies carrying a mtDNA synonymous mutation (mt:CoIsyn) showed no rhabdomere loss (-×-, ey>mitoXhoI [mt:CoIsyn]), demonstrating that overexpression of MitoXhoI per se was not detrimental to photoreceptor cells. Data are presented as means ± SD; n ≥7 flies. (D) Retinal morphology of 2-d or 2-wk-old heteroplasmic flies with or without homoplasmic eyes, as examined by transmission electron microscopy. In addition to the flies used in C, heteroplasmic flies without any nuclear transgene (w1118 [wt+ mt:CoIT300I]) were examined as an additional control. C, cell body; R, rhabdomere. Note the disorganized rhabdomeres (arrowheads) and vesicular structures (arrow) indicating degeneration. Nuclear genotypes are Cg-Gal4/UAS-mitoXhoI (Cg>mitoXhoI), ey-Gal4/UAS-mitoXhoI (ey>mitoXhoI),and ey-Gal4/UAS-mitoXhoI; UAS-letm1/+ (ey >mitoXhoI +letm1).
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Figure 4: Tissue-specific mt:CoIT300I homoplasmy. (A) Tissue-specific expression of MitoXhoI shifted heteroplasmic mt:CoIT300I flies to homoplasmy in fat body and eye disk. A 4.0-kb mtDNA fragment flanking the XhoI site was amplified by PCR from flies with the indicated genotypes (1–6) and further digested by XhoI enzyme. The wt mtDNA carrying the XhoI recognition site could be digested into two fragments (1.6 and 2.4 kb), whereas mutant mtDNA was resistant to XhoI digestion. Here 1–6 denote the genotypes and tissues of different Drosophila lines. Note that tissue-specific expression of MitoXhoI under control of Cg-Gal4 (Cg>mitoXhoI) or eyeless-Gal4 (ey>mitoXhoI) completely eliminated the wt mtDNA and resulted in 100% of mt:CoIT300I in fat body or eye disk, respectively, whereas the whole body remains heteroplasmic. (B–D) mt:CoIT300I homoplasmy in the eye leads to photoreceptor degeneration. (B) Rhabdomeres observed by optical neutralization from 2- or 8-d-old heteroplasmic flies with homoplasmic eyes. Eye tissues were made homoplasmic by eye-specific expression of MitoXhoI under control of eyeless-Gal4 driver (ey>mitoXhoI). Adult flies were kept at 29°C under a 12-h light/12-h dark cycle. (C) Mean number of rhabdomeres per ommatidium, as determined by optical neutralization as a function of age. The numbers of rhabdomeres decreased rapidly in homoplasmic eyes (-o-, ey>mitoXhoI [wt+ mt:CoIT300I]) but were rescued by coexpression of Letm1 (-Δ-, ey >mitoXhoI +letm1 [wt+ mt:CoIT300I]). Expression of MitoXhoI in flies carrying a mtDNA synonymous mutation (mt:CoIsyn) showed no rhabdomere loss (-×-, ey>mitoXhoI [mt:CoIsyn]), demonstrating that overexpression of MitoXhoI per se was not detrimental to photoreceptor cells. Data are presented as means ± SD; n ≥7 flies. (D) Retinal morphology of 2-d or 2-wk-old heteroplasmic flies with or without homoplasmic eyes, as examined by transmission electron microscopy. In addition to the flies used in C, heteroplasmic flies without any nuclear transgene (w1118 [wt+ mt:CoIT300I]) were examined as an additional control. C, cell body; R, rhabdomere. Note the disorganized rhabdomeres (arrowheads) and vesicular structures (arrow) indicating degeneration. Nuclear genotypes are Cg-Gal4/UAS-mitoXhoI (Cg>mitoXhoI), ey-Gal4/UAS-mitoXhoI (ey>mitoXhoI),and ey-Gal4/UAS-mitoXhoI; UAS-letm1/+ (ey >mitoXhoI +letm1).
Mentions: Wild-type mtDNA has an XhoI site, whereas the mt:CoIT300I mitochondrial genome does not and is therefore resistant to XhoI digestion. We reasoned that expression of a mitochondrially targeted form of XhoI, MitoXhoI (Xu et al., 2008), in a heteroplasmic background would destroy wt mtDNA specifically and shift mt:CoIT300I heteroplasmy toward homoplasmy (Supplemental Figure S4A). As a test of principle, we ubiquitously expressed MitoXhoI under control of a tubulin-Gal4 driver (tub-Gal4) in heteroplasmic larvae and quantified mtDNAs based on their sensitivity to XhoI digestion (Supplemental Figure S4B). Expression of MitoXhoI efficiently eliminated wt mtDNA, resulting in nearly 100% mt:CoIT300I. The absolute amount of mtDNA in the heteroplasmic flies expressing MitoXhoI was similar to that in wt flies (Supplemental Figure S4C), suggesting that the copy number of mtDNA remains constant after removal of wt mtDNA. We further tested the efficacy of this approach by generating homoplasmy in specific tissues—larval fat body and eye disk—because of the ease of dissecting homogeneous tissues. Expression of MitoXhoI in fat body driven by Cg-Gal4 or in eye disk by eyeless-Gal4 (ey-Gal4) in the heteroplasmic background completely removed wt mtDNA in the fat body or eye disk, respectively, whereas other tissues remained heteroplasmic (Figure 4A).

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