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Figures
- Fig. 1.
Gene trap screen for mitochondrial phenotypes identifies Sucla2 mutant allele. (A) Wild-type MEFs transfected with mito-YFP and stained with DAPI. (B) Wild-type MEFs stained with DiIC1(5) (HIDC) and analyzed by FACS. Cells pre-treated with CCCP (green line), a proton ionophore, exhibit relative depolarization of the MMP. Cells pre-treated with nigericin (orange line), a K+-selective ionophore, exhibit relative hyperpolarization of the MMP. (C) Localization of ROSAβgeo gene trap integration into intron 4–5 of Sucla2 locus. Red (F), blue (R) and green (G) arrows depict relative primer locations for RT-PCR experiments shown in E. (D) FACS analysis of Sucla2+/− gene trap ES clone for mito-YFP fluorescence. Each line depicts summary of three independent FACS experiments and shows that the Sucla2 gene trap clone exhibits an ~30% reduction in mean YFP fluorescence compared with the parental ES cell line. (E) RT-PCR analysis of RNA isolated from Sucla2 wild-type (+/+; lanes 1–2) and homozygous mutant (−/−; lanes 3–4) MEFs using the primer pairs indicated on the gene map in C. Lanes 1 and 3 represent wild-type allele-specific PCR (FR), generating a 566-bp product. Lanes 2 and 4 represent gene trap allele-specific PCR (FG), generating a 500-bp product from the mutant allele. The smaller second band (380-bp product) in lane 4 is from a gene trap allele-derived transcript from which exon 3 of Sucla2 splices directly onto βgeo, skipping Sucla2 exon 4 (sequenced-verified). (F) Western blot analysis of Sucla2 MEFs. Three independent lines each of Sucla2+/+ and Sucla2−/− MEFs were utilized for western blot analysis of SCS enzyme complex components. Re indicates Sucla2−/− MEF cell line rescued by ectopic expression of wild-type Sucla2 cDNA. (G) Analysis of SCS activities in Sucla2 MEFs. ADP-specific and GDP-specific SCS activities were measured for wild-type and mutant MEFs. Sucla2−/− MEFs exhibit an ADP-specific SCS enzyme deficiency that is rescued by ectopic expression of Sucla2 (+Re). (H) X-gal staining of wild-type (left) and heterozygous mutant (right) e12.5 embryos shows Sucla2 expression pattern, predominantly in brain, heart, developing spinal cord and/or neighboring tissues with little expression in liver.
- Fig. 2.
Sucla2 mutant embryos display growth deficiency and placental mtDNA depletion. (A) Representative photos of wild-type (left) and homozygous mutant (right) e17.5 embryos. (B) Bar graph depicting average wet weight of e17.5 Sucla2 embryos (numbers represent sample size for each genotype), with Sucla2−/− embryos weighing 25% less. (C) Relative mtDNA content for embryonic placentas from e17.5 Sucla2 embryos (numbers represent sample size for each genotype). (D) Western blot analysis of e17.5 Sucla2 placentas. The relative mtDNA copy number for each sample is indicated below each lane of the western blot. COX1 is a mtDNA-encoded subunit of cytochrome c oxidase (respiratory chain complex IV).
- Fig. 3.
Sucla2-deficient placenta exhibit increased mineralization. Sections of wild-type (left) and mutant (right) placentas from e17.5 Sucla2 embryos. Sections were stained with H&E (10× magnification), Prussian Blue (for iron) (10× magnification), von Kossa (10× magnification) and Alizarin Red (4× magnification) for calcium. Arrows in upper right panel indicate potential areas of increased mineralization in mutant placenta. Inset boxes show 40× magnification of areas of increased mineralization.
- Fig. 4.
Sucla2-deficient MEFs exhibit functionally significant mtDNA depletion associated with relative mitochondrial depolarization, cellular respiration defects and respiratory chain deficiencies. (A) Sucla2 mutant MEFs exhibit mtDNA depletion compared with MEFs from wild-type littermates that is rescued by ectopic expression of wild-type Sucla2 cDNA. (B) The relative mitochondrial membrane potential for Sucla2 MEFs was determined by staining cells with DiIC1(5) (HIDC) followed by FACS analysis (three independent lines for each genotype). The graph on the left shows the analysis soon after the establishment of the MEFs. The graph on the right shows the analysis after 5 weeks of culture with multiple passages. The mutant MEFs demonstrate a progressive relative depolarization of the mitochondrial inner membrane. (C) Cellular respiration analysis of Sucla2 MEFs demonstrate that Sucla2−/− cells exhibit defects in basal respiration, oligomycin-sensitive respiration and respiratory capacity. (D) Histochemical staining of MEFs reveals complex IV deficiency in a subset of Sucla2−/− cells. MEFs were stained for SDH (no mtDNA-encoded subunits) and COX (has mtDNA encoded subunits) activities. All cells show uniform staining for SDH, whereas a subset of mutant MEFs exhibit little or no detectable COX activity, in contrast to wild-type cells demonstrating uniform normal staining. (E) Analysis of mitochondrial electron transport chain enzyme activities shows partial deficiency of ETC complex III and a reduction in citrate synthase (CS) activity in Sucla2 mutant MEFs. CS is a TCA cycle enzyme commonly used as a biochemical marker of mitochondrial matrix content.
- Fig. 5.
Sucla2-deficient embryo tissues exhibit progressive, functionally significant mtDNA depletion and elevated MMA. (A) Relative mtDNA content of tissues from e15.5 Sucla2 tissues (n=3 for each genotype). (B) Relative mtDNA content of tissues from e17.5 Sucla2 tissues (n=8 for each genotype) showing progressive, significant mtDNA depletion in brain and skeletal muscle. (C) Western blot analysis of e17.5 Sucla2 brain tissues. The relative mtDNA copy number for each sample is indicated below each lane of the western blot. Severe reduction in COX1 expression is observed when the relative mtDNA copy number falls below 600. The bar graph shows quantification of the band intensities of the blot shown. (D) Complex IV deficiency is proportional to mtDNA content in Sucla2−/− brain. Brains from eight wild-type and eight mutant e17.5 embryos were used for analysis of mitochondrial complex IV activity and for relative mtDNA content. Graph depicts relationship of complex IV activity and mtDNA content. Mutant brains with relative mtDNA copy number below ~600 exhibit proportional loss of complex IV activity. (E) Sucla2−/− brains exhibit increased levels of MMA. Brain lysates from wild-type and mutant e17.5 embryos were used for measurement of MMA levels normalized to total cellular protein content.
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