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Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.

Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC - J. Cell Biol. (2003)

Bottom Line: We find that mice deficient in either Mfn1 or Mfn2 die in midgestation.However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal.Strikingly, a subset of mitochondria in mutant cells lose membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.

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Construction and verification of knockout mice. (A) Genomic targeting of Mfn1. The top bar indicates the wild-type Mfn1 genomic locus with exons aligned above. The dark gray segment contains coding sequences for the G1 and G2 motifs of the GTPase domain. A double crossover with the targeting construct (middle bar) results in a targeted allele (bottom bar) containing a premature stop codon (asterisk) in exon 3 and a substitution of the G1 and G2 encoding genomic sequence with a neomycin- resistance gene (light gray segment labeled Neo; flanking loxP sites indicated by triangles). PGK-DTA, diphtheria toxin subunit A driven by the PGK promoter; Xb, XbaI. (E) Genomic targeting of Mfn2. Drawn as in A. RI, EcoRI. (B and F) Southern blot analyses of targeted embryonic stem clones and offspring. Genomic DNAs were digested with XbaI (B) for Mfn1 and EcoRI (F) for Mfn2 and analyzed with the probes indicated in A and E. The wild-type and knockout bands are indicated as are genotypes. (C and G) PCR genotyping. Three primers (labeled 1, 2, and 3) were used simultaneously to amplify distinct fragments from the wild-type and mutant loci. The DNA samples are identical to those in B and F, respectively. (D and H) Western analyses of wild-type and mutant lysates. Postnuclear embryonic lysates were analyzed with affinity-purified antibodies directed against Mfn1 (D) and Mfn2 (H). β-Actin was used as a loading control.
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fig1: Construction and verification of knockout mice. (A) Genomic targeting of Mfn1. The top bar indicates the wild-type Mfn1 genomic locus with exons aligned above. The dark gray segment contains coding sequences for the G1 and G2 motifs of the GTPase domain. A double crossover with the targeting construct (middle bar) results in a targeted allele (bottom bar) containing a premature stop codon (asterisk) in exon 3 and a substitution of the G1 and G2 encoding genomic sequence with a neomycin- resistance gene (light gray segment labeled Neo; flanking loxP sites indicated by triangles). PGK-DTA, diphtheria toxin subunit A driven by the PGK promoter; Xb, XbaI. (E) Genomic targeting of Mfn2. Drawn as in A. RI, EcoRI. (B and F) Southern blot analyses of targeted embryonic stem clones and offspring. Genomic DNAs were digested with XbaI (B) for Mfn1 and EcoRI (F) for Mfn2 and analyzed with the probes indicated in A and E. The wild-type and knockout bands are indicated as are genotypes. (C and G) PCR genotyping. Three primers (labeled 1, 2, and 3) were used simultaneously to amplify distinct fragments from the wild-type and mutant loci. The DNA samples are identical to those in B and F, respectively. (D and H) Western analyses of wild-type and mutant lysates. Postnuclear embryonic lysates were analyzed with affinity-purified antibodies directed against Mfn1 (D) and Mfn2 (H). β-Actin was used as a loading control.

Mentions: We constructed gene replacement vectors for Mfn1 and Mfn2 using the neomycin resistance gene for positive selection and the diphtheria toxin subunit A gene for negative selection. In both cases, a stop codon was engineered at the very beginning of the GTPase domain near the NH2 terminus (Fig. 1, A and E). In addition, the resulting genomic loci each contain a replacement of the G1 and G2 motifs of the GTPase domain with the neomycin expression cassette. These universal GTPase motifs are crucial for binding of the α and β phosphates of GTP and for Mg+2 coordination (Bourne et al., 1991; Sprang, 1997). Genetic analyses in Drosophila and Saccharomyces cerevisiae (Hales and Fuller, 1997; Hermann et al., 1998), as well as our own studies (see Fig. 7 C and Fig. 8 C), demonstrate that an intact GTPase domain is essential for Fzo function. Therefore, the disrupted Mfn1 and Mfn2 alleles described here should be alleles. Both Southern blot and PCR analysis confirmed germline transmission of the targeted alleles (Fig. 1, B, C, F, and G). Importantly, Western blot analysis using affinity-purified antisera raised against Mfn1 or Mfn2 confirmed loss of the targeted protein in homozygous mutant lysates (Fig. 1, D and H).


Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.

Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC - J. Cell Biol. (2003)

Construction and verification of knockout mice. (A) Genomic targeting of Mfn1. The top bar indicates the wild-type Mfn1 genomic locus with exons aligned above. The dark gray segment contains coding sequences for the G1 and G2 motifs of the GTPase domain. A double crossover with the targeting construct (middle bar) results in a targeted allele (bottom bar) containing a premature stop codon (asterisk) in exon 3 and a substitution of the G1 and G2 encoding genomic sequence with a neomycin- resistance gene (light gray segment labeled Neo; flanking loxP sites indicated by triangles). PGK-DTA, diphtheria toxin subunit A driven by the PGK promoter; Xb, XbaI. (E) Genomic targeting of Mfn2. Drawn as in A. RI, EcoRI. (B and F) Southern blot analyses of targeted embryonic stem clones and offspring. Genomic DNAs were digested with XbaI (B) for Mfn1 and EcoRI (F) for Mfn2 and analyzed with the probes indicated in A and E. The wild-type and knockout bands are indicated as are genotypes. (C and G) PCR genotyping. Three primers (labeled 1, 2, and 3) were used simultaneously to amplify distinct fragments from the wild-type and mutant loci. The DNA samples are identical to those in B and F, respectively. (D and H) Western analyses of wild-type and mutant lysates. Postnuclear embryonic lysates were analyzed with affinity-purified antibodies directed against Mfn1 (D) and Mfn2 (H). β-Actin was used as a loading control.
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Related In: Results  -  Collection

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fig1: Construction and verification of knockout mice. (A) Genomic targeting of Mfn1. The top bar indicates the wild-type Mfn1 genomic locus with exons aligned above. The dark gray segment contains coding sequences for the G1 and G2 motifs of the GTPase domain. A double crossover with the targeting construct (middle bar) results in a targeted allele (bottom bar) containing a premature stop codon (asterisk) in exon 3 and a substitution of the G1 and G2 encoding genomic sequence with a neomycin- resistance gene (light gray segment labeled Neo; flanking loxP sites indicated by triangles). PGK-DTA, diphtheria toxin subunit A driven by the PGK promoter; Xb, XbaI. (E) Genomic targeting of Mfn2. Drawn as in A. RI, EcoRI. (B and F) Southern blot analyses of targeted embryonic stem clones and offspring. Genomic DNAs were digested with XbaI (B) for Mfn1 and EcoRI (F) for Mfn2 and analyzed with the probes indicated in A and E. The wild-type and knockout bands are indicated as are genotypes. (C and G) PCR genotyping. Three primers (labeled 1, 2, and 3) were used simultaneously to amplify distinct fragments from the wild-type and mutant loci. The DNA samples are identical to those in B and F, respectively. (D and H) Western analyses of wild-type and mutant lysates. Postnuclear embryonic lysates were analyzed with affinity-purified antibodies directed against Mfn1 (D) and Mfn2 (H). β-Actin was used as a loading control.
Mentions: We constructed gene replacement vectors for Mfn1 and Mfn2 using the neomycin resistance gene for positive selection and the diphtheria toxin subunit A gene for negative selection. In both cases, a stop codon was engineered at the very beginning of the GTPase domain near the NH2 terminus (Fig. 1, A and E). In addition, the resulting genomic loci each contain a replacement of the G1 and G2 motifs of the GTPase domain with the neomycin expression cassette. These universal GTPase motifs are crucial for binding of the α and β phosphates of GTP and for Mg+2 coordination (Bourne et al., 1991; Sprang, 1997). Genetic analyses in Drosophila and Saccharomyces cerevisiae (Hales and Fuller, 1997; Hermann et al., 1998), as well as our own studies (see Fig. 7 C and Fig. 8 C), demonstrate that an intact GTPase domain is essential for Fzo function. Therefore, the disrupted Mfn1 and Mfn2 alleles described here should be alleles. Both Southern blot and PCR analysis confirmed germline transmission of the targeted alleles (Fig. 1, B, C, F, and G). Importantly, Western blot analysis using affinity-purified antisera raised against Mfn1 or Mfn2 confirmed loss of the targeted protein in homozygous mutant lysates (Fig. 1, D and H).

Bottom Line: We find that mice deficient in either Mfn1 or Mfn2 die in midgestation.However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal.Strikingly, a subset of mitochondria in mutant cells lose membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.

Show MeSH
Related in: MedlinePlus