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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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Stochastic loss of membrane potential in mitochondria of mutant cells. (A) mtDNA is detected by Southern blot analysis using a COX1 probe. (B and C) COXI expression in Mfn1 (B) and Mfn2 (C) mutant cells. (D–F) Staining of mitochondria using dyes sensitive to membrane potential. Wild-type (D), Mfn1 mutant (E), and Mfn2 mutant (F) cells expressing mitochondrially targeted EYFP (green) were stained with the dye MitoTracker Red, whose sequestration into mitochondria is sensitive to membrane potential. In these merged images, note that in the mutant cells (E and F) a subset of mitochondria (arrows) stain poorly with MitoTracker Red and thus appear green.
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fig6: Stochastic loss of membrane potential in mitochondria of mutant cells. (A) mtDNA is detected by Southern blot analysis using a COX1 probe. (B and C) COXI expression in Mfn1 (B) and Mfn2 (C) mutant cells. (D–F) Staining of mitochondria using dyes sensitive to membrane potential. Wild-type (D), Mfn1 mutant (E), and Mfn2 mutant (F) cells expressing mitochondrially targeted EYFP (green) were stained with the dye MitoTracker Red, whose sequestration into mitochondria is sensitive to membrane potential. In these merged images, note that in the mutant cells (E and F) a subset of mitochondria (arrows) stain poorly with MitoTracker Red and thus appear green.

Mentions: We tested whether Mfn1 and Mfn2 mutant cells lose mtDNA because there is complete loss of mtDNA in fzo1Δ yeast (Hermann et al., 1998; Rapaport et al., 1998). Southern blot and PCR analysis showed that both mutant cell lines contain normal levels of mtDNA (Fig. 6 A; unpublished data). In addition, the mitochondria in mutant cells expressed COX1, a mitochondrial protein encoded by mtDNA (Fig. 6, B and C). Therefore, unlike fzo1Δ yeast, Mfn-deficient cells clearly retain mtDNA, and this feature allows a critical assessment of the relationship between respiration and mitochondrial fusion. Like wild-type cells, both Mfn1 and Mfn2 mutant cultures showed high rates of endogenous and coupled respiration (unpublished data), indicating no gross defects in respiration and further confirming that mtDNA products are intact and functional.


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)

Stochastic loss of membrane potential in mitochondria of mutant cells. (A) mtDNA is detected by Southern blot analysis using a COX1 probe. (B and C) COXI expression in Mfn1 (B) and Mfn2 (C) mutant cells. (D–F) Staining of mitochondria using dyes sensitive to membrane potential. Wild-type (D), Mfn1 mutant (E), and Mfn2 mutant (F) cells expressing mitochondrially targeted EYFP (green) were stained with the dye MitoTracker Red, whose sequestration into mitochondria is sensitive to membrane potential. In these merged images, note that in the mutant cells (E and F) a subset of mitochondria (arrows) stain poorly with MitoTracker Red and thus appear green.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172648&req=5

fig6: Stochastic loss of membrane potential in mitochondria of mutant cells. (A) mtDNA is detected by Southern blot analysis using a COX1 probe. (B and C) COXI expression in Mfn1 (B) and Mfn2 (C) mutant cells. (D–F) Staining of mitochondria using dyes sensitive to membrane potential. Wild-type (D), Mfn1 mutant (E), and Mfn2 mutant (F) cells expressing mitochondrially targeted EYFP (green) were stained with the dye MitoTracker Red, whose sequestration into mitochondria is sensitive to membrane potential. In these merged images, note that in the mutant cells (E and F) a subset of mitochondria (arrows) stain poorly with MitoTracker Red and thus appear green.
Mentions: We tested whether Mfn1 and Mfn2 mutant cells lose mtDNA because there is complete loss of mtDNA in fzo1Δ yeast (Hermann et al., 1998; Rapaport et al., 1998). Southern blot and PCR analysis showed that both mutant cell lines contain normal levels of mtDNA (Fig. 6 A; unpublished data). In addition, the mitochondria in mutant cells expressed COX1, a mitochondrial protein encoded by mtDNA (Fig. 6, B and C). Therefore, unlike fzo1Δ yeast, Mfn-deficient cells clearly retain mtDNA, and this feature allows a critical assessment of the relationship between respiration and mitochondrial fusion. Like wild-type cells, both Mfn1 and Mfn2 mutant cultures showed high rates of endogenous and coupled respiration (unpublished data), indicating no gross defects in respiration and further confirming that mtDNA products are intact and functional.

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