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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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Mitochondrial fusion assay. PEG fusion of cells containing mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 (E) mutant cells displaying predominantly unfused mitochondria. (C and F) Magnified views of boxed portions in B and E, respectively. (D) Sectoring effect in Mfn1 mutant cell.
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fig5: Mitochondrial fusion assay. PEG fusion of cells containing mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 (E) mutant cells displaying predominantly unfused mitochondria. (C and F) Magnified views of boxed portions in B and E, respectively. (D) Sectoring effect in Mfn1 mutant cell.

Mentions: To definitively show that lack of fusion is the basis for the fragmented mitochondria in mutant cells, we measured mitochondrial fusion activity using a polyethylene glycol (PEG) cell fusion assay. A cell line expressing mitochondrially targeted dsRed was cocultured with a cell line expressing mitochondrially targeted GFP, and PEG was transiently applied to fuse the cells. Cycloheximide was included to prevent synthesis of new fluorescent molecules in the fused cells. When wild-type cells were examined 7 h after PEG treatment, all of the fused cells (n = 200) contained extensively fused mitochondria (Fig. 5 A) as demonstrated by colocalization of red and green fluorescent signals. In contrast, when Mfn1 mutant cells were examined 7 h after PEG fusion 57% (n = 364) of the fused cells contained predominantly unfused mitochondria (Fig. 5, B and C) even when red and green mitochondria were dispersed throughout the fused cell. 35% of cells showed extensive mitochondrial fusion, and 8% showed partial fusion. Similarly, 69% (n = 202) of fused Mfn2 mutant cells showed predominantly unfused mitochondria after 7 h (Fig. 5, E and F). 1% showed extensive fusion, and 30% showed partial fusion. Mfn1 and Mfn2 mutant cells with unfused mitochondria were observed even 24 h after PEG treatment (unpublished data). Thus, mutant cells have severely reduced levels of mitochondrial fusion. Interestingly, in 10% of fused Mfn1 mutant cells, the mitochondria did not readily spread throughout the cytoplasm as shown by discrete sectors of red and green fluorescence (Fig. 5 D). Only 1% of fused Mfn2 mutant cells exhibited this sectoring effect. Therefore, it seems that the mobility of Mfn1-deficient mitochondria is impaired to a greater extent than that of Mfn2-deficient mitochondria, perhaps due to their more severely fragmented morphology.


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)

Mitochondrial fusion assay. PEG fusion of cells containing mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 (E) mutant cells displaying predominantly unfused mitochondria. (C and F) Magnified views of boxed portions in B and E, respectively. (D) Sectoring effect in Mfn1 mutant cell.
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Related In: Results  -  Collection

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fig5: Mitochondrial fusion assay. PEG fusion of cells containing mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 (E) mutant cells displaying predominantly unfused mitochondria. (C and F) Magnified views of boxed portions in B and E, respectively. (D) Sectoring effect in Mfn1 mutant cell.
Mentions: To definitively show that lack of fusion is the basis for the fragmented mitochondria in mutant cells, we measured mitochondrial fusion activity using a polyethylene glycol (PEG) cell fusion assay. A cell line expressing mitochondrially targeted dsRed was cocultured with a cell line expressing mitochondrially targeted GFP, and PEG was transiently applied to fuse the cells. Cycloheximide was included to prevent synthesis of new fluorescent molecules in the fused cells. When wild-type cells were examined 7 h after PEG treatment, all of the fused cells (n = 200) contained extensively fused mitochondria (Fig. 5 A) as demonstrated by colocalization of red and green fluorescent signals. In contrast, when Mfn1 mutant cells were examined 7 h after PEG fusion 57% (n = 364) of the fused cells contained predominantly unfused mitochondria (Fig. 5, B and C) even when red and green mitochondria were dispersed throughout the fused cell. 35% of cells showed extensive mitochondrial fusion, and 8% showed partial fusion. Similarly, 69% (n = 202) of fused Mfn2 mutant cells showed predominantly unfused mitochondria after 7 h (Fig. 5, E and F). 1% showed extensive fusion, and 30% showed partial fusion. Mfn1 and Mfn2 mutant cells with unfused mitochondria were observed even 24 h after PEG treatment (unpublished data). Thus, mutant cells have severely reduced levels of mitochondrial fusion. Interestingly, in 10% of fused Mfn1 mutant cells, the mitochondria did not readily spread throughout the cytoplasm as shown by discrete sectors of red and green fluorescence (Fig. 5 D). Only 1% of fused Mfn2 mutant cells exhibited this sectoring effect. Therefore, it seems that the mobility of Mfn1-deficient mitochondria is impaired to a greater extent than that of Mfn2-deficient mitochondria, perhaps due to their more severely fragmented morphology.

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