CoChat
Sign Up
Cited by 2,601Mitochondrial Function and PathologyAutophagy in Disease and TherapyCell death mechanisms and regulation

The DOI matches its Crossref record; 53 of the 53 references it cites were found in a registry or an index.

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

Open access
Chen, Hsiuchen, Detmer, Scott A., Ewald, Andrew J., Griffin, Erik E., Fraser, Scott E., Chan, David C.·2003·The Journal of Cell Biology
DOI 10.1083/jcb.200211046PMC PMC2172648
Open this paper in CoChat — free account

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.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1083/jcb.200211046

Cites

53 of 53 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1. 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.
Figure 1. 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.CC BY-NC-SA · PubMed Central
Figure 2. Defective giant cell layer of mutant placentae. (A–D) DAPI-stained sections of placentae from e10.5 wild-type (A and C) and mutant (B and D) littermate embryos. The boxed areas of A and B are enlarged in C and D. Arrows and arrowheads indicate trophoblast giant cells. Note that the giant cells in D are sparser and have smaller nuclei. (E and F) Hematoxylin-eosin–stained sections from the placentae above. (G and H) PL-I (giant cell marker) RNA in situ analysis of placentae from e9.5 wild-type (G) and mutant (H) littermates.
Figure 2. Defective giant cell layer of mutant placentae. (A–D) DAPI-stained sections of placentae from e10.5 wild-type (A and C) and mutant (B and D) littermate embryos. The boxed areas of A and B are enlarged in C and D. Arrows and arrowheads indicate trophoblast giant cells. Note that the giant cells in D are sparser and have smaller nuclei. (E and F) Hematoxylin-eosin–stained sections from the placentae above. (G and H) PL-I (giant cell marker) RNA in situ analysis of placentae from e9.5 wild-type (G) and mutant (H) littermates.CC BY-NC-SA · PubMed Central
Figure 3. Morphological defects in mitochondria of mutant cells. (A–F) Mitochondrial morphology in wild-type (A and B), Mfn1 mutant (C and D), and Mfn2 mutant (E and F) MEF cells. MEFs expressing mitochondrial EYFP (green) were counterstained with rhodamine-phalloidin (red). (B, D, and F) Higher magnification images of the boxed areas in A, C, and E, respectively. Arrow indicates a tubule >10 μm in length. (G and H) Mitochondrial morphology in live wild-type (G) and mutant (H) TS cells. The mitochondria were stained with MitoTracker Red, and the nuclei were stained with Syto16 (green). Several cells are tightly clustered.
Figure 3. Morphological defects in mitochondria of mutant cells. (A–F) Mitochondrial morphology in wild-type (A and B), Mfn1 mutant (C and D), and Mfn2 mutant (E and F) MEF cells. MEFs expressing mitochondrial EYFP (green) were counterstained with rhodamine-phalloidin (red). (B, D, and F) Higher magnification images of the boxed areas in A, C, and E, respectively. Arrow indicates a tubule >10 μm in length. (G and H) Mitochondrial morphology in live wild-type (G) and mutant (H) TS cells. The mitochondria were stained with MitoTracker Red, and the nuclei were stained with Syto16 (green). Several cells are tightly clustered.CC BY-NC-SA · PubMed Central
Figure 4. Dynamics of mitochondria in wild-type and mutant cells. Still frames from time-lapse confocal microscopy. (A) In a wild-type cell, two pairs of mitochondria can be seen moving toward each other. These pairs contact end-to-end and fuse immediately. Note that mitochondria move along their long axes. (B) In a Mfn1 mutant cell, the mitochondria move in an undirected manner. (C) In a Mfn2 mutant cell, two ovoid mitochondria contact each other but do not fuse until much later. Note also the lack of directed movement in most mitochondria. (D) One spherical Mfn2-deficient mitochondrion protrudes a tubular extension that separates and then migrates away along its long axis. Images were processed in Adobe Photoshop® with the emboss filter, and selected mitochondria were manually highlighted in blue. See also videos 1–3 available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1.
Figure 4. Dynamics of mitochondria in wild-type and mutant cells. Still frames from time-lapse confocal microscopy. (A) In a wild-type cell, two pairs of mitochondria can be seen moving toward each other. These pairs contact end-to-end and fuse immediately. Note that mitochondria move along their long axes. (B) In a Mfn1 mutant cell, the mitochondria move in an undirected manner. (C) In a Mfn2 mutant cell, two ovoid mitochondria contact each other but do not fuse until much later. Note also the lack of directed movement in most mitochondria. (D) One spherical Mfn2-deficient mitochondrion protrudes a tubular extension that separates and then migrates away along its long axis. Images were processed in Adobe Photoshop® with the emboss filter, and selected mitochondria were manually highlighted in blue. See also videos 1–3 available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1.CC BY-NC-SA · PubMed Central
Figure 5. 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.
Figure 5. 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.CC BY-NC-SA · PubMed Central
Figure 6. 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.
Figure 6. 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.CC BY-NC-SA · PubMed Central
Figure 7. Rescue of Mfn1-deficient cells Mfn1 mutant cells. Mfn1 mutant cells (A) were infected with a retrovirus expressing Myc epitope-tagged versions of Mfn1 (B), Mfn1(K88T) (C), Mfn2 (D), or dominant- negative Drp1(K38A) (E). In the merged images, mitochondrial morphology is revealed by MitoTracker Red staining, and infected cells are identified by immunofluorescence with an anti-Myc antibody (green). In E, the signals are largely nonoverlapping because most of the Drp1 resides in a cytosolic pool. The results are summarized in F, which depicts the percentage of infected cells belonging to each of four morphological classifications. 600 cells were scored for each infection.
Figure 7. Rescue of Mfn1-deficient cells Mfn1 mutant cells. Mfn1 mutant cells (A) were infected with a retrovirus expressing Myc epitope-tagged versions of Mfn1 (B), Mfn1(K88T) (C), Mfn2 (D), or dominant- negative Drp1(K38A) (E). In the merged images, mitochondrial morphology is revealed by MitoTracker Red staining, and infected cells are identified by immunofluorescence with an anti-Myc antibody (green). In E, the signals are largely nonoverlapping because most of the Drp1 resides in a cytosolic pool. The results are summarized in F, which depicts the percentage of infected cells belonging to each of four morphological classifications. 600 cells were scored for each infection.CC BY-NC-SA · PubMed Central
Figure 8. Rescue of Mfn2-deficient cells Mfn2 mutant cells. Mfnz mutant cells (A) were infected with a retrovirus expressing Myc epitope-tagged versions of Mfn2 (B), Mfn2(K109A) (C), Mfn1 (D), or dominant-negative Drp1(K38A) (E). The cells were stained as in Fig. 7. The results are summarized in F. 200 cells were scored for each infection.
Figure 8. Rescue of Mfn2-deficient cells Mfn2 mutant cells. Mfnz mutant cells (A) were infected with a retrovirus expressing Myc epitope-tagged versions of Mfn2 (B), Mfn2(K109A) (C), Mfn1 (D), or dominant-negative Drp1(K38A) (E). The cells were stained as in Fig. 7. The results are summarized in F. 200 cells were scored for each infection.CC BY-NC-SA · PubMed Central
Figure 9. Immunoprecipitation of Mfn complexes. (A) Wild-type cells were infected with retroviruses expressing Myc- or HA-tagged Mfn1 (labeled 1), Mfn2 (labeled 2), or Drp1 (labeled D) as indicated on top. Anti-Myc immunoprecipitates (top) and total cell lysates (bottom) were analyzed by Western blotting against the HA epitope. The total cell lysate samples contain one sixth cell equivalents compared with the immunoprecipitates. (B) Anti-Myc immunoprecipitates (top) and total cell lysates (bottom) from Mfn1 or Mfn2 mutant cells (indicated on top) were used in an analysis similar to A.
Figure 9. Immunoprecipitation of Mfn complexes. (A) Wild-type cells were infected with retroviruses expressing Myc- or HA-tagged Mfn1 (labeled 1), Mfn2 (labeled 2), or Drp1 (labeled D) as indicated on top. Anti-Myc immunoprecipitates (top) and total cell lysates (bottom) were analyzed by Western blotting against the HA epitope. The total cell lysate samples contain one sixth cell equivalents compared with the immunoprecipitates. (B) Anti-Myc immunoprecipitates (top) and total cell lysates (bottom) from Mfn1 or Mfn2 mutant cells (indicated on top) were used in an analysis similar to A.CC BY-NC-SA · PubMed Central
Figure 10. Models. (A) The protective role of mitochondrial fusion. At a low rate, individual mitochondria stochastically lose function. In wild-type cells, a defective mitochondrion (shaded) undergoes fusion with functional mitochondria and regains activity. In Mfn- deficient cells, such rescue occurs at a much reduced rate. (B) Three modes of mitofusin action. Mitofusins form homotypic and heterotypic complexes that lead to three activities (I, II, III) involving fusion. See Discussion for details. Mfn1 mutant cells contain only activity III; Mfn2 mutant cells contain only activity I. Since disruption of either Mfn1 or Mfn2 fragments mitochondria and results in distinct phenotypes, MEFs appear to use all three activities (indicated by asterisks). In contrast, trophoblast giant cells predominantly use activity III because they are affected in Mfn2 mutants and not Mfn1 mutants.
Figure 10. Models. (A) The protective role of mitochondrial fusion. At a low rate, individual mitochondria stochastically lose function. In wild-type cells, a defective mitochondrion (shaded) undergoes fusion with functional mitochondria and regains activity. In Mfn- deficient cells, such rescue occurs at a much reduced rate. (B) Three modes of mitofusin action. Mitofusins form homotypic and heterotypic complexes that lead to three activities (I, II, III) involving fusion. See Discussion for details. Mfn1 mutant cells contain only activity III; Mfn2 mutant cells contain only activity I. Since disruption of either Mfn1 or Mfn2 fragments mitochondria and results in distinct phenotypes, MEFs appear to use all three activities (indicated by asterisks). In contrast, trophoblast giant cells predominantly use activity III because they are affected in Mfn2 mutants and not Mfn1 mutants.CC BY-NC-SA · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 52 references

Research works printed but not linked: 3

  1. 1981, Eur. J. Cell Biol., 25, 175
  2. 1986, Mol. Cell. Biol., 6, 1204
  3. 1998, Mol. Med. Today., 4, 286

Cited by

Sorted by citations

Items 801–900 of 1,010

Related

See all

Metadata

Authors
Chen, H., Detmer, S. A., Ewald, A. J., Griffin, E. E., Fraser, S. E., & Chan, D. C.
Affiliations
California Institute of Technology (US)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2003-01-13
Volume
160
Issue
2
Pages
189-200
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.200211046
PMC
PMC2172648
OpenAlex
W2073095545
Cited by
2,601
References
52
Citations by year
2026: 111 · 2025: 132 · 2024: 143 · 2023: 149 · 2022: 173 · 2021: 192 · 2020: 170 · 2019: 149 · 2018: 160 · 2017: 127 · 2016: 121 · 2015: 115 · 2014: 126 · 2013: 103 · 2012: 112
FWCI
14.48
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • CaltechAUTHORS (California Institute of Technology) (submittedVersion, cc-by-nc-sa)
  • Warwick Research Archive Portal (University of Warwick) (submittedVersion)
Funders
National Institute of General Medical Sciences (1 R01 GM62967-01, R01GM062967, T32GM007616, GM07616)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
mitofusins, Mfn1, mitofusin 2, mitochondrial fusion, mitochondrial fission, mitochondrial morphology, embryonic development, mitochondrial membrane potential, mitochondrial fragmentation, embryonic fibroblasts, mouse mutants
MeSH terms
Animals, Cell Movement, Cells, Cultured, Cytoskeletal Proteins, Embryo, Mammalian, Female, Embryonic and Fetal Development, Fetus, Genes, Lethal, Intracellular Membranes, Male, Membrane Potentials, Membrane Proteins, Mitochondria, Placenta, Trophoblasts, Mice, Knockout, Gene Expression Regulation, Developmental, GTP Phosphohydrolases, Embryo Loss, Mitochondrial Proteins, Membrane Transport Proteins, Mitochondrial Membrane Transport Proteins, Macromolecular Substances, Utrophin, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review