PMID:15899901

Citation	Chen, H, Chomyn, A and Chan, DC (2005) Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J. Biol. Chem. 280:26185-92
Abstract	Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.
Links	PubMed Online version:10.1074/jbc.M503062200
Keywords	Alleles; Animals; Blotting, Western; Cell Proliferation; Fibroblasts/metabolism; GTP Phosphohydrolases/genetics; GTP Phosphohydrolases/physiology; Genotype; Green Fluorescent Proteins/metabolism; Membrane Potentials; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria/metabolism; Mitochondria/physiology; Mutation; Oxygen/chemistry; Oxygen Consumption; Polyethylene Glycols/chemistry; RNA/chemistry; RNA Interference; Recombinant Fusion Proteins/chemistry; Time Factors
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MOUSE:MFN1		GO:0008053: mitochondrial fusion	ECO:0000315:		P		Mfn1 clearly demonstrates positive regulation amount of mitochondrial fusion, as seen and inferred from Table 1 Uniprot Nomenclature: Mitofusin-1 (MFN1_MOUSE) Uniprot ID: Q811U4 Organism: Mus musculus (Mouse)	complete CACAO 12464
MOUSE:MFN1		GO:0010918: positive regulation of mitochondrial membrane potential	ECO:0000316:	UniProtKB:Q80U63	P		MFn1 reveals that it is a positive regulator of mitochondrial membrane potential as loss of membrane potential was reversible when MFN-null cells when they began to over express Mfn1. Figure 4A-C Uniprot Nomenclature: Mitofusin-1 (MFN1_MOUSE) Organism: Mus musculus (Mouse) "Consistent with our previous study (3), we found that both Mfn1-null and Mfn2-null cells have mild heterogeneity in mitochondrial ΔΨ.2 Most Mfn1-null cells showed some mitochondria with weak MitoTracker Red staining, but this was restricted to only a few mitochondria (1–10/cell). With Mfn2-null cells, only a minority of cells showed heterogeneity in MitoTracker staining, and again this defect was restricted to a few mitochondria. In contrast, 100% of Mfn-dm cells showed widespread heterogeneity of mitochondrial ΔΨ, with a large fraction of the mitochondrial population failing to stain well with MitoTracker Red (Fig. 4, A–C). The loss of mitochondrial ΔΨ was reversible with complete restoration of ΔΨ in Mfn-null cells overexpressing Mfn1.2 Depletion of OPA1 by RNAi similarly led to wide-spread loss of mitochondrial ΔΨ (Fig. 4, D–F). Cells overexpressing OPA1 showed no ΔΨ defects (Fig. 4, G–I). Taken together with the PEG fusion data, these results indicate that the severity of mitochondrial ΔΨ loss correlates with the severity of fusion deficiency. "	complete CACAO 12467
MOUSE:MFN1		GO:0030307: positive regulation of cell growth	ECO:0000315:		P		Mfn1 is needed for cell growth, as seen in Supplemental Fig. S1 and as Mfn2-null cells depleted of Mfn1 by shRNA grew to only 18% of the parental Mfn2-null control. Figure 3B also showed growth rescue by reintroduction of Mfn1. Uniprot Nomenclature: Mitofusin-1 (MFN1_MOUSE) Uniprot ID: Q811U4 Organism: Mus musculus (Mouse)	complete CACAO 12468
MOUSE:MFN1		GO:0070584: mitochondrion morphogenesis	ECO:0000316:	UniProtKB:Q80U63	P		Mfn-dm (double mutant) cells revealed the necessity of Mfn1 and Mfn2 for mitochondrial morphology, Therefore through this double mutant, the reliance of Mfn2 and Mfn1 for Mitochondrial morphology is confirmed. Seen in FIG. 1. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12711
MOUSE:MFN1		GO:0008053: mitochondrial fusion	ECO:0000316:	UniProtKB:Q80U63	P		Mfn-dm (double mutant) cells had 0% mitochondrial fusion, as seen on Table I. Table 1 and FIG. 2 illustrate the importance of both Mfn1 and Mfn2 for mitochondrial fusion. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12712
MOUSE:MFN1		GO:0016049: cell growth	ECO:0000316:	UniProtKB:Q80U63	P		Mfn-dm (double mutant) cells showed growth defects, as seen on FIG. 3. Thus, Mfn1 and Mfn2 are required for appropriate cell growth. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12713
MOUSE:MFN1		GO:0009060: aerobic respiration	ECO:0000316:	UniProtKB:Q80U63	P		Mfn-dm (double mutant) showed respiratory defects, as seen on FIG. 5. These defects were reversible, as seen in Mfn1 and Mfn2, thus showing the importance of Mfn1 and 2 for aerobic respiration. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12714
MOUSE:MFN2		GO:0008053: mitochondrial fusion	ECO:0000315:		P		Mfn2 demonstrates positive regulation of mitochondrial fusion, as seen and inferred from Table 1 Uniprot Nomenclature: Mitofusin-2 (MFN2_MOUSE) Organism: Mus musculus (Mouse)	complete CACAO 12465
MOUSE:MFN2		GO:0030307: positive regulation of cell growth	ECO:0000316:	UniProtKB:Q811U4 UniProtKB:P10415	P		Reintroduction of Mfn2 and Mfn1 in growth defected cells, showed cell growth rescue (as seen at Figure 3B) with human cells. Uniprot Nomenclature: Mitofusin-2 (MFN2_MOUSE) Uniprot ID: Q80U63 Uniprot Nomenclature: Apoptosis regulator Bcl-2 (Human) Uniprot ID: P10415 Organism: Homo sapiens (Human) Uniprot Nomenclature: Mitofusin-1 (MFN1_MOUSE) Uniprot ID: Q811U4 Organism: Mus musculus (Mouse)	complete CACAO 12470
MOUSE:MFN2		GO:0070584: mitochondrion morphogenesis	ECO:0000316:	UniProtKB:Q811U4	P		Mfn-dm (double mutant) cells revealed the necessity of Mfn1 and Mfn2 for mitochondrial morphology, Therefore through this double mutant, the reliance of Mfn2 and Mfn1 for Mitochondrial morphology is confirmed. Seen in FIG. 1. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12707
MOUSE:MFN2		GO:0008053: mitochondrial fusion	ECO:0000316:	UniProtKB:Q811U4	P		Mfn-dm (double mutant) cells had 0% mitochondrial fusion, as seen on Table I. Table 1 and FIG. 2 illustrate the importance of both Mfn1 and Mfn2 for mitochondrial fusion. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12708
MOUSE:MFN2		GO:0016049: cell growth	ECO:0000316:	UniProtKB:Q811U4	P		Mfn-dm (double mutant) cells showed growth defects, as seen on FIG. 3. Thus, Mfn1 and Mfn2 are needed for appropriate cell growth. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12709
MOUSE:MFN2		GO:0009060: aerobic respiration	ECO:0000316:	UniProtKB:Q811U4	P		Mfn-dm (double mutant) showed respiratory defects, as seen on FIG. 5. These defects were reversible, as seen in Mfn1 and Mfn2, thus showing the importance of Mfn1 and 2 for aerobic respiration. Mitofusin-2 and Mitofusin-1 Mus musculus (Mouse)	complete CACAO 12710
MOUSE:OPA1		GO:0030307: positive regulation of cell growth	ECO:0000315:		P		OPA1 is needed for cell growth, as OPA1-RNAi cells grew much more slowly and attained only 15% of the density of mock-infected control cells, as seen in Figure 3C. Uniprot Nomenclature: Dynamin-like 120 kDa protein, mitochondrial (OPA1_MOUSE) Uniprot ID: P58281 Organism: Mus musculus (Mouse)	complete CACAO 12469
MOUSE:OPA1		GO:0070584: mitochondrion morphogenesis	ECO:0000315:		P		OPA1-RNAi cells showed a lack of mitochondrial morphologies. OPA1 is thus needed for any mitochondrial morphology, as seen in FIG. 1. Dynamin-like 120 kDa protein, mitochondrial Mouse	complete CACAO 12715
MOUSE:OPA1		GO:0008053: mitochondrial fusion	ECO:0000315:		P		OPA1-RNAi cells had 0% mitochondrial fusion as seen in TABLE 1. Moreover, these results (TABLE 1 and FIGURE 2) demonstrate that OPA1 is strictly required for mitochondrial fusion. Dynamin-like 120 kDa protein, mitochondrial Mouse	complete CACAO 12716
MOUSE:OPA1		GO:0016049: cell growth	ECO:0000315:		P		As seen in Figure 3, growth defects were seen in OPA1-RNAi cells. Indicating that OPA1 is needed for proper cell growth. Dynamin-like 120 kDa protein, mitochondrial Mouse	complete CACAO 12717
MOUSE:OPA1		GO:0009060: aerobic respiration	ECO:0000315:		P		Severe aerobic respiratory defects are observable in OPA1-RNAi cells. These were reversible when restoration of OPA1 was done, as seen in FIG 5, F and G. Dynamin-like 120 kDa protein, mitochondrial Mouse	complete CACAO 12718

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