Identification of murine Fzo homologues
We identified two murine homologues of fzo
from a mouse cDNA library. In accordance with the nomenclature for the human mitofusins (Santel and Fuller, 2001
), we designate these murine homologues as Mfn1 and Mfn2. Linkage analysis placed Mfn1
at the proximal end of mouse chromosome 3 (~12–13 cM) in a region syntenic to human 3q25-26. Mfn2
was localized to the distal end of mouse chromosome 4 (~70–80 cM) in a region syntenic to human 1p36.
As with the fzo
genes from Drosophila melanogaster
(Santel and Fuller, 2001
; Hwa et al., 2002
), Homo sapiens
(Santel and Fuller, 2001
), and Saccharomyces cerevisiae
(Hermann et al., 1998
; Rapaport et al., 1998
), each murine Mfn
gene encodes a predicted transmembrane GTPase. The transmembrane segment is flanked by two regions containing hydrophobic heptad repeats, hallmarks of coiled-coil regions (Lupas, 1996
). Mfn1 and Mfn2 are 81% similar to each other and are both 52% similar to Drosophila
Generation of knockout mice deficient in Mfn1 and Mfn2
We constructed gene replacement vectors for Mfn1
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 (). 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 C and C), demonstrate that an intact GTPase domain is essential for Fzo function. Therefore, the disrupted Mfn1
alleles described here should be null alleles. Both Southern blot and PCR analysis confirmed germline transmission of the targeted alleles (). Importantly, Western blot analysis using affinity-purified antisera raised against Mfn1 or Mfn2 confirmed loss of the targeted protein in homozygous mutant lysates ().
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. (more ...)
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 (more ...)
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 (more ...)
Embryonic lethality in homozygous mutant mice
Both the Mfn1 and Mfn2 knockouts demonstrate full viability and fertility in heterozygous animals but result in embryonic lethality of homozygous mutants. For the Mfn1 knockout line, normal frequencies of live mutant embryos were obtained up to embryonic day (e)10.5 (noon of the day a copulatory plug was detected is designated e0.5). However, by e11.5, 20% of the mutant embryos were resorbed, indicating inviability. At e12.5, most identifiable mutant embryos were resorbed (86%), and additional resorptions were so advanced that they could not be genotyped. Although resorptions were not evident until e11.5, by e8.5 all mutant embryos were significantly smaller and showed pronounced developmental delay. In addition, mutant embryos were often deformed.
In the Mfn2 knockout line, normal numbers of live homozygous mutant embryos were recovered up to e9.5. However, starting at e10.5, 29% of homozygous mutant embryos were in the process of resorption. By e11.5, 87% of homozygous mutant embryos were resorbed. The live homozygous mutant embryos at e9.5 and e10.5 were slightly smaller than their littermates but were otherwise well developed and showed no obvious malformations.
Preliminary studies indicate that double homozygous mutant embryos die earlier than either single mutant and show greater developmental delay (unpublished results). This observation suggests that Mfn1 and Mfn2 are both required, and may have a cooperative relationship, in a particular developmental event early in embryogenesis.
Placental defects in Mfn2 mutant mice
We examined placental development in detail because placental insufficiency is one of the most common causes of midgestation lethality (Copp, 1995
). Hematoxylin-eosin–stained histological sections of placentae from wild-type and heterozygous embryos showed the typical trilaminar structure composed of a proximal labyrinthine layer, a middle spongiotrophoblast layer, and a distal, circumferential giant cell layer ( E). Trophoblast giant cells are polyploid cells (derived from endoreplication, a process where DNA replication proceeds repeatedly without associated cytokinesis) that lie at the critical interface between fetal and maternal tissues and play important roles in hormone production, recruitment of blood vessels, and invasion of the conceptus into the uterine lining (Cross, 2000
). Deficiencies in these cells lead to midgestation lethality (Kraut et al., 1998
; Riley et al., 1998
; Hesse et al., 2000
; Scott et al., 2000
). Strikingly, placentae from Mfn2 mutant embryos reproducibly show an impaired giant cell layer with two defects. The giant cells are deficient in quantity, and the few that are observed contain smaller nuclei, implying a reduction in the number of endoreplication cycles ( F). These observations were confirmed with DAPI staining, which highlights the giant cells because of their high DNA content (, A–D). No defects in placental development were detected in Mfn1 mutant embryos (unpublished data) despite expression of Mfn1 in the placenta ( D).
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 (more ...)
Using RNA in situ hybridization, we examined the expression of molecular markers specific for each of the three placental layers. With the giant cell marker PL-I (Faria et al., 1991
), wild-type placentae showed an intense full ring of giant cell staining with multiple layers of giant cells packed in the region distal to the spongiotrophoblast layer ( G). In contrast, Mfn2 mutant placentae showed an incomplete ring of weakly staining giant cells ( H). Moreover, the giant cell layer was generally only one cell layer thick. Similar results were seen in placentae from e8.5 through e10.5. These observations were confirmed with RNA in situ hybridizations (unpublished data) using the additional giant cell markers, proliferin (Lee et al., 1988
) and Hand1 (Riley et al., 1998
). Interestingly, our in situ hybridizations also revealed somewhat weaker staining with the ectoplacental cone and spongiotrophoblast marker 4311 (Lescisin et al., 1988
), suggesting an additional but more subtle defect in this placental layer (unpublished data). No differences in staining between wild-type and Mfn2 mutant sections were observed using TEF5 (unpublished data), a marker for the labyrinthine layer (Jacquemin et al., 1998
). Together, these findings suggest that the Mfn2 mutant embryos are dying in midgestation secondary to an inadequate placenta.
Both Mfn1- and Mfn2-deficient cells have aberrant mitochondrial morphology
To examine mitochondrial morphology in Mfn-deficient cells, we derived mouse embryonic fibroblasts (MEFs) from e10.5 embryos. The MEF cultures were infected with a retrovirus expressing mitochondrially targeted enhanced yellow fluorescent protein (EYFP). Wild-type MEFs showed a range of mitochondrial morphologies. The major morphological class, which encompassed >90% of wild-type cells, was characterized by a network of extended wavy tubules distributed in a roughly radial manner throughout the cytoplasm. Such cells had no or only a few spherical mitochondria. The length of these mitochondrial tubules ranged from several microns to >10 μm (, arrow). A much smaller morphological class, which encompassed only ~6% of wild-type cells, had mitochondria that were mostly spherical and were termed as “fragmented.”
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 (more ...)
In contrast, Mfn1 mutant MEFs had dramatically fragmented mitochondria (). Greater than 95% of these cells contained only severely fragmented mitochondria, whereas only 1–2% of cells contained any significant tubules. Similarly, in 85% of Mfn2 mutant fibroblasts the mitochondria appeared mostly as spheres or ovals (). Only a small minority of mutant cells (4.5%) contained significant tubules. Neither Mfn1 nor Mfn2 mutant cells ever displayed the networks of long extended tubules that characterize the largest morphological class in wild-type cells. Thin section EM revealed that in spite of their aberrant dimensions the spherical mitochondria in these mutant cells contained both cristae and the typical double membrane (unpublished data). These results are consistent with the hypothesis that Mfn1 and Mfn2 play major roles in mitochondrial fusion.
To determine whether mitochondrial defects underlie the placental insufficiency of Mfn2 mutant embryos, we derived trophoblast stem (TS) cell lines from wild-type and mutant blastocysts. With MitoTracker Red staining, the cells within wild-type TS colonies displayed a network of long mitochondrial tubules ( G). In contrast, Mfn2 mutant TS cells showed only spherical mitochondria ( H).
Although loss of either Mfn1 or Mfn2 results in fragmentation of mitochondrial tubules, the two mutations lead to characteristic mitochondrial morphologies that are readily distinguishable. Loss of Mfn1 leads to a greater degree of fragmentation, resulting in either very short mitochondrial tubules or very small spheres that are uniform in size, with diameters no larger than that of a normal tubule. In contrast, many Mfn2 mutant cells exhibit mitochondrial spheres of widely varying sizes. Interestingly, the diameters of some of the spherical mitochondria in both Mfn2 mutant MEF and TS cells are several times larger than the diameters of mitochondrial tubules in wild-type cells. Because a simple defect in mitochondrial fusion would be expected to result in shorter tubules and small spheres with the diameter of normal tubules, these observations suggest that in addition to controlling mitochondrial fusion, Mfn2 is involved in maintaining tubular shape. Together, these results suggest that even though both Mfn1 and Mfn2 are expressed in fibroblasts, each protein is essential for mitochondrial fusion and may have some distinct functions.
Altered mitochondrial dynamics in Mfn-deficient cells
Because mitochondria are such dynamic organelles, we used time-lapse confocal microscopy to determine whether mutant MEFs display aberrations in mitochondrial dynamics. In wild-type cells, the mitochondria are highly motile, and at least one apparent fusion or fission event was observed for most mitochondria during 20-min recordings ( A; video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1
). However, in both Mfn1 and Mfn2 mutant cells the ovoid or spherical mitochondria undergo fusion events much less frequently (videos 2 and 3, available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1
), although a few such events can be found ( C).
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 (more ...)
In addition to reduced fusion, our time-lapse videos revealed striking defects in the mobility of mitochondria in mutant cells. Most mitochondria in wild-type cells were tubular, were directed radially, and moved back and forth along their long axis on radial tracks ( A). This movement along defined tracks is consistent with reports that mitochondria can be anchored along microtubule or actin filaments, depending on the cell type (Nangaku et al., 1994
; Morris and Hollenbeck, 1995
; Tanaka et al., 1998b
). The mitochondria of Mfn-deficient cells display a dramatic alteration in mobility; the spherical or ovoid mitochondria in mutant cells show random “Brownian-like” movements (). This lack of directed movement is not due to disorganization of the cytoskeleton because staining with an antitubulin mAb or rhodamine-phalloidin revealed no defects (unpublished data; ). Strikingly, in Mfn2 mutant cells with both spherical and short tubular mitochondria, the tubular mitochondria can still move longitudinally along radial tracks, whereas the spherical mitochondria do not. However, the two morphological classes are interchangeable because tubules can project out of spheres and subsequently move away ( D), or conversely tubules can merge with spheres and lose directed movement. These observations suggest that there is no intrinsic defect in mobilizing Mfn2-deficient mitochondria. Instead, the hampered mobility of spherical mitochondria is secondary to their altered morphology resulting from reduced fusion.
Decreased mitochondrial fusion rates in cells lacking Mfn1 or Mfn2
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 ( 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 () 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 (). 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 ( 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.
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 (more ...)
Stochastic defects in mitochondrial membrane potential
We tested whether Mfn1 and Mfn2 mutant cells lose mtDNA because there is complete loss of mtDNA in fzo1
Δ 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 ( A; unpublished data). In addition, the mitochondria in mutant cells expressed COX1, a mitochondrial protein encoded by mtDNA (). Therefore, unlike fzo1
Δ 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.
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 (more ...)
Although the mutant cells are capable of respiration, we reasoned that functional defects may exist at the level of individual mitochondria. To test this hypothesis, we used MitoTracker Red, a lipophilic cationic dye which is sensitive to the mitochondrial membrane potential, to stain MEFs expressing EYFP targeted to the mitochondrial matrix. In wild-type cells, EYFP-marked mitochondria were typically uniformly stained with MitoTracker Red, resulting in colocalization of the two fluorophores ( D). In 7% of wild-type cells, there existed isolated mitochondria that were labeled with EYFP but failed to sequester MitoTracker Red dye. Such mitochondria were invariably small and spherical as opposed to the tubular mitochondria that predominate in these cells. In Mfn1 mutant cultures, almost every cell contained a subset of mitochondria that showed poor staining with MitoTracker Red ( E). Likewise, in Mfn2 mutant cultures over one third of the cells showed some mitochondria that were marked by EYFP but not by MitoTracker Red ( F). It is likely that the compromised mitochondria detected by this assay initially contained membrane potential because the matrix-targeted EYFP requires an intact membrane potential for import. Because of the high stability of EYFP, mitochondria that subsequently lose or reduce their membrane potential would still contain EYFP fluorescence. These results indicate that while bulk cultures display respiratory activity, the function of individual mitochondria is compromised in the absence of either Mfn1 or Mfn2.
Rescue of mitochondrial tubules by modulation of fusion or fission
To unequivocally demonstrate that the mitochondrial morphological defects observed in mutant cells are due to loss of Mfn, we tested whether these defects could be rescued by restoration of Mfn function and whether such rescue depended on an intact GTPase domain. In uninfected Mfn1 mutant cultures or mutant cultures infected with an empty virus, >95% of the cells showed highly fragmented mitochondria that lacked interconnections (). However, when Mfn1 mutant cultures were infected with a retrovirus expressing Mfn1-Myc >90% of the infected cells showed extensive mitochondrial networks consisting of long tubules (). Similarly, >80% of Mfn2 mutant cells infected with a Mfn2-expressing retrovirus showed predominantly tubular mitochondria (). In contrast, constructs carrying mutations in the GTPase G1 motif (Mfn1[K88T] and Mfn2[K109A]) had greatly reduced ability to restore tubular structure (, and ). These results show that both Mfn1 and Mfn2 promote mitochondrial fusion in a GTPase-dependent manner.
Although both Mfn1 and Mfn2 are clearly required for normal mitochondrial tubules in MEFs, we were able to rescue the morphological defects by overexpression of a single mitofusin. Overexpression of Mfn1 in Mfn2-deficient cells was sufficient for rescue of mitochondrial morphology (). In addition, overexpression of Mfn2 in Mfn1-deficient cells was sufficient to restore mitochondrial tubules (). These results show that although both Mfn1 and Mfn2 are required in vivo for normal mitochondrial morphology in MEFs, when overexpressed, each protein is capable of acting alone to promote mitochondrial fusion. Interestingly, these experiments also reveal differential activities of the two homologues. With Mfn2-deficient cells, both Mfn1-Myc and Mfn2-Myc were equally effective in restoring extensive mitochondrial networks, resulting in rescue in 75–80% of expressing cells ( F). In contrast, with Mfn1-deficient cells, 91% of Mfn1-Myc–infected cells showed entirely tubular networks, but only 25% of Mfn2-Myc–infected cells showed such a phenotype ( F).
Because the morphological defects in mutant cells were clearly reversible, we tested whether inhibition of mitochondrial fission could also restore tubular structure. Drp1 plays a key role in mediating mitochondrial fission. To inhibit fission, we constructed an allele of mouse Drp1 with the same mutation (K38A) as in a dominant-negative allele of human Drp1 (Smirnova et al., 2001
). Infection of either Mfn1 or Mfn2 mutant cells with a retrovirus expressing Drp1(K38A) resulted in a striking restoration of mitochondrial tubules in 90% () and 50% () of the cells, respectively. Although the mitochondrial networks in these infected cells were tubular, they were imperfect compared with the networks obtained by rescue with Mfn1 or Mfn2. First, the thickness of the tubules promoted by Drp1(K38A) were less uniform, often showing regions of thinning attached to thicker knobs. Second, the mitochondrial networks were generally less dispersed, with an increase in density near the cell nucleus that often gave the appearance of circumferential rings. Nevertheless, these results indicate that a reduction in fusion caused by loss of Mfn1 or Mfn2 can be compensated, at least partially, by a reduction in fission.
Mfn1 and Mfn2 form homotypic and heterotypic oligomers
Because both Mfn1 and Mfn2 are essential for normal mitochondrial morphology, it is possible that they act in concert to promote mitochondrial fusion. To explore this idea, we tested whether these two proteins form a complex when expressed in wild-type MEF cells ( A). Our results revealed three types of intermolecular interactions. First, both Mfn1 and Mfn2 can form homotypic complexes. Mfn1-HA is coimmunoprecipitated with Mfn1-Myc; analogously, Mfn2-HA is coimmunoprecipitated with Mfn2-Myc. Second, Mfn1 and Mfn2 form heterotypic complexes. Mfn2-HA is coimmunoprecipitated with Mfn1-Myc; conversely, Mfn1-HA is coimmunoprecipitated with Mfn2-Myc. All of these interactions are specific because no HA-tagged protein is found in the anti-Myc immunoprecipitate when the Myc-tagged protein is omitted (unpublished data) or when a control Drp1-Myc is used.
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 (more ...)
It is a formal possibility that the homotypic interactions detected might not be strictly homotypic due to the expression of endogenous Mfn1 and Mfn2 in the parental cells. To relieve these concerns, we also performed the immunoprecipitation assay in mutant MEFs ( B). We find that Mfn1-Mfn1 homotypic complexes are formed in Mfn2-deficient cells, thereby showing that this interaction is strictly homotypic. Likewise, we also detect Mfn2-Mfn2 homotypic complexes in Mfn1-deficient cells.