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

Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for... JCB Article Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development 1 1 1,2 1 1,2 1 Hsiuchen Chen, Scott A. Detmer, Andrew J. Ewald, Erik E. Griffin, Scott E. Fraser, and David C. Chan 1 2 Division of Biology and Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125 itochondrial morphology is determined by a dy- in mitochondrial fusion. Moreover, we find that Mfn1 and namic equilibrium between organelle fusion and Mfn2 form homotypic and heterotypic complexes and fission, but the significance of these processes in show, by rescue of mutant cells, that the homotypic com- vertebrates is unknown. The mitofusins, Mfn1 and plexes are functional for fusion. We conclude that Mfn1 Mfn2, have been shown to affect mitochondrial morphol- and Mfn2 have both redundant and distinct functions and ogy when overexpressed. We find that mice deficient in ei- act in three separate molecular complexes to promote ther Mfn1 or Mfn2 die in midgestation. However, whereas mitochondrial fusion. Strikingly, a subset of mitochondria Mfn2 mutant embryos have a specific and severe disruption in mutant cells lose membrane potential. Therefore, mito- of the placental trophoblast giant cell layer, Mfn1-deficient chondrial fusion is essential for embryonic development, giant cells are normal. Embryonic fibroblasts lacking Mfn1 and by enabling cooperation between mitochondria, has or Mfn2 display distinct types of fragmented mitochondria, protective effects on the mitochondrial population. a phenotype we determine to be due to a severe reduction Introduction Mitochondria are remarkably dynamic organelles. Time- fzo1 yeast form “petite” colonies that lack mitochondrial lapse microscopy of living cells reveals that mitochondria DNA (mtDNA) (Hermann et al., 1998; Rapaport et al., undergo constant migration and morphological changes 1998). Furthermore, there is a disruption of mating-induced (Bereiter-Hahn and Voth, 1994; Nunnari et al., 1997; mitochondrial fusion (Hermann et al., 1998). Humans contain Rizzuto et al., 1998). Even in cells with a seemingly “stable” two Fzo homologues, termed mitofusin (Mfn)1 and Mfn2, that network of mitochondrial tubules, there are frequent and can alter mitochondrial morphology when overexpressed in cell continual cycles of mitochondrial fusion and fission, opposing lines (Santel and Fuller, 2001; Rojo et al., 2002). Both mito- processes that exist in equilibrium and serve to maintain the fusins are broadly expressed (Rojo et al., 2002), and therefore overall architecture of these organelles (Bereiter-Hahn and their functional redundancy is unclear. Voth, 1994; Nunnari et al., 1997). Despite our increasing knowledge about the importance In both yeast and flies, mitochondrial fusion is controlled by of mitochondrial fusion in lower eukaryotes, there is disagree- the nuclearly encoded mitochondrial transmembrane GTPase, ment about its physiological role in vertebrates. Doubts have fuzzy onions (Fzo).* In Drosophila, Fzo is specifically and been raised about the frequency and importance of mito- transiently expressed in spermatids. Disruption of Fzo prevents chondrial fusion in cultured mammalian cells (Enriquez et developmentally regulated mitochondrial fusion in postmeiotic al., 2000). However, several observations suggest that this spermatids and results in male sterility (Hales and Fuller, basic cellular process indeed plays a significant role in vertebrate 1997). In budding yeast, deletion of FZO1 disrupts the highly cells. First, ultrastructural studies of mitochondria show that branched, tubular mitochondrial network typical of normal dramatic transitions occur during the development of certain cells and results in numerous small spherical mitochondria. tissues. For example, the mitochondria of rat cardiac muscle and diaphragm skeletal muscle appear as isolated ellipses or The online version of this article contains supplemental material. tubules in embryonic stages but then reorganize into reticular Address correspondence to David C. Chan, 1200 East California Blvd., networks in the adult (Bakeeva et al., 1978, 1981, 1983). It MC114-96, Pasadena, CA 91125. Tel.: (626) 395-2670. Fax: (626) 395- is likely that these progressive morphological changes occur 8826. E-mail: [email protected] through mitochondrial fusion. Second, time-lapse fluores- *Abbreviations used in this paper: Drp, dynamin-related protein; e, embry- cence microscopy of cultured HeLa cells shows that the mi- onic day; EYFP, enhanced YFP; Fzo, fuzzy onions; MEF, mouse embry- tochondria are organized into extensive tubular networks onic fibroblast; Mfn, mitofusin; mtDNA, mitochondrial DNA; PEG, polyethylene glycol; TS, trophoblast stem. that undergo frequent fusion and fission (Rizzuto et al., Key words: membrane fusion; mitochondria; GTPase; mice; knockout 1998). Third, experimentally induced cell hybrids demon-  The Rockefeller University Press, 0021-9525/2003/01/189/12 $8.00 The Journal of Cell Biology, Volume 160, Number 2, January 20, 2003 189–200 http://www.jcb.org/cgi/doi/10.1083/jcb.200211046 189 The Journal of Cell Biology 190 The Journal of Cell Biology | Volume 160, Number 2, 2003 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). -Actin was used as a loading control. strate rapid mtDNA mixing (Hayashi et al., 1994) and com- cDNA library. In accordance with the nomenclature for the plementation of mtDNA gene products (Nakada et al., human mitofusins (Santel and Fuller, 2001), we designate 2001b; Ono et al., 2001). Finally, mitochondrial dynamics these murine homologues as Mfn1 and Mfn2. Linkage anal- have been implicated in the regulation of apoptosis. Induc- ysis placed Mfn1 at the proximal end of mouse chromosome tion of cell death is sometimes associated with fragmentation 3 (12–13 cM) in a region syntenic to human 3q25-26. of the mitochondrial network. This fragmentation requires Mfn2 was localized to the distal end of mouse chromosome dynamin-related protein (Drp)1, which is involved in mito- 4 (70–80 cM) in a region syntenic to human 1p36. chondrial fission (Frank et al., 2001). As with the fzo genes from Drosophila melanogaster (Santel To assess the physiological role of mitochondrial fusion in and Fuller, 2001; Hwa et al., 2002), Homo sapiens (Santel vertebrates, we have generated knockout mice for Mfn1 and and Fuller, 2001), and Saccharomyces cerevisiae (Hermann et Mfn2. Our analysis reveals that Mfn1 and Mfn2 are each es- al., 1998; Rapaport et al., 1998), each murine Mfn gene en- sential for embryonic development and mitochondrial fu- codes a predicted transmembrane GTPase. The transmem- sion. These mitofusins can exist as both homotypic and het- brane segment is flanked by two regions containing hydro- erotypic oligomers and therefore can cooperate as well as act phobic heptad repeats, hallmarks of coiled-coil regions individually to promote mitochondrial fusion. In addition, (Lupas, 1996). Mfn1 and Mfn2 are 81% similar to each our results suggest that mitochondrial fusion functions to al- other and are both 52% similar to Drosophila Fzo. low cooperation between mitochondria, thereby protecting mitochondria from respiratory dysfunction. Generation of knockout mice deficient in Mfn1 and Mfn2 We constructed gene replacement vectors for Mfn1 and Mfn2 using the neomycin resistance gene for positive selection and Results the diphtheria toxin subunit A gene for negative selection. In Identification of murine Fzo homologues both cases, a stop codon was engineered at the very beginning We identified two murine homologues of fzo from a mouse of the GTPase domain near the NH terminus (Fig. 1, A and The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 191 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. E). In addition, the resulting genomic loci each contain a re- out line, normal frequencies of live mutant embryos were placement of the G1 and G2 motifs of the GTPase domain obtained up to embryonic day (e)10.5 (noon of the day a cop- with the neomycin expression cassette. These universal GTP- ulatory plug was detected is designated e0.5). However, by ase motifs are crucial for binding of the  and  phosphates e11.5, 20% of the mutant embryos were resorbed, indicating of GTP and for Mg coordination (Bourne et al., 1991; inviability. At e12.5, most identifiable mutant embryos were Sprang, 1997). Genetic analyses in Drosophila and Saccharo- resorbed (86%), and additional resorptions were so advanced myces cerevisiae (Hales and Fuller, 1997; Hermann et al., that they could not be genotyped. Although resorptions were 1998), as well as our own studies (see Fig. 7 C and Fig. 8 C), not evident until e11.5, by e8.5 all mutant embryos were sig- demonstrate that an intact GTPase domain is essential for nificantly smaller and showed pronounced developmental de- Fzo function. Therefore, the disrupted Mfn1 and Mfn2 alleles lay. In addition, mutant embryos were often deformed. described here should be null alleles. Both Southern blot and In the Mfn2 knockout line, normal numbers of live ho- PCR analysis confirmed germline transmission of the targeted mozygous mutant embryos were recovered up to e9.5. How- alleles (Fig. 1, B, C, F, and G). Importantly, Western blot ever, starting at e10.5, 29% of homozygous mutant embryos analysis using affinity-purified antisera raised against Mfn1 or were in the process of resorption. By e11.5, 87% of homozy- Mfn2 confirmed loss of the targeted protein in homozygous gous mutant embryos were resorbed. The live homozygous mutant lysates (Fig. 1, D and H). mutant embryos at e9.5 and e10.5 were slightly smaller than their littermates but were otherwise well developed and Embryonic lethality in homozygous mutant mice showed no obvious malformations. Both the Mfn1 and Mfn2 knockouts demonstrate full viabil- Preliminary studies indicate that double homozygous mu- ity and fertility in heterozygous animals but result in embry- tant embryos die earlier than either single mutant and show onic lethality of homozygous mutants. For the Mfn1 knock- greater developmental delay (unpublished results). This ob- The Journal of Cell Biology 192 The Journal of Cell Biology | Volume 160, Number 2, 2003 servation suggests that Mfn1 and Mfn2 are both required, and may have a cooperative relationship, in a particular de- velopmental event early in embryogenesis. Placental defects in Mfn2 mutant mice We examined placental development in detail because placen- tal insufficiency is one of the most common causes of midges- tation lethality (Copp, 1995). Hematoxylin-eosin–stained histological sections of placentae from wild-type and heterozy- gous embryos showed the typical trilaminar structure com- posed of a proximal labyrinthine layer, a middle spongiotro- phoblast layer, and a distal, circumferential giant cell layer (Fig. 2 E). Trophoblast giant cells are polyploid cells (derived from endoreplication, a process where DNA replication pro- ceeds 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 quan- tity, and the few that are observed contain smaller nuclei, im- plying a reduction in the number of endoreplication cycles (Fig. 2 F). These observations were confirmed with DAPI staining, which highlights the giant cells because of their high DNA content (Fig. 2, A–D). No defects in placental develop- ment were detected in Mfn1 mutant embryos (unpublished data) despite expression of Mfn1 in the placenta (Fig. 1 D). Using RNA in situ hybridization, we examined the ex- pression 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 gi- ant cell staining with multiple layers of giant cells packed in the region distal to the spongiotrophoblast layer (Fig. 2 G). In contrast, Mfn2 mutant placentae showed an incomplete ring of weakly staining giant cells (Fig. 2 H). Moreover, the giant cell layer was generally only one cell layer thick. Simi- lar results were seen in placentae from e8.5 through e10.5. These observations were confirmed with RNA in situ hy- Figure 3. Morphological defects in mitochondria of mutant cells. bridizations (unpublished data) using the additional giant (A–F) Mitochondrial morphology in wild-type (A and B), Mfn1 mutant cell markers, proliferin (Lee et al., 1988) and Hand1 (Riley (C and D), and Mfn2 mutant (E and F) MEF cells. MEFs expressing et al., 1998). Interestingly, our in situ hybridizations also re- mitochondrial EYFP (green) were counterstained with rhodamine- phalloidin (red). (B, D, and F) Higher magnification images of the vealed somewhat weaker staining with the ectoplacental boxed areas in A, C, and E, respectively. Arrow indicates a tubule cone and spongiotrophoblast marker 4311 (Lescisin et al., 10 m in length. (G and H) Mitochondrial morphology in live 1988), suggesting an additional but more subtle defect in wild-type (G) and mutant (H) TS cells. The mitochondria were this placental layer (unpublished data). No differences in stained with MitoTracker Red, and the nuclei were stained with staining between wild-type and Mfn2 mutant sections were Syto16 (green). Several cells are tightly clustered. 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 logical class, which encompassed 90% of wild-type cells, midgestation secondary to an inadequate placenta. was characterized by a network of extended wavy tubules distributed in a roughly radial manner throughout the cyto- Both Mfn1- and Mfn2-deficient cells have aberrant plasm. Such cells had no or only a few spherical mitochon- mitochondrial morphology dria. The length of these mitochondrial tubules ranged from To examine mitochondrial morphology in Mfn-deficient several microns to 10 m (Fig. 3, A and B, arrow). A cells, we derived mouse embryonic fibroblasts (MEFs) from much smaller morphological class, which encompassed only e10.5 embryos. The MEF cultures were infected with a ret- 6% of wild-type cells, had mitochondria that were mostly rovirus expressing mitochondrially targeted enhanced yellow spherical and were termed as “fragmented.” fluorescent protein (EYFP). Wild-type MEFs showed a In contrast, Mfn1 mutant MEFs had dramatically frag- range of mitochondrial morphologies. The major morpho- mented mitochondria (Fig. 3, C and D). Greater than 95% The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 193 of these cells contained only severely fragmented mitochon- dria, whereas only 1–2% of cells contained any significant tu- bules. Similarly, in 85% of Mfn2 mutant fibroblasts the mi- tochondria appeared mostly as spheres or ovals (Fig. 3, E and F). 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 charac- terize 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 mito- chondrial tubules (Fig. 3 G). In contrast, Mfn2 mutant TS cells showed only spherical mitochondria (Fig. 3 H). Although loss of either Mfn1 or Mfn2 results in fragmen- tation of mitochondrial tubules, the two mutations lead to characteristic mitochondrial morphologies that are readily distinguishable. Loss of Mfn1 leads to a greater degree Figure 4. Dynamics of mitochondria in wild-type and mutant of fragmentation, resulting in either very short mitochon- cells. Still frames from time-lapse confocal microscopy. (A) In a wild-type cell, two pairs of mitochondria can be seen moving toward drial tubules or very small spheres that are uniform in size, each other. These pairs contact end-to-end and fuse immediately. with diameters no larger than that of a normal tubule. In Note that mitochondria move along their long axes. (B) In a Mfn1 contrast, many Mfn2 mutant cells exhibit mitochondrial mutant cell, the mitochondria move in an undirected manner. (C) In spheres of widely varying sizes. Interestingly, the diameters a Mfn2 mutant cell, two ovoid mitochondria contact each other of some of the spherical mitochondria in both Mfn2 mutant but do not fuse until much later. Note also the lack of directed MEF and TS cells are several times larger than the diameters movement in most mitochondria. (D) One spherical Mfn2-deficient of mitochondrial tubules in wild-type cells. Because a simple mitochondrion protrudes a tubular extension that separates and defect in mitochondrial fusion would be expected to result then migrates away along its long axis. Images were processed in Adobe Photoshop with the emboss filter, and selected mitochondria in shorter tubules and small spheres with the diameter of were manually highlighted in blue. See also videos 1–3 available at normal tubules, these observations suggest that in addition http://www.jcb.org/cgi/content/full/jcb.200211046/DC1. 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 fi- Morris and Hollenbeck, 1995; Tanaka et al., 1998b). The broblasts, each protein is essential for mitochondrial fusion mitochondria of Mfn-deficient cells display a dramatic alter- and may have some distinct functions. ation in mobility; the spherical or ovoid mitochondria in mutant cells show random “Brownian-like” movements (Fig. 4, B and C). This lack of directed movement is not due Altered mitochondrial dynamics in Mfn-deficient cells to disorganization of the cytoskeleton because staining with Because mitochondria are such dynamic organelles, we used an antitubulin mAb or rhodamine-phalloidin revealed no time-lapse confocal microscopy to determine whether mu- defects (unpublished data; Fig. 3). Strikingly, in Mfn2 mu- tant MEFs display aberrations in mitochondrial dynamics. tant cells with both spherical and short tubular mitochon- In wild-type cells, the mitochondria are highly motile, and dria, the tubular mitochondria can still move longitudinally at least one apparent fusion or fission event was observed for along radial tracks, whereas the spherical mitochondria do most mitochondria during 20-min recordings (Fig. 4 A; not. However, the two morphological classes are inter- video 1, available at http://www.jcb.org/cgi/content/full/ changeable because tubules can project out of spheres and jcb.200211046/DC1). However, in both Mfn1 and Mfn2 subsequently move away (Fig. 4 D), or conversely tubules mutant cells the ovoid or spherical mitochondria undergo can merge with spheres and lose directed movement. These fusion events much less frequently (videos 2 and 3, available observations suggest that there is no intrinsic defect in mobi- at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1), lizing Mfn2-deficient mitochondria. Instead, the hampered although a few such events can be found (Fig. 4 C). mobility of spherical mitochondria is secondary to their al- In addition to reduced fusion, our time-lapse videos re- tered morphology resulting from reduced fusion. vealed striking defects in the mobility of mitochondria in mutant cells. Most mitochondria in wild-type cells were tu- Decreased mitochondrial fusion rates in cells lacking bular, were directed radially, and moved back and forth Mfn1 or Mfn2 along their long axis on radial tracks (Fig. 4 A). This move- ment along defined tracks is consistent with reports that mi- To definitively show that lack of fusion is the basis for the tochondria can be anchored along microtubule or actin fila- fragmented mitochondria in mutant cells, we measured ments, depending on the cell type (Nangaku et al., 1994; mitochondrial fusion activity using a polyethylene glycol The Journal of Cell Biology 194 The Journal of Cell Biology | Volume 160, Number 2, 2003 (PEG) cell fusion assay. A cell line expressing mitochondri- ally targeted dsRed was cocultured with a cell line expressing mitochondrially targeted GFP, and PEG was transiently ap- plied to fuse the cells. Cycloheximide was included to pre- vent 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 exten- sively fused mitochondria (Fig. 5 A) as demonstrated by colocalization of red and green fluorescent signals. In con- trast, when Mfn1 mutant cells were examined 7 h after PEG fusion 57% (n  364) of the fused cells contained predomi- nantly 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 fu- sion, and 8% showed partial fusion. Similarly, 69% (n 202) of fused Mfn2 mutant cells showed predominantly un- fused 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 ob- served even 24 h after PEG treatment (unpublished data). Thus, mutant cells have severely reduced levels of mitochon- drial 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. Figure 5. Mitochondrial fusion assay. PEG fusion of cells containing Stochastic defects in mitochondrial membrane potential mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing We tested whether Mfn1 and Mfn2 mutant cells lose extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 mtDNA because there is complete loss of mtDNA in fzo1 (E) mutant cells displaying predominantly unfused mitochondria. yeast (Hermann et al., 1998; Rapaport et al., 1998). South- (C and F) Magnified views of boxed portions in B and E, respectively. ern blot and PCR analysis showed that both mutant cell (D) Sectoring effect in Mfn1 mutant cell. lines contain normal levels of mtDNA (Fig. 6 A; unpub- lished data). In addition, the mitochondria in mutant cells cultures over one third of the cells showed some mitochon- expressed COX1, a mitochondrial protein encoded by dria that were marked by EYFP but not by MitoTracker Red mtDNA (Fig. 6, B and C). Therefore, unlike fzo1 yeast, (Fig. 6 F). It is likely that the compromised mitochondria Mfn-deficient cells clearly retain mtDNA, and this feature detected by this assay initially contained membrane poten- allows a critical assessment of the relationship between respi- tial because the matrix-targeted EYFP requires an intact ration and mitochondrial fusion. Like wild-type cells, both membrane potential for import. Because of the high stability Mfn1 and Mfn2 mutant cultures showed high rates of en- of EYFP, mitochondria that subsequently lose or reduce dogenous and coupled respiration (unpublished data), indi- their membrane potential would still contain EYFP fluores- cating no gross defects in respiration and further confirming cence. These results indicate that while bulk cultures display that mtDNA products are intact and functional. respiratory activity, the function of individual mitochondria Although the mutant cells are capable of respiration, we is compromised in the absence of either Mfn1 or Mfn2. reasoned that functional defects may exist at the level of in- dividual mitochondria. To test this hypothesis, we used Mi- Rescue of mitochondrial tubules by modulation of toTracker Red, a lipophilic cationic dye which is sensitive to fusion or fission the mitochondrial membrane potential, to stain MEFs ex- To unequivocally demonstrate that the mitochondrial mor- pressing EYFP targeted to the mitochondrial matrix. In phological defects observed in mutant cells are due to loss of wild-type cells, EYFP-marked mitochondria were typically Mfn, we tested whether these defects could be rescued by res- uniformly stained with MitoTracker Red, resulting in colo- toration of Mfn function and whether such rescue depended calization of the two fluorophores (Fig. 6 D). In 7% of wild- on an intact GTPase domain. In uninfected Mfn1 mutant type cells, there existed isolated mitochondria that were la- cultures or mutant cultures infected with an empty virus, beled with EYFP but failed to sequester MitoTracker Red 95% of the cells showed highly fragmented mitochondria dye. Such mitochondria were invariably small and spherical that lacked interconnections (Fig. 7, A and F). However, when as opposed to the tubular mitochondria that predominate in Mfn1 mutant cultures were infected with a retrovirus express- these cells. In Mfn1 mutant cultures, almost every cell con- ing Mfn1-Myc 90% of the infected cells showed extensive tained a subset of mitochondria that showed poor staining mitochondrial networks consisting of long tubules (Fig. 7, B with MitoTracker Red (Fig. 6 E). Likewise, in Mfn2 mutant The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 195 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. and F). Similarly, 80% of Mfn2 mutant cells infected with Because the morphological defects in mutant cells were a Mfn2-expressing retrovirus showed predominantly tubular clearly reversible, we tested whether inhibition of mitochon- mitochondria (Fig. 8, B and F). In contrast, constructs car- drial fission could also restore tubular structure. Drp1 plays a rying mutations in the GTPase G1 motif (Mfn1[K88T] and key role in mediating mitochondrial fission. To inhibit fis- Mfn2[K109A]) had greatly reduced ability to restore tubular sion, we constructed an allele of mouse Drp1 with the same structure (Fig. 7, C and F, and Fig. 8, C and F). These re- mutation (K38A) as in a dominant-negative allele of human sults show that both Mfn1 and Mfn2 promote mitochon- Drp1 (Smirnova et al., 2001). Infection of either Mfn1 or drial fusion in a GTPase-dependent manner. Mfn2 mutant cells with a retrovirus expressing Drp1(K38A) Although both Mfn1 and Mfn2 are clearly required for resulted in a striking restoration of mitochondrial tubules in normal mitochondrial tubules in MEFs, we were able to 90% (Fig. 7, E and F) and 50% (Fig. 8, E and F) of the cells, rescue the morphological defects by overexpression of a respectively. Although the mitochondrial networks in these single mitofusin. Overexpression of Mfn1 in Mfn2-defi- infected cells were tubular, they were imperfect compared cient cells was sufficient for rescue of mitochondrial mor- with the networks obtained by rescue with Mfn1 or Mfn2. phology (Fig. 8, D and F). In addition, overexpression of First, the thickness of the tubules promoted by Drp1(K38A) Mfn2 in Mfn1-deficient cells was sufficient to restore mi- were less uniform, often showing regions of thinning at- tochondrial tubules (Fig. 7, D and F). These results show tached to thicker knobs. Second, the mitochondrial networks that although both Mfn1 and Mfn2 are required in vivo for were generally less dispersed, with an increase in density near normal mitochondrial morphology in MEFs, when overex- the cell nucleus that often gave the appearance of circumfer- pressed, each protein is capable of acting alone to promote ential rings. Nevertheless, these results indicate that a reduc- mitochondrial fusion. Interestingly, these experiments also tion in fusion caused by loss of Mfn1 or Mfn2 can be com- reveal differential activities of the two homologues. With pensated, at least partially, by a reduction in fission. Mfn2-deficient cells, both Mfn1-Myc and Mfn2-Myc were equally effective in restoring extensive mitochondrial net- Mfn1 and Mfn2 form homotypic and works, resulting in rescue in 75–80% of expressing cells heterotypic oligomers (Fig. 8 F). In contrast, with Mfn1-deficient cells, 91% of Mfn1-Myc–infected cells showed entirely tubular net- Because both Mfn1 and Mfn2 are essential for normal mito- works, but only 25% of Mfn2-Myc–infected cells showed chondrial morphology, it is possible that they act in concert such a phenotype (Fig. 7 F). to promote mitochondrial fusion. To explore this idea, we The Journal of Cell Biology 196 The Journal of Cell Biology | Volume 160, Number 2, 2003 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. tested whether these two proteins form a complex when ex- Discussion pressed in wild-type MEF cells (Fig. 9 A). Our results re- An essential role for mitochondrial fusion vealed three types of intermolecular interactions. First, both in mouse development Mfn1 and Mfn2 can form homotypic complexes. Mfn1-HA is coimmunoprecipitated with Mfn1-Myc; analogously, Although mitochondrial dynamics involving fusion and fission Mfn2-HA is coimmunoprecipitated with Mfn2-Myc. Sec- has been previously demonstrated in vertebrates, the physio- ond, Mfn1 and Mfn2 form heterotypic complexes. Mfn2- logical importance of these processes has remained unclear. HA is coimmunoprecipitated with Mfn1-Myc; conversely, Our analysis of Mfn1 and Mfn2 mutant mice demonstrates an Mfn1-HA is coimmunoprecipitated with Mfn2-Myc. All of essential role for mitochondrial fusion in vertebrate develop- these interactions are specific because no HA-tagged protein ment. Both lines of mice die in midgestation. For Mfn2-defi- is found in the anti-Myc immunoprecipitate when the Myc- cient mice, we observe a dramatic disruption in placental de- tagged protein is omitted (unpublished data) or when a con- velopment, most obviously in the paucity of trophoblast giant trol Drp1-Myc is used. cells. Trophoblast cell lines cultured from Mfn2 mutant blas- It is a formal possibility that the homotypic interactions tocysts show fragmentation of mitochondrial tubules, consis- detected might not be strictly homotypic due to the expres- tent with a defect in mitochondrial fusion. These results show sion of endogenous Mfn1 and Mfn2 in the parental cells. To that despite its broad expression pattern (unpublished data) relieve these concerns, we also performed the immunopre- Mfn2 is only required for selective developmental transitions. cipitation assay in mutant MEFs (Fig. 9 B). We find that Trophoblast giant cells are polyploid cells that arise from Mfn1-Mfn1 homotypic complexes are formed in Mfn2- endoreplication, a process often associated with highly meta- deficient cells, thereby showing that this interaction is strictly bolically active cells (Edgar and Orr-Weaver, 2001). Because homotypic. Likewise, we also detect Mfn2-Mfn2 homotypic of their high metabolic rate, we speculate that, in vivo, tro- complexes in Mfn1-deficient cells. phoblast giant cells may be particularly vulnerable to pertur- The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 197 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. bations in mitochondrial dynamics and presumably func- al., 2000; Tieu and Nunnari, 2000), what is the advantage of tion. In fact, preeclampsia, the leading cause of fetal and maintaining mitochondria in a highly dynamic state? maternal morbidity in the United States, is marked by shal- Our analysis of Mfn-deficient cells suggests an answer to this low trophoblast invasion, often resulting in fetal growth re- question. Although bulk cultures of Mfn-deficient fibroblasts tardation. Genetic and cellular evidence suggests that the show normal levels of endogenous and coupled respiration by underlying cause may be a mitochondrial defect (Widsch- oxygen electrode measurements, when individual mitochondria wendter et al., 1998; Talosi et al., 2000). are examined we find that many cells contain a percentage of We anticipate that there are other developmental pro- nonfunctional mitochondria as evidenced by loss of membrane cesses in which the precise regulation of mitochondrial dy- potential. In other cell culture systems, the use of dyes sensitive namics is essential. Thus, we have constructed conditional to mitochondrial membrane potential has revealed occasional alleles of Mfn1 and Mfn2 that should facilitate the analysis and transient losses of membrane potential within small regions of mitochondrial dynamics in adult tissues under both phys- of a single mitochondrial tubule (Loew, 1999). We suggest that iological and experimental conditions. the dynamic nature of mitochondria protects these organelles by ensuring that regional losses of membrane potential, caused perhaps by local depletion of metabolic substrates or mtDNA, A model for the role of mitochondrial fusion in are always transient (Fig. 10 A). Mitochondrial fusion enables protecting mitochondrial function intermitochondrial cooperation by allowing exchange of both It has been paradoxical why eukaryotes have invested in fusion membrane and matrix contents and therefore may help to re- and fission pathways in cells that have “stable” mitochondrial store local depletions and maintain mitochondrial function networks. Given that mitochondrial tubules can be maintained (Nakada et al., 2001a). Although there is no gross loss of by reducing fusion and fission simultaneously (Bleazard et al., mtDNA in mutant cells, we currently do not know if the indi- 1999; Sesaki and Jensen, 1999; Fekkes et al., 2000; Mozdy et vidual, defective mitochondria have lost mtDNA. The Journal of Cell Biology 198 The Journal of Cell Biology | Volume 160, Number 2, 2003 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 immuno- precipitates (top) and total cell lysates (bottom) from Mfn1 or Mfn2 mutant cells (indicated on top) were used in an analysis similar to A. Multiple molecular modes of mitofusin action Given their broad and overlapping expression, it has been Figure 10. Models. (A) The protective role of mitochondrial fusion. unclear why there are two separate mammalian mitofusins. At a low rate, individual mitochondria stochastically lose function. Our results show that the two mitofusins form three distinct In wild-type cells, a defective mitochondrion (shaded) undergoes molecular complexes that are capable of promoting mito- fusion with functional mitochondria and regains activity. In Mfn- deficient cells, such rescue occurs at a much reduced rate. (B) Three chondrial fusion–Mfn1 homotypic oligomers, Mfn2 homo- modes of mitofusin action. Mitofusins form homotypic and heterotypic typic oligomers, and Mfn1-Mfn2 heterotypic oligomers. We complexes that lead to three activities (I, II, III) involving fusion. See propose that the relative importance of each of these modes Discussion for details. Mfn1 mutant cells contain only activity III; Mfn2 of mitofusin action can change depending on the cell type mutant cells contain only activity I. Since disruption of either Mfn1 or (Fig. 10 B). In mouse fibroblasts, all three oligomeric com- Mfn2 fragments mitochondria and results in distinct phenotypes, MEFs plexes are likely to play important roles because disruption appear to use all three activities (indicated by asterisks). In contrast, of either Mfn1 or Mfn2 leads to severe mitochondrial frag- trophoblast giant cells predominantly use activity III because they are affected in Mfn2 mutants and not Mfn1 mutants. mentation. Nevertheless, overexpression of Mfn1 homotypic oligomers or Mfn2 homotypic oligomers is sufficient to re- store mitochondrial tubules, clearly demonstrating that the ally homogeneous. However, at any given time point indi- homotypic complexes are functional for fusion. In tropho- vidual mitochondria are functionally distinct entities (Col- blast giant cells, it appears that Mfn2 homotypic oligomers lins et al., 2002). Therefore, without mitochondrial fusion, are most important. As a result, this cell type is affected in the stochastic differences between distinct mitochondria can Mfn2-deficient embryos but not Mfn1-deficient embryos. accumulate to affect the well-being of the cell. The mamma- It remains to be determined whether these three com- lian mitofusins, Mfn1 and Mfn2, function in three distinct plexes function in the same or distinct types of mitochon- molecular complexes to promote mitochondrial fusion and drial fusion. It is also interesting to note that loss of Mfn2 thus protect mitochondrial function. leads to the formation of both large and small mitochondrial spheres. This phenotype is easily distinguished from loss of Materials and methods Mfn1. The larger mitochondrial fragments may be due to a higher residual fusion activity in Mfn2 mutant cells than in Cloning of Mfn cDNA and genomic constructs Homology searches using the Drosophila Fzo sequence (Hales and Fuller, Mfn1 mutant cells. The loss of tubular shape may simply re- 1997) identified a murine EST (IMAGE Consortium Clone ID 733269) that flect the loss of cytoskeletal interactions as noted in Fig. 4. encodes a highly homologous polypeptide. This cDNA was used to screen Alternatively, it may be that Mfn2, and by extension Mfn2 a mouse kidney cDNA library, resulting in isolation of cDNAs for both Mfn1 and Mfn2. These sequences are available from GenBank/EMBL/ homotypic complexes, play a more direct role in maintain- DDBJ under accession nos. AY174062 and AY123975. ing mitochondrial tubular shape. Thus, although each of the Genomic clones of Mfn1 and Mfn2 were retrieved from a lambda 129/ Mfn complexes is involved in fusion, it is possible that they SvJ mouse genomic library (Stratagene). Genomic fragments were sub- cloned into the targeting vector pPGKneobpAlox2PGKDTA (a gift from F. have distinct functions in addition to fusion. Gertler and L. Jackson-Grusby, Massachusetts Institute of Technology, In conclusion, in cells with continual cycles of fusion and Cambridge, MA) as the right arms. To insert the left arms, genomic frag- ments were amplified by PCR, which also introduced a stop codon just be- fission, the mitochondrial population is essentially function- The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 199 fore the conserved GKS sequence in each GTPase domain. All constructs TS cells from e3.5 blastocysts were derived using established protocols were verified by DNA sequence analysis. The sequences of all oligonucle- (Tanaka et al., 1998a). Live cells were stained with MitoTracker Red (150 otides used in this study are available from the authors. nM) and Syto16 (100 nM; Molecular Probes). To identify the chromosomal locations of Mfn1 and Mfn2, the Jackson Lab- oratory (C57BL/6JEi SPRET/Ei) F1 SPRET/Ei backcross panel was used. PEG fusion Linkage analysis was performed by the Jackson Laboratory Mapping Resource. 40,000 cells expressing mitochondrially targeted GFP were cultured over- night on 25-mm coverslips with 40,000 cells expressing mitochondrially Construction of knockout mice targeted dsRed. The next morning, cells were fused for 60 s with 50% PEG Each gene replacement vector was linearized with SacII and electroporated 1500 (Roche). The cells were washed and grown for 7 h or 24 h in me- into low passage 129/SvEv embryonic stem cells using established proce- dium containing 30 g/ml cycloheximide before fixation. dures (Chester et al., 1998). Neomycin-resistant colonies were screened by Southern blot analysis. Two independently isolated clones for each targeted Immunofluorescence allele were injected into C57BL/6 blastocysts to generate chimeric mice. Cells grown on polylysine-coated coverslips were fixed with prewarmed Excision of the neomycin cassette had no effect on phenotypes. (37 C) 3.7% formaldehyde and permeabilized in PBS/0.1% Triton X-100. In some experiments, cells were incubated with 150 nM MitoTracker Red for 30 Confirmation of targeting event min before fixation and then permeabilized with acetone at 20 C. Cells For Southern blot analysis of targeted alleles, genomic DNAs were di- were blocked with 5% bovine calf serum and incubated with primary anti- gested with XbaI (Mfn1 mutant line) or EcoRI (Mfn2 mutant line) and hy- body. For Myc epitope–tagged proteins, the mouse monoclonal antibody bridized with a flanking genomic probe. PCR was used for routine geno- 9E10 was used. For COX1, mouse monoclonal 1D6-E1-A8 (Molecular typing of offspring. A forward genomic primer, a reverse genomic primer, Probes) was used. For detection, Cy3- or Alexa Fluor 488–conjugated second- and a reverse neomycin primer were used to detect both the targeted and ary antibodies (Jackson ImmunoResearch Laboratories and Molecular Probes) wild-type alleles in a single reaction. were used. Cells were imaged with a Plan NeoFluar 63 objective on a Zeiss For Western blot analysis, chicken antisera was generated against Mfn1 410 laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc.). (residues 348–579) or Mfn2 (residues 369–598) fused to an NH -terminal histidine tag. IgY purified from chicken eggs was affinity purified on a col- Analysis of mtDNA umn coupled to a maltose-binding protein fusion protein containing either Southern analysis of mtDNA was performed by linearizing with XhoI and Mfn1 residues 348–579 or Mfn2 residues 369–598. probing with a radio-labeled PCR fragment containing the COX1 gene. In situ hybridization Immunoprecipitation Individual implantation sites were fixed overnight at 4 C in 4% parafor- Cell lines were infected with various combinations of retroviruses express- maldehyde, dehydrated through an ethanol series, treated with xylenes, ing Myc- and HA-tagged Mfn1, Mfn2, and Drp1(K38A). Monolayers were and embedded into paraffin blocks. For staining, 10-m sections were cut resuspended in lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton in a transverse plane with respect to the placenta. Slides were processed X-100, and a protease inhibitor cocktail) and immunoprecipitated with for hematoxylin-eosin staining or in situ hybridization as described previ- 9E10 antibody coupled to protein A–Sepharose beads. After washing, sam- ously (Vortkamp et al., 1996). All riboprobe templates were derived from ples were immunoblotted with HA.11 (Covance). RT-PCR using e11.5 placental RNA as template. No specific staining was detected using sense probes. For genotyping, embryonic tissue was scraped off unstained slides. DNA was recovered and genotyped by PCR. Online supplemental material Confocal time-lapse videos of EYFP-labeled mitochondria in wild-type Retroviral and plasmid constructs (video 1), Mfn1 mutant (video 2), and Mfn2 mutant (video 3) MEFs are available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1. Vid- To construct Mfn1 or Mfn2 with Myc epitope tags, a BamHI site was intro- eos 1–3 show mitochondrial dynamics in wild-type and mutant MEFs. Vid- duced immediately before the stop codon. The cDNA was subcloned into eos 1 and 3 are 20-min recordings; video 2 is a 10-min recording. The mi- the vector pcDNA3.1( )/Myc-His A (Invitrogen). Myc epitope tag cassettes croscopic field is 96 m 96 m in video 1, 68 m 68 m in video 2, derived from pMMHb-3 Myc were then inserted into the BamHI site to and 75 m 75 m in video 3. form pMfn-Myc. Mfn1(K88T) and Mfn2(K109A) were constructed by site- directed mutagenesis (Kunkel et al., 1991). To make the Mfn-HA constructs, Mfn1 and Mfn2 cDNAs were subcloned into a pcDNA3.1( ) vector contain- We thank Dr. Philip Leder for his support in the early stages of this work. ing a COOH-terminal 3 HA tag. Murine Drp1 was amplified from mouse We are grateful to M. Michelman for embryonic stem cell culture assis- placental RNA, and Drp1(K38A) was constructed by site-directed mutagene- tance and A. Harrington for blastocyst injections. We thank Drs. M. Rojo sis. The Drp1(K38A) insert was subcloned into a modified pcDNA3.1( )/ Myc-HisA vector containing seven Myc epitope tags at the COOH terminus. and A. Lombes for stimulating discussions about the PEG fusion assay. To generate retroviral expression constructs for each of the above, the H. Chen is supported by an Alcott Postdoctoral fellowship. S.A. Detmer epitope-tagged cDNAs were recloned into the retroviral vector pCLW (a and E.E. Griffin are supported by a National Institutes of Health training gift from C. Lois, California Institute of Technology). The retroviral expres- grant NIHGM07616 and E.E. Griffin is funded by a Ferguson fellowship. sion vectors were cotransfected with the ecotropic retroviral packaging A.J. Ewald is a participant in the Initiative in Computational Molecular Bi- vector pCLEco (a gift from C. Lois, California Institute of Technology) into ology funded by the Burroughs Wellcome Fund Interfaces program. D.C. 293T cells. Retroviral stocks were harvested 48 h after transfection and Chan is a Bren scholar, Rita Allen scholar, Beckman Young investigator, used to infect MEF cultures. Fragments encoding mitochondrially targeted and recipient of a Burroughs Wellcome Fund Career Development award GFP and dsRed (from plasmids pHS1 and pHS51; gifts from H. Sesaki and in Biomedical Sciences. This research was supported by the National Insti- R. Jensen, Johns Hopkins University, Baltimore, MD) were subcloned into pCLW to generate retroviral expression vectors. tutes of Health (grant 1 RO1 GM62967-01). Submitted: 12 November 2002 MEF and TS cell lines Revised: 12 December 2002 MEFs were derived from e10.5 embryos. Embryos were mechanically dis- Accepted: 12 December 2002 persed by repeated passage through a P1000 pipette tip and plated with MEF media (DME, 10% FCS, 1 nonessential amino acids, 1 mM L-glu- tamine, penicillin/streptomycin [Life Technologies/GIBCO BRL]). 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Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development

Chen, Hsiuchen; Detmer, Scott A.; Ewald, Andrew J.; Griffin, Erik E.; Fraser, Scott E.; Chan, David C.
The Journal of Cell Biology , Volume 160 (2) – Jan 20, 2003
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Copyright © 2003, The Rockefeller University Press
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0021-9525
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10.1083/jcb.200211046
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Abstract

JCB Article Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development 1 1 1,2 1 1,2 1 Hsiuchen Chen, Scott A. Detmer, Andrew J. Ewald, Erik E. Griffin, Scott E. Fraser, and David C. Chan 1 2 Division of Biology and Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125 itochondrial morphology is determined by a dy- in mitochondrial fusion. Moreover, we find that Mfn1 and namic equilibrium between organelle fusion and Mfn2 form homotypic and heterotypic complexes and fission, but the significance of these processes in show, by rescue of mutant cells, that the homotypic com- vertebrates is unknown. The mitofusins, Mfn1 and plexes are functional for fusion. We conclude that Mfn1 Mfn2, have been shown to affect mitochondrial morphol- and Mfn2 have both redundant and distinct functions and ogy when overexpressed. We find that mice deficient in ei- act in three separate molecular complexes to promote ther Mfn1 or Mfn2 die in midgestation. However, whereas mitochondrial fusion. Strikingly, a subset of mitochondria Mfn2 mutant embryos have a specific and severe disruption in mutant cells lose membrane potential. Therefore, mito- of the placental trophoblast giant cell layer, Mfn1-deficient chondrial fusion is essential for embryonic development, giant cells are normal. Embryonic fibroblasts lacking Mfn1 and by enabling cooperation between mitochondria, has or Mfn2 display distinct types of fragmented mitochondria, protective effects on the mitochondrial population. a phenotype we determine to be due to a severe reduction Introduction Mitochondria are remarkably dynamic organelles. Time- fzo1 yeast form “petite” colonies that lack mitochondrial lapse microscopy of living cells reveals that mitochondria DNA (mtDNA) (Hermann et al., 1998; Rapaport et al., undergo constant migration and morphological changes 1998). Furthermore, there is a disruption of mating-induced (Bereiter-Hahn and Voth, 1994; Nunnari et al., 1997; mitochondrial fusion (Hermann et al., 1998). Humans contain Rizzuto et al., 1998). Even in cells with a seemingly “stable” two Fzo homologues, termed mitofusin (Mfn)1 and Mfn2, that network of mitochondrial tubules, there are frequent and can alter mitochondrial morphology when overexpressed in cell continual cycles of mitochondrial fusion and fission, opposing lines (Santel and Fuller, 2001; Rojo et al., 2002). Both mito- processes that exist in equilibrium and serve to maintain the fusins are broadly expressed (Rojo et al., 2002), and therefore overall architecture of these organelles (Bereiter-Hahn and their functional redundancy is unclear. Voth, 1994; Nunnari et al., 1997). Despite our increasing knowledge about the importance In both yeast and flies, mitochondrial fusion is controlled by of mitochondrial fusion in lower eukaryotes, there is disagree- the nuclearly encoded mitochondrial transmembrane GTPase, ment about its physiological role in vertebrates. Doubts have fuzzy onions (Fzo).* In Drosophila, Fzo is specifically and been raised about the frequency and importance of mito- transiently expressed in spermatids. Disruption of Fzo prevents chondrial fusion in cultured mammalian cells (Enriquez et developmentally regulated mitochondrial fusion in postmeiotic al., 2000). However, several observations suggest that this spermatids and results in male sterility (Hales and Fuller, basic cellular process indeed plays a significant role in vertebrate 1997). In budding yeast, deletion of FZO1 disrupts the highly cells. First, ultrastructural studies of mitochondria show that branched, tubular mitochondrial network typical of normal dramatic transitions occur during the development of certain cells and results in numerous small spherical mitochondria. tissues. For example, the mitochondria of rat cardiac muscle and diaphragm skeletal muscle appear as isolated ellipses or The online version of this article contains supplemental material. tubules in embryonic stages but then reorganize into reticular Address correspondence to David C. Chan, 1200 East California Blvd., networks in the adult (Bakeeva et al., 1978, 1981, 1983). It MC114-96, Pasadena, CA 91125. Tel.: (626) 395-2670. Fax: (626) 395- is likely that these progressive morphological changes occur 8826. E-mail: [email protected] through mitochondrial fusion. Second, time-lapse fluores- *Abbreviations used in this paper: Drp, dynamin-related protein; e, embry- cence microscopy of cultured HeLa cells shows that the mi- onic day; EYFP, enhanced YFP; Fzo, fuzzy onions; MEF, mouse embry- tochondria are organized into extensive tubular networks onic fibroblast; Mfn, mitofusin; mtDNA, mitochondrial DNA; PEG, polyethylene glycol; TS, trophoblast stem. that undergo frequent fusion and fission (Rizzuto et al., Key words: membrane fusion; mitochondria; GTPase; mice; knockout 1998). Third, experimentally induced cell hybrids demon-  The Rockefeller University Press, 0021-9525/2003/01/189/12 $8.00 The Journal of Cell Biology, Volume 160, Number 2, January 20, 2003 189–200 http://www.jcb.org/cgi/doi/10.1083/jcb.200211046 189 The Journal of Cell Biology 190 The Journal of Cell Biology | Volume 160, Number 2, 2003 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). -Actin was used as a loading control. strate rapid mtDNA mixing (Hayashi et al., 1994) and com- cDNA library. In accordance with the nomenclature for the plementation of mtDNA gene products (Nakada et al., human mitofusins (Santel and Fuller, 2001), we designate 2001b; Ono et al., 2001). Finally, mitochondrial dynamics these murine homologues as Mfn1 and Mfn2. Linkage anal- have been implicated in the regulation of apoptosis. Induc- ysis placed Mfn1 at the proximal end of mouse chromosome tion of cell death is sometimes associated with fragmentation 3 (12–13 cM) in a region syntenic to human 3q25-26. of the mitochondrial network. This fragmentation requires Mfn2 was localized to the distal end of mouse chromosome dynamin-related protein (Drp)1, which is involved in mito- 4 (70–80 cM) in a region syntenic to human 1p36. chondrial fission (Frank et al., 2001). As with the fzo genes from Drosophila melanogaster (Santel To assess the physiological role of mitochondrial fusion in and Fuller, 2001; Hwa et al., 2002), Homo sapiens (Santel vertebrates, we have generated knockout mice for Mfn1 and and Fuller, 2001), and Saccharomyces cerevisiae (Hermann et Mfn2. Our analysis reveals that Mfn1 and Mfn2 are each es- al., 1998; Rapaport et al., 1998), each murine Mfn gene en- sential for embryonic development and mitochondrial fu- codes a predicted transmembrane GTPase. The transmem- sion. These mitofusins can exist as both homotypic and het- brane segment is flanked by two regions containing hydro- erotypic oligomers and therefore can cooperate as well as act phobic heptad repeats, hallmarks of coiled-coil regions individually to promote mitochondrial fusion. In addition, (Lupas, 1996). Mfn1 and Mfn2 are 81% similar to each our results suggest that mitochondrial fusion functions to al- other and are both 52% similar to Drosophila Fzo. low cooperation between mitochondria, thereby protecting mitochondria from respiratory dysfunction. Generation of knockout mice deficient in Mfn1 and Mfn2 We constructed gene replacement vectors for Mfn1 and Mfn2 using the neomycin resistance gene for positive selection and Results the diphtheria toxin subunit A gene for negative selection. In Identification of murine Fzo homologues both cases, a stop codon was engineered at the very beginning We identified two murine homologues of fzo from a mouse of the GTPase domain near the NH terminus (Fig. 1, A and The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 191 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. E). In addition, the resulting genomic loci each contain a re- out line, normal frequencies of live mutant embryos were placement of the G1 and G2 motifs of the GTPase domain obtained up to embryonic day (e)10.5 (noon of the day a cop- with the neomycin expression cassette. These universal GTP- ulatory plug was detected is designated e0.5). However, by ase motifs are crucial for binding of the  and  phosphates e11.5, 20% of the mutant embryos were resorbed, indicating of GTP and for Mg coordination (Bourne et al., 1991; inviability. At e12.5, most identifiable mutant embryos were Sprang, 1997). Genetic analyses in Drosophila and Saccharo- resorbed (86%), and additional resorptions were so advanced myces cerevisiae (Hales and Fuller, 1997; Hermann et al., that they could not be genotyped. Although resorptions were 1998), as well as our own studies (see Fig. 7 C and Fig. 8 C), not evident until e11.5, by e8.5 all mutant embryos were sig- demonstrate that an intact GTPase domain is essential for nificantly smaller and showed pronounced developmental de- Fzo function. Therefore, the disrupted Mfn1 and Mfn2 alleles lay. In addition, mutant embryos were often deformed. described here should be null alleles. Both Southern blot and In the Mfn2 knockout line, normal numbers of live ho- PCR analysis confirmed germline transmission of the targeted mozygous mutant embryos were recovered up to e9.5. How- alleles (Fig. 1, B, C, F, and G). Importantly, Western blot ever, starting at e10.5, 29% of homozygous mutant embryos analysis using affinity-purified antisera raised against Mfn1 or were in the process of resorption. By e11.5, 87% of homozy- Mfn2 confirmed loss of the targeted protein in homozygous gous mutant embryos were resorbed. The live homozygous mutant lysates (Fig. 1, D and H). mutant embryos at e9.5 and e10.5 were slightly smaller than their littermates but were otherwise well developed and Embryonic lethality in homozygous mutant mice showed no obvious malformations. Both the Mfn1 and Mfn2 knockouts demonstrate full viabil- Preliminary studies indicate that double homozygous mu- ity and fertility in heterozygous animals but result in embry- tant embryos die earlier than either single mutant and show onic lethality of homozygous mutants. For the Mfn1 knock- greater developmental delay (unpublished results). This ob- The Journal of Cell Biology 192 The Journal of Cell Biology | Volume 160, Number 2, 2003 servation suggests that Mfn1 and Mfn2 are both required, and may have a cooperative relationship, in a particular de- velopmental event early in embryogenesis. Placental defects in Mfn2 mutant mice We examined placental development in detail because placen- tal insufficiency is one of the most common causes of midges- tation lethality (Copp, 1995). Hematoxylin-eosin–stained histological sections of placentae from wild-type and heterozy- gous embryos showed the typical trilaminar structure com- posed of a proximal labyrinthine layer, a middle spongiotro- phoblast layer, and a distal, circumferential giant cell layer (Fig. 2 E). Trophoblast giant cells are polyploid cells (derived from endoreplication, a process where DNA replication pro- ceeds 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 quan- tity, and the few that are observed contain smaller nuclei, im- plying a reduction in the number of endoreplication cycles (Fig. 2 F). These observations were confirmed with DAPI staining, which highlights the giant cells because of their high DNA content (Fig. 2, A–D). No defects in placental develop- ment were detected in Mfn1 mutant embryos (unpublished data) despite expression of Mfn1 in the placenta (Fig. 1 D). Using RNA in situ hybridization, we examined the ex- pression 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 gi- ant cell staining with multiple layers of giant cells packed in the region distal to the spongiotrophoblast layer (Fig. 2 G). In contrast, Mfn2 mutant placentae showed an incomplete ring of weakly staining giant cells (Fig. 2 H). Moreover, the giant cell layer was generally only one cell layer thick. Simi- lar results were seen in placentae from e8.5 through e10.5. These observations were confirmed with RNA in situ hy- Figure 3. Morphological defects in mitochondria of mutant cells. bridizations (unpublished data) using the additional giant (A–F) Mitochondrial morphology in wild-type (A and B), Mfn1 mutant cell markers, proliferin (Lee et al., 1988) and Hand1 (Riley (C and D), and Mfn2 mutant (E and F) MEF cells. MEFs expressing et al., 1998). Interestingly, our in situ hybridizations also re- mitochondrial EYFP (green) were counterstained with rhodamine- phalloidin (red). (B, D, and F) Higher magnification images of the vealed somewhat weaker staining with the ectoplacental boxed areas in A, C, and E, respectively. Arrow indicates a tubule cone and spongiotrophoblast marker 4311 (Lescisin et al., 10 m in length. (G and H) Mitochondrial morphology in live 1988), suggesting an additional but more subtle defect in wild-type (G) and mutant (H) TS cells. The mitochondria were this placental layer (unpublished data). No differences in stained with MitoTracker Red, and the nuclei were stained with staining between wild-type and Mfn2 mutant sections were Syto16 (green). Several cells are tightly clustered. 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 logical class, which encompassed 90% of wild-type cells, midgestation secondary to an inadequate placenta. was characterized by a network of extended wavy tubules distributed in a roughly radial manner throughout the cyto- Both Mfn1- and Mfn2-deficient cells have aberrant plasm. Such cells had no or only a few spherical mitochon- mitochondrial morphology dria. The length of these mitochondrial tubules ranged from To examine mitochondrial morphology in Mfn-deficient several microns to 10 m (Fig. 3, A and B, arrow). A cells, we derived mouse embryonic fibroblasts (MEFs) from much smaller morphological class, which encompassed only e10.5 embryos. The MEF cultures were infected with a ret- 6% of wild-type cells, had mitochondria that were mostly rovirus expressing mitochondrially targeted enhanced yellow spherical and were termed as “fragmented.” fluorescent protein (EYFP). Wild-type MEFs showed a In contrast, Mfn1 mutant MEFs had dramatically frag- range of mitochondrial morphologies. The major morpho- mented mitochondria (Fig. 3, C and D). Greater than 95% The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 193 of these cells contained only severely fragmented mitochon- dria, whereas only 1–2% of cells contained any significant tu- bules. Similarly, in 85% of Mfn2 mutant fibroblasts the mi- tochondria appeared mostly as spheres or ovals (Fig. 3, E and F). 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 charac- terize 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 mito- chondrial tubules (Fig. 3 G). In contrast, Mfn2 mutant TS cells showed only spherical mitochondria (Fig. 3 H). Although loss of either Mfn1 or Mfn2 results in fragmen- tation of mitochondrial tubules, the two mutations lead to characteristic mitochondrial morphologies that are readily distinguishable. Loss of Mfn1 leads to a greater degree Figure 4. Dynamics of mitochondria in wild-type and mutant of fragmentation, resulting in either very short mitochon- cells. Still frames from time-lapse confocal microscopy. (A) In a wild-type cell, two pairs of mitochondria can be seen moving toward drial tubules or very small spheres that are uniform in size, each other. These pairs contact end-to-end and fuse immediately. with diameters no larger than that of a normal tubule. In Note that mitochondria move along their long axes. (B) In a Mfn1 contrast, many Mfn2 mutant cells exhibit mitochondrial mutant cell, the mitochondria move in an undirected manner. (C) In spheres of widely varying sizes. Interestingly, the diameters a Mfn2 mutant cell, two ovoid mitochondria contact each other of some of the spherical mitochondria in both Mfn2 mutant but do not fuse until much later. Note also the lack of directed MEF and TS cells are several times larger than the diameters movement in most mitochondria. (D) One spherical Mfn2-deficient of mitochondrial tubules in wild-type cells. Because a simple mitochondrion protrudes a tubular extension that separates and defect in mitochondrial fusion would be expected to result then migrates away along its long axis. Images were processed in Adobe Photoshop with the emboss filter, and selected mitochondria in shorter tubules and small spheres with the diameter of were manually highlighted in blue. See also videos 1–3 available at normal tubules, these observations suggest that in addition http://www.jcb.org/cgi/content/full/jcb.200211046/DC1. 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 fi- Morris and Hollenbeck, 1995; Tanaka et al., 1998b). The broblasts, each protein is essential for mitochondrial fusion mitochondria of Mfn-deficient cells display a dramatic alter- and may have some distinct functions. ation in mobility; the spherical or ovoid mitochondria in mutant cells show random “Brownian-like” movements (Fig. 4, B and C). This lack of directed movement is not due Altered mitochondrial dynamics in Mfn-deficient cells to disorganization of the cytoskeleton because staining with Because mitochondria are such dynamic organelles, we used an antitubulin mAb or rhodamine-phalloidin revealed no time-lapse confocal microscopy to determine whether mu- defects (unpublished data; Fig. 3). Strikingly, in Mfn2 mu- tant MEFs display aberrations in mitochondrial dynamics. tant cells with both spherical and short tubular mitochon- In wild-type cells, the mitochondria are highly motile, and dria, the tubular mitochondria can still move longitudinally at least one apparent fusion or fission event was observed for along radial tracks, whereas the spherical mitochondria do most mitochondria during 20-min recordings (Fig. 4 A; not. However, the two morphological classes are inter- video 1, available at http://www.jcb.org/cgi/content/full/ changeable because tubules can project out of spheres and jcb.200211046/DC1). However, in both Mfn1 and Mfn2 subsequently move away (Fig. 4 D), or conversely tubules mutant cells the ovoid or spherical mitochondria undergo can merge with spheres and lose directed movement. These fusion events much less frequently (videos 2 and 3, available observations suggest that there is no intrinsic defect in mobi- at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1), lizing Mfn2-deficient mitochondria. Instead, the hampered although a few such events can be found (Fig. 4 C). mobility of spherical mitochondria is secondary to their al- In addition to reduced fusion, our time-lapse videos re- tered morphology resulting from reduced fusion. vealed striking defects in the mobility of mitochondria in mutant cells. Most mitochondria in wild-type cells were tu- Decreased mitochondrial fusion rates in cells lacking bular, were directed radially, and moved back and forth Mfn1 or Mfn2 along their long axis on radial tracks (Fig. 4 A). This move- ment along defined tracks is consistent with reports that mi- To definitively show that lack of fusion is the basis for the tochondria can be anchored along microtubule or actin fila- fragmented mitochondria in mutant cells, we measured ments, depending on the cell type (Nangaku et al., 1994; mitochondrial fusion activity using a polyethylene glycol The Journal of Cell Biology 194 The Journal of Cell Biology | Volume 160, Number 2, 2003 (PEG) cell fusion assay. A cell line expressing mitochondri- ally targeted dsRed was cocultured with a cell line expressing mitochondrially targeted GFP, and PEG was transiently ap- plied to fuse the cells. Cycloheximide was included to pre- vent 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 exten- sively fused mitochondria (Fig. 5 A) as demonstrated by colocalization of red and green fluorescent signals. In con- trast, when Mfn1 mutant cells were examined 7 h after PEG fusion 57% (n  364) of the fused cells contained predomi- nantly 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 fu- sion, and 8% showed partial fusion. Similarly, 69% (n 202) of fused Mfn2 mutant cells showed predominantly un- fused 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 ob- served even 24 h after PEG treatment (unpublished data). Thus, mutant cells have severely reduced levels of mitochon- drial 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. Figure 5. Mitochondrial fusion assay. PEG fusion of cells containing Stochastic defects in mitochondrial membrane potential mitochondrially targeted dsRed and GFP. (A) Wild-type cell showing We tested whether Mfn1 and Mfn2 mutant cells lose extensive mitochondrial fusion. (B and E) Mfn1 (B) and Mfn2 mtDNA because there is complete loss of mtDNA in fzo1 (E) mutant cells displaying predominantly unfused mitochondria. yeast (Hermann et al., 1998; Rapaport et al., 1998). South- (C and F) Magnified views of boxed portions in B and E, respectively. ern blot and PCR analysis showed that both mutant cell (D) Sectoring effect in Mfn1 mutant cell. lines contain normal levels of mtDNA (Fig. 6 A; unpub- lished data). In addition, the mitochondria in mutant cells cultures over one third of the cells showed some mitochon- expressed COX1, a mitochondrial protein encoded by dria that were marked by EYFP but not by MitoTracker Red mtDNA (Fig. 6, B and C). Therefore, unlike fzo1 yeast, (Fig. 6 F). It is likely that the compromised mitochondria Mfn-deficient cells clearly retain mtDNA, and this feature detected by this assay initially contained membrane poten- allows a critical assessment of the relationship between respi- tial because the matrix-targeted EYFP requires an intact ration and mitochondrial fusion. Like wild-type cells, both membrane potential for import. Because of the high stability Mfn1 and Mfn2 mutant cultures showed high rates of en- of EYFP, mitochondria that subsequently lose or reduce dogenous and coupled respiration (unpublished data), indi- their membrane potential would still contain EYFP fluores- cating no gross defects in respiration and further confirming cence. These results indicate that while bulk cultures display that mtDNA products are intact and functional. respiratory activity, the function of individual mitochondria Although the mutant cells are capable of respiration, we is compromised in the absence of either Mfn1 or Mfn2. reasoned that functional defects may exist at the level of in- dividual mitochondria. To test this hypothesis, we used Mi- Rescue of mitochondrial tubules by modulation of toTracker Red, a lipophilic cationic dye which is sensitive to fusion or fission the mitochondrial membrane potential, to stain MEFs ex- To unequivocally demonstrate that the mitochondrial mor- pressing EYFP targeted to the mitochondrial matrix. In phological defects observed in mutant cells are due to loss of wild-type cells, EYFP-marked mitochondria were typically Mfn, we tested whether these defects could be rescued by res- uniformly stained with MitoTracker Red, resulting in colo- toration of Mfn function and whether such rescue depended calization of the two fluorophores (Fig. 6 D). In 7% of wild- on an intact GTPase domain. In uninfected Mfn1 mutant type cells, there existed isolated mitochondria that were la- cultures or mutant cultures infected with an empty virus, beled with EYFP but failed to sequester MitoTracker Red 95% of the cells showed highly fragmented mitochondria dye. Such mitochondria were invariably small and spherical that lacked interconnections (Fig. 7, A and F). However, when as opposed to the tubular mitochondria that predominate in Mfn1 mutant cultures were infected with a retrovirus express- these cells. In Mfn1 mutant cultures, almost every cell con- ing Mfn1-Myc 90% of the infected cells showed extensive tained a subset of mitochondria that showed poor staining mitochondrial networks consisting of long tubules (Fig. 7, B with MitoTracker Red (Fig. 6 E). Likewise, in Mfn2 mutant The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 195 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. and F). Similarly, 80% of Mfn2 mutant cells infected with Because the morphological defects in mutant cells were a Mfn2-expressing retrovirus showed predominantly tubular clearly reversible, we tested whether inhibition of mitochon- mitochondria (Fig. 8, B and F). In contrast, constructs car- drial fission could also restore tubular structure. Drp1 plays a rying mutations in the GTPase G1 motif (Mfn1[K88T] and key role in mediating mitochondrial fission. To inhibit fis- Mfn2[K109A]) had greatly reduced ability to restore tubular sion, we constructed an allele of mouse Drp1 with the same structure (Fig. 7, C and F, and Fig. 8, C and F). These re- mutation (K38A) as in a dominant-negative allele of human sults show that both Mfn1 and Mfn2 promote mitochon- Drp1 (Smirnova et al., 2001). Infection of either Mfn1 or drial fusion in a GTPase-dependent manner. Mfn2 mutant cells with a retrovirus expressing Drp1(K38A) Although both Mfn1 and Mfn2 are clearly required for resulted in a striking restoration of mitochondrial tubules in normal mitochondrial tubules in MEFs, we were able to 90% (Fig. 7, E and F) and 50% (Fig. 8, E and F) of the cells, rescue the morphological defects by overexpression of a respectively. Although the mitochondrial networks in these single mitofusin. Overexpression of Mfn1 in Mfn2-defi- infected cells were tubular, they were imperfect compared cient cells was sufficient for rescue of mitochondrial mor- with the networks obtained by rescue with Mfn1 or Mfn2. phology (Fig. 8, D and F). In addition, overexpression of First, the thickness of the tubules promoted by Drp1(K38A) Mfn2 in Mfn1-deficient cells was sufficient to restore mi- were less uniform, often showing regions of thinning at- tochondrial tubules (Fig. 7, D and F). These results show tached to thicker knobs. Second, the mitochondrial networks that although both Mfn1 and Mfn2 are required in vivo for were generally less dispersed, with an increase in density near normal mitochondrial morphology in MEFs, when overex- the cell nucleus that often gave the appearance of circumfer- pressed, each protein is capable of acting alone to promote ential rings. Nevertheless, these results indicate that a reduc- mitochondrial fusion. Interestingly, these experiments also tion in fusion caused by loss of Mfn1 or Mfn2 can be com- reveal differential activities of the two homologues. With pensated, at least partially, by a reduction in fission. Mfn2-deficient cells, both Mfn1-Myc and Mfn2-Myc were equally effective in restoring extensive mitochondrial net- Mfn1 and Mfn2 form homotypic and works, resulting in rescue in 75–80% of expressing cells heterotypic oligomers (Fig. 8 F). In contrast, with Mfn1-deficient cells, 91% of Mfn1-Myc–infected cells showed entirely tubular net- Because both Mfn1 and Mfn2 are essential for normal mito- works, but only 25% of Mfn2-Myc–infected cells showed chondrial morphology, it is possible that they act in concert such a phenotype (Fig. 7 F). to promote mitochondrial fusion. To explore this idea, we The Journal of Cell Biology 196 The Journal of Cell Biology | Volume 160, Number 2, 2003 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. tested whether these two proteins form a complex when ex- Discussion pressed in wild-type MEF cells (Fig. 9 A). Our results re- An essential role for mitochondrial fusion vealed three types of intermolecular interactions. First, both in mouse development Mfn1 and Mfn2 can form homotypic complexes. Mfn1-HA is coimmunoprecipitated with Mfn1-Myc; analogously, Although mitochondrial dynamics involving fusion and fission Mfn2-HA is coimmunoprecipitated with Mfn2-Myc. Sec- has been previously demonstrated in vertebrates, the physio- ond, Mfn1 and Mfn2 form heterotypic complexes. Mfn2- logical importance of these processes has remained unclear. HA is coimmunoprecipitated with Mfn1-Myc; conversely, Our analysis of Mfn1 and Mfn2 mutant mice demonstrates an Mfn1-HA is coimmunoprecipitated with Mfn2-Myc. All of essential role for mitochondrial fusion in vertebrate develop- these interactions are specific because no HA-tagged protein ment. Both lines of mice die in midgestation. For Mfn2-defi- is found in the anti-Myc immunoprecipitate when the Myc- cient mice, we observe a dramatic disruption in placental de- tagged protein is omitted (unpublished data) or when a con- velopment, most obviously in the paucity of trophoblast giant trol Drp1-Myc is used. cells. Trophoblast cell lines cultured from Mfn2 mutant blas- It is a formal possibility that the homotypic interactions tocysts show fragmentation of mitochondrial tubules, consis- detected might not be strictly homotypic due to the expres- tent with a defect in mitochondrial fusion. These results show sion of endogenous Mfn1 and Mfn2 in the parental cells. To that despite its broad expression pattern (unpublished data) relieve these concerns, we also performed the immunopre- Mfn2 is only required for selective developmental transitions. cipitation assay in mutant MEFs (Fig. 9 B). We find that Trophoblast giant cells are polyploid cells that arise from Mfn1-Mfn1 homotypic complexes are formed in Mfn2- endoreplication, a process often associated with highly meta- deficient cells, thereby showing that this interaction is strictly bolically active cells (Edgar and Orr-Weaver, 2001). Because homotypic. Likewise, we also detect Mfn2-Mfn2 homotypic of their high metabolic rate, we speculate that, in vivo, tro- complexes in Mfn1-deficient cells. phoblast giant cells may be particularly vulnerable to pertur- The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 197 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. bations in mitochondrial dynamics and presumably func- al., 2000; Tieu and Nunnari, 2000), what is the advantage of tion. In fact, preeclampsia, the leading cause of fetal and maintaining mitochondria in a highly dynamic state? maternal morbidity in the United States, is marked by shal- Our analysis of Mfn-deficient cells suggests an answer to this low trophoblast invasion, often resulting in fetal growth re- question. Although bulk cultures of Mfn-deficient fibroblasts tardation. Genetic and cellular evidence suggests that the show normal levels of endogenous and coupled respiration by underlying cause may be a mitochondrial defect (Widsch- oxygen electrode measurements, when individual mitochondria wendter et al., 1998; Talosi et al., 2000). are examined we find that many cells contain a percentage of We anticipate that there are other developmental pro- nonfunctional mitochondria as evidenced by loss of membrane cesses in which the precise regulation of mitochondrial dy- potential. In other cell culture systems, the use of dyes sensitive namics is essential. Thus, we have constructed conditional to mitochondrial membrane potential has revealed occasional alleles of Mfn1 and Mfn2 that should facilitate the analysis and transient losses of membrane potential within small regions of mitochondrial dynamics in adult tissues under both phys- of a single mitochondrial tubule (Loew, 1999). We suggest that iological and experimental conditions. the dynamic nature of mitochondria protects these organelles by ensuring that regional losses of membrane potential, caused perhaps by local depletion of metabolic substrates or mtDNA, A model for the role of mitochondrial fusion in are always transient (Fig. 10 A). Mitochondrial fusion enables protecting mitochondrial function intermitochondrial cooperation by allowing exchange of both It has been paradoxical why eukaryotes have invested in fusion membrane and matrix contents and therefore may help to re- and fission pathways in cells that have “stable” mitochondrial store local depletions and maintain mitochondrial function networks. Given that mitochondrial tubules can be maintained (Nakada et al., 2001a). Although there is no gross loss of by reducing fusion and fission simultaneously (Bleazard et al., mtDNA in mutant cells, we currently do not know if the indi- 1999; Sesaki and Jensen, 1999; Fekkes et al., 2000; Mozdy et vidual, defective mitochondria have lost mtDNA. The Journal of Cell Biology 198 The Journal of Cell Biology | Volume 160, Number 2, 2003 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 immuno- precipitates (top) and total cell lysates (bottom) from Mfn1 or Mfn2 mutant cells (indicated on top) were used in an analysis similar to A. Multiple molecular modes of mitofusin action Given their broad and overlapping expression, it has been Figure 10. Models. (A) The protective role of mitochondrial fusion. unclear why there are two separate mammalian mitofusins. At a low rate, individual mitochondria stochastically lose function. Our results show that the two mitofusins form three distinct In wild-type cells, a defective mitochondrion (shaded) undergoes molecular complexes that are capable of promoting mito- fusion with functional mitochondria and regains activity. In Mfn- deficient cells, such rescue occurs at a much reduced rate. (B) Three chondrial fusion–Mfn1 homotypic oligomers, Mfn2 homo- modes of mitofusin action. Mitofusins form homotypic and heterotypic typic oligomers, and Mfn1-Mfn2 heterotypic oligomers. We complexes that lead to three activities (I, II, III) involving fusion. See propose that the relative importance of each of these modes Discussion for details. Mfn1 mutant cells contain only activity III; Mfn2 of mitofusin action can change depending on the cell type mutant cells contain only activity I. Since disruption of either Mfn1 or (Fig. 10 B). In mouse fibroblasts, all three oligomeric com- Mfn2 fragments mitochondria and results in distinct phenotypes, MEFs plexes are likely to play important roles because disruption appear to use all three activities (indicated by asterisks). In contrast, of either Mfn1 or Mfn2 leads to severe mitochondrial frag- trophoblast giant cells predominantly use activity III because they are affected in Mfn2 mutants and not Mfn1 mutants. mentation. Nevertheless, overexpression of Mfn1 homotypic oligomers or Mfn2 homotypic oligomers is sufficient to re- store mitochondrial tubules, clearly demonstrating that the ally homogeneous. However, at any given time point indi- homotypic complexes are functional for fusion. In tropho- vidual mitochondria are functionally distinct entities (Col- blast giant cells, it appears that Mfn2 homotypic oligomers lins et al., 2002). Therefore, without mitochondrial fusion, are most important. As a result, this cell type is affected in the stochastic differences between distinct mitochondria can Mfn2-deficient embryos but not Mfn1-deficient embryos. accumulate to affect the well-being of the cell. The mamma- It remains to be determined whether these three com- lian mitofusins, Mfn1 and Mfn2, function in three distinct plexes function in the same or distinct types of mitochon- molecular complexes to promote mitochondrial fusion and drial fusion. It is also interesting to note that loss of Mfn2 thus protect mitochondrial function. leads to the formation of both large and small mitochondrial spheres. This phenotype is easily distinguished from loss of Materials and methods Mfn1. The larger mitochondrial fragments may be due to a higher residual fusion activity in Mfn2 mutant cells than in Cloning of Mfn cDNA and genomic constructs Homology searches using the Drosophila Fzo sequence (Hales and Fuller, Mfn1 mutant cells. The loss of tubular shape may simply re- 1997) identified a murine EST (IMAGE Consortium Clone ID 733269) that flect the loss of cytoskeletal interactions as noted in Fig. 4. encodes a highly homologous polypeptide. This cDNA was used to screen Alternatively, it may be that Mfn2, and by extension Mfn2 a mouse kidney cDNA library, resulting in isolation of cDNAs for both Mfn1 and Mfn2. These sequences are available from GenBank/EMBL/ homotypic complexes, play a more direct role in maintain- DDBJ under accession nos. AY174062 and AY123975. ing mitochondrial tubular shape. Thus, although each of the Genomic clones of Mfn1 and Mfn2 were retrieved from a lambda 129/ Mfn complexes is involved in fusion, it is possible that they SvJ mouse genomic library (Stratagene). Genomic fragments were sub- cloned into the targeting vector pPGKneobpAlox2PGKDTA (a gift from F. have distinct functions in addition to fusion. Gertler and L. Jackson-Grusby, Massachusetts Institute of Technology, In conclusion, in cells with continual cycles of fusion and Cambridge, MA) as the right arms. To insert the left arms, genomic frag- ments were amplified by PCR, which also introduced a stop codon just be- fission, the mitochondrial population is essentially function- The Journal of Cell Biology Essential role of mitochondrial fusion in mice | Chen et al. 199 fore the conserved GKS sequence in each GTPase domain. All constructs TS cells from e3.5 blastocysts were derived using established protocols were verified by DNA sequence analysis. The sequences of all oligonucle- (Tanaka et al., 1998a). Live cells were stained with MitoTracker Red (150 otides used in this study are available from the authors. nM) and Syto16 (100 nM; Molecular Probes). To identify the chromosomal locations of Mfn1 and Mfn2, the Jackson Lab- oratory (C57BL/6JEi SPRET/Ei) F1 SPRET/Ei backcross panel was used. PEG fusion Linkage analysis was performed by the Jackson Laboratory Mapping Resource. 40,000 cells expressing mitochondrially targeted GFP were cultured over- night on 25-mm coverslips with 40,000 cells expressing mitochondrially Construction of knockout mice targeted dsRed. The next morning, cells were fused for 60 s with 50% PEG Each gene replacement vector was linearized with SacII and electroporated 1500 (Roche). The cells were washed and grown for 7 h or 24 h in me- into low passage 129/SvEv embryonic stem cells using established proce- dium containing 30 g/ml cycloheximide before fixation. dures (Chester et al., 1998). Neomycin-resistant colonies were screened by Southern blot analysis. Two independently isolated clones for each targeted Immunofluorescence allele were injected into C57BL/6 blastocysts to generate chimeric mice. Cells grown on polylysine-coated coverslips were fixed with prewarmed Excision of the neomycin cassette had no effect on phenotypes. (37 C) 3.7% formaldehyde and permeabilized in PBS/0.1% Triton X-100. In some experiments, cells were incubated with 150 nM MitoTracker Red for 30 Confirmation of targeting event min before fixation and then permeabilized with acetone at 20 C. Cells For Southern blot analysis of targeted alleles, genomic DNAs were di- were blocked with 5% bovine calf serum and incubated with primary anti- gested with XbaI (Mfn1 mutant line) or EcoRI (Mfn2 mutant line) and hy- body. For Myc epitope–tagged proteins, the mouse monoclonal antibody bridized with a flanking genomic probe. PCR was used for routine geno- 9E10 was used. For COX1, mouse monoclonal 1D6-E1-A8 (Molecular typing of offspring. A forward genomic primer, a reverse genomic primer, Probes) was used. For detection, Cy3- or Alexa Fluor 488–conjugated second- and a reverse neomycin primer were used to detect both the targeted and ary antibodies (Jackson ImmunoResearch Laboratories and Molecular Probes) wild-type alleles in a single reaction. were used. Cells were imaged with a Plan NeoFluar 63 objective on a Zeiss For Western blot analysis, chicken antisera was generated against Mfn1 410 laser scanning confocal microscope (Carl Zeiss MicroImaging, Inc.). (residues 348–579) or Mfn2 (residues 369–598) fused to an NH -terminal histidine tag. IgY purified from chicken eggs was affinity purified on a col- Analysis of mtDNA umn coupled to a maltose-binding protein fusion protein containing either Southern analysis of mtDNA was performed by linearizing with XhoI and Mfn1 residues 348–579 or Mfn2 residues 369–598. probing with a radio-labeled PCR fragment containing the COX1 gene. In situ hybridization Immunoprecipitation Individual implantation sites were fixed overnight at 4 C in 4% parafor- Cell lines were infected with various combinations of retroviruses express- maldehyde, dehydrated through an ethanol series, treated with xylenes, ing Myc- and HA-tagged Mfn1, Mfn2, and Drp1(K38A). Monolayers were and embedded into paraffin blocks. For staining, 10-m sections were cut resuspended in lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton in a transverse plane with respect to the placenta. Slides were processed X-100, and a protease inhibitor cocktail) and immunoprecipitated with for hematoxylin-eosin staining or in situ hybridization as described previ- 9E10 antibody coupled to protein A–Sepharose beads. After washing, sam- ously (Vortkamp et al., 1996). All riboprobe templates were derived from ples were immunoblotted with HA.11 (Covance). RT-PCR using e11.5 placental RNA as template. No specific staining was detected using sense probes. For genotyping, embryonic tissue was scraped off unstained slides. DNA was recovered and genotyped by PCR. Online supplemental material Confocal time-lapse videos of EYFP-labeled mitochondria in wild-type Retroviral and plasmid constructs (video 1), Mfn1 mutant (video 2), and Mfn2 mutant (video 3) MEFs are available at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1. Vid- To construct Mfn1 or Mfn2 with Myc epitope tags, a BamHI site was intro- eos 1–3 show mitochondrial dynamics in wild-type and mutant MEFs. Vid- duced immediately before the stop codon. The cDNA was subcloned into eos 1 and 3 are 20-min recordings; video 2 is a 10-min recording. The mi- the vector pcDNA3.1( )/Myc-His A (Invitrogen). Myc epitope tag cassettes croscopic field is 96 m 96 m in video 1, 68 m 68 m in video 2, derived from pMMHb-3 Myc were then inserted into the BamHI site to and 75 m 75 m in video 3. form pMfn-Myc. Mfn1(K88T) and Mfn2(K109A) were constructed by site- directed mutagenesis (Kunkel et al., 1991). To make the Mfn-HA constructs, Mfn1 and Mfn2 cDNAs were subcloned into a pcDNA3.1( ) vector contain- We thank Dr. Philip Leder for his support in the early stages of this work. ing a COOH-terminal 3 HA tag. Murine Drp1 was amplified from mouse We are grateful to M. Michelman for embryonic stem cell culture assis- placental RNA, and Drp1(K38A) was constructed by site-directed mutagene- tance and A. Harrington for blastocyst injections. We thank Drs. M. Rojo sis. The Drp1(K38A) insert was subcloned into a modified pcDNA3.1( )/ Myc-HisA vector containing seven Myc epitope tags at the COOH terminus. and A. Lombes for stimulating discussions about the PEG fusion assay. To generate retroviral expression constructs for each of the above, the H. Chen is supported by an Alcott Postdoctoral fellowship. S.A. Detmer epitope-tagged cDNAs were recloned into the retroviral vector pCLW (a and E.E. Griffin are supported by a National Institutes of Health training gift from C. Lois, California Institute of Technology). The retroviral expres- grant NIHGM07616 and E.E. Griffin is funded by a Ferguson fellowship. sion vectors were cotransfected with the ecotropic retroviral packaging A.J. Ewald is a participant in the Initiative in Computational Molecular Bi- vector pCLEco (a gift from C. Lois, California Institute of Technology) into ology funded by the Burroughs Wellcome Fund Interfaces program. D.C. 293T cells. Retroviral stocks were harvested 48 h after transfection and Chan is a Bren scholar, Rita Allen scholar, Beckman Young investigator, used to infect MEF cultures. Fragments encoding mitochondrially targeted and recipient of a Burroughs Wellcome Fund Career Development award GFP and dsRed (from plasmids pHS1 and pHS51; gifts from H. Sesaki and in Biomedical Sciences. This research was supported by the National Insti- R. Jensen, Johns Hopkins University, Baltimore, MD) were subcloned into pCLW to generate retroviral expression vectors. tutes of Health (grant 1 RO1 GM62967-01). Submitted: 12 November 2002 MEF and TS cell lines Revised: 12 December 2002 MEFs were derived from e10.5 embryos. Embryos were mechanically dis- Accepted: 12 December 2002 persed by repeated passage through a P1000 pipette tip and plated with MEF media (DME, 10% FCS, 1 nonessential amino acids, 1 mM L-glu- tamine, penicillin/streptomycin [Life Technologies/GIBCO BRL]). 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References