Mitofusins Mfn1 and Mfn2 Coordinately Regulate Mitochondrial Fusion And

The Journal of Cell Biology

JCB

Introduction spermatids andresultsinmalesterility(HalesFuller, developmentally regulatedmitochondrialfusioninpostmeiotic transiently expressedinspermatids.DisruptionofFzoprevents cells andresultsinnumeroussmallsphericalmitochondria. branched, tubularmitochondrialnetworktypicalofnormal ther Mfn1orMfn2dieinmidgestation.However, whereas ogy when overexpressed. We findthatmicedeficient inei- Mfn2, have beenshown toaffectmitochondrial morphol- http://www.jcb.org/cgi/doi/10.1083/jcb.200211046 The Journal ofCellBiology  1997). Inbuddingyeast,deletionof fuzzy onions the nuclearlyencodedmitochondrialtransmembraneGTPase, Voth, 1994;Nunnarietal.,1997). overall architectureoftheseorganelles(Bereiter-Hahnand processes thatexistinequilibriumandservetomaintainthe continual cyclesofmitochondrialfusionandfission,opposing network ofmitochondrialtubules,therearefrequentand Rizzuto et (Bereiter-Hahn andVoth,1994;Nunnarietal.,1997; undergo constantmigrationandmorphologicalchanges lapse microscopyoflivingcellsrevealsthatmitochondria Mitochondria areremarkablydynamicorganelles.Time- a phenotypewedeterminetobeduesevere reduction or Mfn2display distincttypesoffragmented mitochondria, giant cellsarenormal.Embryonic fibroblastslacking Mfn1 of theplacentaltrophoblastgiantcelllayer, Mfn1-deficient Mfn2 mutantembryos have aspecificandsevere disruption M 1 Key words: membranefusion;mitochondria; GTPase; mice;knockout polyethylene glycol;TS,trophoblast stem. onic fibroblast;Mfn,mitofusin;mtDNA, mitochondrialDNA;PEG, onic *Abbreviations usedinthispaper:Drp, dynamin-relatedprotein;e,embry- 8826. E-mail:[email protected] MC114-96, Pasadena,CA91125.Tel.:(626)395-2670.Fax:395- Address correspondencetoDavidC.Chan,1200EastCaliforniaBlvd., The onlineversionofthisarticlecontainssupplementalmaterial. Hsiuchen Chen, embryonic development mitochondrial fusion andare essentialfor Mitofusins Mfn1andMfn2coordinately regulate

Division ofBiology and

TheRockefeller University Press, 0021-9525

Article In bothyeastandflies,mitochondrialfusioniscontrolledby

day; EYFP,enhancedYFP;Fzo,fuzzy onions;MEF,mouseembry-

vertebrates isunknown. The mitofusins,Mfn1and ﬁssion, butthesigniﬁcance oftheseprocessesin namic equilibriumbetweenorganellefusionand itochondrial morphologyisdeterminedby ady-

al., 1998).Evenincellswithaseemingly“stable”

(Fzo).*In

1 ScottA.Detmer, , Volume 160,Number 2,January 20,2003189–200 2 Drosophila Biological Imaging Center, Beckman Institute, CaliforniaInstitute of Technology, Pasadena, CA91125 , Fzoisspecificallyand FZO1 /2003/01/189/12 $8.00 1 Andrew J.Ewald, disruptsthehighly 1,2 Erik E.Grifﬁn, protective effectsonthemitochondrial population. and by enablingcooperation betweenmitochondria, has chondrial fusionisessentialforembryonic development, in mutantcellslosemembrane potential. Therefore, mito- mitochondrial act inthreeseparate molecularcomplexestopromote and Mfn2have bothredundantanddistinctfunctions plexes arefunctionalforfusion. We concludethatMfn1 show, by rescueofmutantcells,thatthehomotypic com- Mfn2 formhomotypicandheterotypiccomplexes in mitochondrial fusion.Moreover, weﬁndthatMfn1and fzo1 1998). Third,experimentally inducedcellhybridsdemon- that undergofrequentfusion andfission(Rizzutoetal., tochondria areorganizedinto extensivetubularnetworks cence microscopyofcultured HeLacellsshowsthatthemi- through mitochondrialfusion. Second,time-lapsefluores- is likelythattheseprogressive morphologicalchangesoccur networks intheadult(Bakeevaetal.,1978,1981,1983). It tubules inembryonicstagesbutthenreorganizeintoreticular and diaphragmskeletalmuscleappearasisolatedellipsesor tissues. Forexample,themitochondriaofratcardiacmuscle dramatic transitionsoccurduringthedevelopmentofcertain cells. First,ultrastructuralstudiesofmitochondriashowthat basic cellularprocessindeedplaysasignificantroleinvertebrate al., 2000).However,severalobservationssuggestthatthis chondrial fusioninculturedmammaliancells(Enriquez et been raisedaboutthefrequencyandimportanceofmito- ment of mitochondrialfusioninlowereukaryotes,thereisdisagree- their functionalredundancyisunclear. fusins arebroadlyexpressed(Rojoetal.,2002),andtherefore lines (SantelandFuller,2001;Rojoetal.,2002).Bothmito- can altermitochondrialmorphologywhenoverexpressedincell two Fzohomologues,termedmitofusin(Mfn)1andMfn2,that mitochondrial fusion(Hermannetal.,1998).Humanscontain 1998). Furthermore,thereisadisruptionofmating-induced DNA (mtDNA)(Hermannetal.,1998;Rapaport Despite ourincreasingknowledgeabouttheimportance about itsphysiologicalroleinvertebrates.Doubtshave yeastform“petite”coloniesthatlackmitochondrial http://www.jcb.org/cgi/content/full/jcb.200211046/DC1 Supplemental Material canbefoundat: 1 fusion. Strikingly, asubsetofmitochondria ScottE.Fraser, 1,2 andDavid C.Chan

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on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology of promoter; Xb,XbaI.(E)Genomictargeting toxin subunit AdrivenbythePGK by triangles).PGK-DTA,diphtheria was usedasaloadingcontrol. against Mfn1(D)andMfn2(H). affinity-purified antibodiesdirected embryonic lysateswereanalyzedwith and mutantlysates.Postnuclear (D andH)Westernanalysesofwild-type identical tothoseinBandF,respectively. and mutantloci.TheDNAsamplesare distinct fragmentsfromthewild-type were usedsimultaneouslytoamplify Three primers(labeled1,2,and3) genotypes. (CandG)PCRgenotyping. knockout bandsareindicatedas indicated inAandE.Thewild-type Mfn2 andanalyzedwiththeprobes with XbaI(B)forMfn1andEcoRI(F) offspring. GenomicDNAsweredigested targeted embryonicstemclonesand (B andF)Southernblotanalysesof Results We identified two murinehomologuesof Identification ofmurineFzohomologues mitochondria fromrespiratory dysfunction. low cooperationbetweenmitochondria, therebyprotecting our resultssuggestthatmitochondrialfusionfunctionstoal- individually topromotemitochondrialfusion.Inaddition, erotypic oligomersandthereforecancooperateaswellact sion. Thesemitofusinscanexistasbothhomotypicandhet- sential forembryonicdevelopmentandmitochondrialfu- Mfn2. OuranalysisrevealsthatMfn1andMfn2areeaches- vertebrates, wehavegeneratedknockoutmiceforMfn1and chondrial fission(Franketal.,2001). dynamin-related protein(Drp)1,whichisinvolvedinmito- of themitochondrialnetwork.Thisfragmentationrequires tion ofcelldeathissometimesassociatedwithfragmentation have beenimplicatedintheregulationofapoptosis.Induc- 2001b; Onoetal.,2001).Finally,mitochondrialdynamics plementation ofmtDNAgeneproducts(Nakadaetal., strate rapidmtDNAmixing(Hayashietal.,1994)andcom- of knockoutmice. Figure 1. 190 labeled Neo;flanking resistance gene(lightgraysegment genomic sequencewithaneomycin- substitution oftheG1andG2encoding stop codon(asterisk)inexon3anda allele (bottombar)containingapremature construct (middlebar)resultsinatargeted double crossoverwiththetargeting and G2motifsoftheGTPasedomain.A contains codingsequencesfortheG1 aligned above.Thedarkgraysegment of wild-type To assessthephysiologicalroleofmitochondrialfusionin Mfn2 Mfn1 The JournalofCellBiology . DrawnasinA.RI,EcoRI. . Thetopbarindicatesthe Mfn1 Construction andverification genomiclocuswithexons

(A)

loxP Genomic targeting

sitesindicated

Volume 160,Number2,2003

-Actin

fzo fromamouse al., 1998;Rapaportet1998),eachmurine other andareboth52%similarto (Lupas, 1996).Mfn1andMfn2are81%similartoeach phobic heptadrepeats,hallmarksofcoiled-coilregions brane segmentisflankedbytworegionscontaininghydro- codes apredictedtransmembraneGTPase.Thetransmem- of theGTPase domain neartheNH both cases,astop codonwasengineeredatthe verybeginning the diphtheriatoxinsubunitA genefornegativeselection.In using theneomycinresistance geneforpositiveselectionand We constructedgenereplacement vectorsfor Generation ofknockoutmice deficientinMfn1andMfn2 and Fuller,2001), Mfn2 ysis placed these murinehomologuesasMfn1andMfn2.Linkageanal- human mitofusins(SantelandFuller,2001),wedesignate cDNA library.Inaccordancewiththenomenclaturefor and Fuller,2001;Hwaetal.,2002), 3 ( 4 ( As withthe 70–80 cM)inaregionsyntenictohuman1p36. 12–13 cM)inaregionsyntenictohuman3q25-26. waslocalizedtothedistalendofmousechromosome Mfn1 fzo attheproximalendofmousechromosome genesfrom Saccharomyces cerevisiae Drosophila melanogaster Drosophila 2 terminus(Fig. 1,Aand Homo sapiens Mfn1 Fzo. (Hermannet Mfn and geneen- (Santel (Santel

Mfn2

on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology Fzo function.Therefore,thedisrupted demonstrate thatanintactGTPasedomainisessentialfor Fig. 7Cand8C), 1998), aswellourownstudies(see mutant lysates(Fig.1,Dand H). Mfn2 confirmedlossofthetargeted proteininhomozygous analysis usingaffinity-purified antiseraraisedagainstMfn1or alleles (Fig.1,B,C,F,andG).Importantly,Westernblot PCR analysisconfirmedgermlinetransmissionofthetargeted described hereshouldbenullalleles.BothSouthernblotand of GTPandforMg myces cerevisiae onic lethalityof homozygousmutants.Forthe Mfn1knock- ity andfertility inheterozygousanimalsbut result inembry- Both theMfn1andMfn2knockouts demonstratefullviabil- Embryonic lethalityinhomozygous mutantmice ase motifsarecrucialforbindingofthe with theneomycinexpressioncassette.TheseuniversalGTP- placement oftheG1andG2motifsGTPasedomain E). Inaddition,theresultinggenomiclocieachcontainare- Sprang, 1997).Geneticanalysesin (HalesandFuller,1997;Hermannetal., 2 coordination(Bourneetal.,1991; Drosophila Mfn1 and and and Mfn2 phosphates

Saccharo-

alleles

greater developmental delay(unpublishedresults). Thisob- tant embryosdie earlierthaneithersinglemutant andshow showed noobviousmalformations. their littermatesbutwereotherwise welldevelopedand mutant embryosate9.5ande10.5 wereslightlysmallerthan gous mutantembryoswereresorbed. Thelivehomozygous were intheprocessofresorption. Bye11.5,87%ofhomozy- ever, startingate10.5,29%ofhomozygousmutantembryos mozygous mutantembryoswererecovereduptoe9.5.How- lay. Inaddition,mutantembryoswereoftendeformed. nificantly smallerandshowedpronounceddevelopmentalde- not evidentuntile11.5,bye8.5allmutantembryosweresig- that theycouldnotbegenotyped.Althoughresorptionswere resorbed (86%),andadditionalresorptionsweresoadvanced inviability. Ate12.5,mostidentifiablemutantembryoswere e11.5, 20%ofthemutantembryoswereresorbed,indicating ulatory plugwasdetectedisdesignatede0.5).However,by obtained uptoembryonicday(e)10.5(noonoftheacop- out line,normalfrequenciesoflivemutantembryoswere Preliminary studiesindicatethat doublehomozygousmu- In theMfn2knockoutline,normalnumbersofliveho-

Essential roleofmitochondrialfusioninmice| littermates. of placentaefrome9.5wild-type(G)andmutant(H) (G andH)PL-I(giantcellmarker)RNAinsituanalysis eosin–stained sectionsfromtheplacentaeabove. sparser andhavesmallernuclei.(EF)Hematoxylin- trophoblast giantcells.NotethatthecellsinDare enlarged inCandD.Arrowsarrowheadsindicate littermate embryos.TheboxedareasofAandBare from e10.5wild-type(AandC)mutant(BD) placentae. Figure 2. (A–D) DAPI-stainedsectionsofplacentae Defective giantcelllayerofmutant Chenetal.

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on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology range ofmitochondrial morphologies.The major morpho- fluorescent protein (EYFP).Wild-typeMEFs showeda rovirus expressingmitochondrially targetedenhancedyellow e10.5 embryos.TheMEFcultures wereinfectedwitharet- cells, wederivedmouseembryonic fibroblasts(MEFs)from To examinemitochondrial morphology inMfn-deficient mitochondrial morphology Both Mfn1-andMfn2-deficientcellshaveaberrant midgestation secondarytoaninadequateplacenta. findings suggestthattheMfn2mutantembryosaredyingin labyrinthine layer(Jacqueminetal.,1998).Together,these observed usingTEF5(unpublisheddata),amarkerforthe staining betweenwild-typeandMfn2mutantsectionswere this placentallayer(unpublisheddata).Nodifferencesin 1988), suggestinganadditionalbutmoresubtledefectin cone andspongiotrophoblastmarker4311(Lescisinetal., vealed somewhatweakerstainingwiththeectoplacental et al.,1998).Interestingly,ourinsituhybridizationsalsore- cell markers,proliferin(Leeetal.,1988)andHand1(Riley bridizations (unpublisheddata)usingtheadditionalgiant These observationswereconfirmedwithRNAinsituhy- lar resultswereseeninplacentaefrome8.5throughe10.5. giant celllayerwasgenerallyonlyonethick.Simi- ring ofweaklystaininggiantcells(Fig.2H).Moreover,the In contrast,Mfn2mutantplacentaeshowedanincomplete the regiondistaltospongiotrophoblastlayer(Fig.2G). ant cellstainingwithmultiplelayersofgiantcellspackedin 1991), wild-typeplacentaeshowedanintensefullringofgi- placental layers.WiththegiantcellmarkerPL-I(Fariaetal., pression ofmolecularmarkersspecificforeachthethree data) despiteexpressionofMfn1intheplacenta(Fig.1D). ment weredetectedinMfn1mutantembryos(unpublished DNA content(Fig.2,A–D).Nodefectsinplacentaldevelop- staining, whichhighlightsthegiantcellsbecauseoftheirhigh (Fig. 2F).TheseobservationswereconfirmedwithDAPI plying areductioninthenumberofendoreplicationcycles tity, andthefewthatareobservedcontainsmallernuclei,im- layer withtwodefects.Thegiantcellsaredeficientinquan- mutant embryosreproduciblyshowanimpairedgiantcell 2000; Scottetal.,2000).Strikingly,placentaefromMfn2 lethality (Krautetal.,1998;RileyHesse (Cross, 2000).Deficienciesinthesecellsleadtomidgestation vessels, andinvasionoftheconceptusintouterinelining important rolesinhormoneproduction,recruitmentofblood critical interfacebetweenfetalandmaternaltissuesplay ceeds repeatedlywithoutassociatedcytokinesis)thatlieatthe from endoreplication,aprocesswhereDNAreplicationpro- (Fig. 2E).Trophoblastgiantcellsarepolyploid(derived phoblast layer,andadistal,circumferentialgiantcelllayer posed ofaproximallabyrinthinelayer,middlespongiotro- gous embryosshowedthetypicaltrilaminarstructurecom- histological sectionsofplacentaefromwild-typeandheterozy- tation lethality(Copp,1995).Hematoxylin-eosin–stained tal insufficiencyisoneofthemostcommoncausesmidges- We examinedplacentaldevelopmentindetailbecauseplacen- Placental defectsinMfn2mutantmice velopmental eventearlyinembryogenesis. and mayhaveacooperativerelationship,inparticularde- servation suggeststhatMfn1andMfn2arebothrequired, 192 Using RNAinsituhybridization,weexaminedtheex- The JournalofCellBiology

Volume 160,Number2,2003

logical class,whichencompassed mented mitochondria (Fig.3,CandD).Greater than95% spherical andweretermedas “fragmented.” boxed areasinA,C,andE,respectively.Arrowindicatesatubule phalloidin (red).(B,D,andF)Highermagnificationimagesofthe mitochondrial EYFP(green)werecounterstainedwithrhodamine- (C andD),Mfn2mutant(EF)MEFcells.MEFsexpressing (A–F) Mitochondrialmorphologyinwild-type(AandB),Mfn1mutant Figure 3. Syto16 (green).Severalcellsaretightlyclustered. stained withMitoTrackerRed,andthenucleiwere wild-type (G)andmutant(H)TScells.Themitochondriawere several micronsto dria. Thelengthofthesemitochondrial tubulesrangedfrom plasm. Suchcellshadnooronly afewsphericalmitochon- distributed inaroughlyradialmannerthroughoutthecyto- was characterizedbyanetworkofextendedwavytubules much smallermorphological class, whichencompassedonly

10 6% ofwild-typecells,hadmitochondria thatweremostly

In contrast,Mfn1 mutantMEFshaddramatically frag-

m inlength.(GandH)Mitochondrialmorphologylive

Morphological defectsinmitochondriaofmutantcells. 10 m (Fig.3,AandB,arrow). A 90% ofwild-typecells,

on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology ments, depending onthecelltype(Nangaku etal.,1994; tochondria can beanchoredalongmicrotubule oractinfila- ment alongdefinedtracksisconsistent withreportsthatmi- along theirlongaxisonradial tracks(Fig.4A).Thismove- bular, weredirectedradially, andmovedbackforth mutant cells.Mostmitochondria inwild-typecellsweretu- vealed strikingdefectsinthe mobilityofmitochondriain although afewsucheventscanbefound(Fig.4C). at http://www.jcb.org/cgi/content/full/jcb.200211046/DC1), fusion eventsmuchlessfrequently(videos2and3,available mutant cellstheovoidorsphericalmitochondriaundergo jcb.200211046/DC1). However,inbothMfn1andMfn2 video 1,availableathttp://www.jcb.org/cgi/content/full/ most mitochondriaduring20-minrecordings(Fig.4A; at leastoneapparentfusionorfissioneventwasobservedfor In wild-typecells,themitochondriaarehighlymotile,and tant MEFsdisplayaberrationsinmitochondrialdynamics. time-lapse confocalmicroscopytodeterminewhethermu- Because mitochondriaaresuchdynamicorganelles,weused Altered mitochondrialdynamicsinMfn-deficientcells and mayhavesomedistinctfunctions. broblasts, eachproteinisessentialformitochondrialfusion that eventhoughbothMfn1andMfn2areexpressedinfi- maintaining tubularshape.Together,theseresultssuggest to controllingmitochondrialfusion,Mfn2isinvolvedin normal tubules,theseobservationssuggestthatinaddition in shortertubulesandsmallsphereswiththediameterof defect inmitochondrialfusionwouldbeexpectedtoresult of mitochondrialtubulesinwild-typecells.Becauseasimple MEF andTScellsareseveraltimeslargerthanthediameters of somethesphericalmitochondriainbothMfn2mutant spheres ofwidelyvaryingsizes.Interestingly,thediameters contrast, manyMfn2mutantcellsexhibitmitochondrial with diametersnolargerthanthatofanormaltubule.In drial tubulesorverysmallspheresthatareuniforminsize, of fragmentation,resultingineitherveryshortmitochon- distinguishable. LossofMfn1leadstoagreaterdegree characteristic mitochondrialmorphologiesthatarereadily tation ofmitochondrialtubules,thetwomutationsleadto cells showedonlysphericalmitochondria(Fig.3H). chondrial tubules(Fig.3G).Incontrast,Mfn2mutantTS wild-type TScoloniesdisplayedanetworkoflongmito- blastocysts. WithMitoTrackerRedstaining,thecellswithin trophoblast stem(TS)celllinesfromwild-typeandmutant placental insufficiencyofMfn2mutantembryos,wederived Mfn1 andMfn2playmajorrolesinmitochondrialfusion. data). Theseresultsareconsistentwiththehypothesisthat both cristaeandthetypicaldoublemembrane(unpublished the sphericalmitochondriainthesemutantcellscontained section EMrevealedthatinspiteoftheiraberrantdimensions terize thelargestmorphologicalclassinwild-typecells.Thin displayed thenetworksoflongextendedtubulesthatcharac- significant tubules.NeitherMfn1norMfn2mutantcellsever F). Onlyasmallminorityofmutantcells(4.5%)contained tochondria appearedmostlyasspheresorovals(Fig.3,Eand bules. Similarly,in85%ofMfn2mutantfibroblaststhemi- dria, whereasonly1–2%ofcellscontainedanysignificanttu- of thesecellscontainedonlyseverelyfragmentedmitochon- In additiontoreducedfusion, ourtime-lapsevideosre- Although lossofeitherMfn1orMfn2resultsinfragmen- To determinewhethermitochondrialdefectsunderliethe http://www.jcb.org/cgi/content/full/jcb.200211046/DC1. were manuallyhighlightedinblue.Seealsovideos1–3availableat Adobe Photoshop then migratesawayalongitslongaxis.Imageswereprocessedin mitochondrion protrudesatubularextensionthatseparatesand movement inmostmitochondria.(D)OnesphericalMfn2-deficient but donotfuseuntilmuchlater.Notealsothelackofdirected a Mfn2mutantcell,twoovoidmitochondriacontacteachother mutant cell,themitochondriamoveinanundirectedmanner.(C)In Note thatmitochondriamovealongtheirlongaxes.(B)InaMfn1 each other.Thesepairscontactend-to-endandfuseimmediately. wild-type cell,twopairsofmitochondriacanbeseenmovingtoward cells. Figure 4. mitochondrial fusion activityusingapolyethylene glycol fragmented mitochondria inmutantcells, wemeasured To definitivelyshowthatlack offusionisthebasisfor Mfn1 orMfn2 Decreased mitochondrialfusion ratesincellslacking tered morphologyresultingfrom reducedfusion. mobility ofsphericalmitochondriaissecondarytotheiral- lizing Mfn2-deficientmitochondria.Instead,thehampered observations suggestthatthereisnointrinsicdefectinmobi- can mergewithspheresandlosedirectedmovement.These subsequently moveaway(Fig.4D),orconverselytubules changeable becausetubulescanprojectoutofspheresand not. However,thetwomorphologicalclassesareinter- along radialtracks,whereasthesphericalmitochondriado dria, thetubularmitochondriacanstillmovelongitudinally tant cellswithbothsphericalandshorttubularmitochon- defects (unpublisheddata;Fig.3).Strikingly,inMfn2mu- an antitubulinmAborrhodamine-phalloidinrevealedno to disorganizationofthecytoskeletonbecausestainingwith (Fig. 4,BandC).Thislackofdirectedmovementisnotdue mutant cellsshowrandom“Brownian-like”movements ation inmobility;thesphericalorovoidmitochondria mitochondria ofMfn-deficientcellsdisplayadramaticalter- Morris andHollenbeck,1995;Tanakaetal.,1998b).The Still framesfromtime-lapseconfocalmicroscopy.

Essential roleofmitochondrialfusioninmice| Dynamics ofmitochondriainwild-typeandmutant

withtheembossfilter,andselectedmitochondria

Chenetal.

(A) Ina

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on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology 194 fusion 57%( trast, whenMfn1mutantcellswereexamined7hafterPEG colocalization ofredandgreenfluorescentsignals.Incon- sively fusedmitochondria(Fig.5A)asdemonstratedby with MitoTracker Red(Fig.6E).Likewise, in Mfn2mutant tained asubset ofmitochondriathatshowed poorstaining these cells.InMfn1mutantcultures, almosteverycellcon- as opposedtothetubularmitochondria thatpredominatein dye. Suchmitochondriawere invariablysmallandspherical beled withEYFPbutfailed to sequesterMitoTrackerRed type cells,thereexistedisolated mitochondriathatwerela- calization ofthetwofluorophores (Fig.6D).In7%ofwild- uniformly stainedwithMitoTrackerRed,resultingincolo- wild-type cells,EYFP-markedmitochondriaweretypically pressing EYFPtargetedtothemitochondrialmatrix.In the mitochondrialmembranepotential,tostainMEFsex- toTracker Red,alipophiliccationicdyewhichissensitiveto dividual mitochondria.Totestthishypothesis,weusedMi- reasoned thatfunctionaldefectsmayexistatthelevelofin- that mtDNAproductsareintactandfunctional. cating nogrossdefectsinrespirationandfurtherconfirming dogenous andcoupledrespiration(unpublisheddata),indi- Mfn1 andMfn2mutantculturesshowedhighratesofen- ration andmitochondrialfusion.Likewild-typecells,both allows acriticalassessmentoftherelationshipbetweenrespi- Mfn-deficient cellsclearlyretainmtDNA,andthisfeature mtDNA (Fig.6,BandC).Therefore,unlike expressed COX1,amitochondrialproteinencodedby lished data).Inaddition,themitochondriainmutantcells lines containnormallevelsofmtDNA(Fig.6A;unpub- ern blotandPCRanalysisshowedthatbothmutantcell yeast (Hermannetal.,1998;Rapaport1998).South- mtDNA becausethereiscompletelossofin We testedwhetherMfn1andMfn2mutantcellslose Stochastic defectsinmitochondrialmembranepotential perhaps duetotheirmoreseverelyfragmentedmorphology. a greaterextentthanthatofMfn2-deficientmitochondria, the mobilityofMfn1-deficientmitochondriaisimpairedto cells exhibitedthissectoringeffect.Therefore,itseemsthat fluorescence (Fig.5D).Only1%offusedMfn2mutant the cytoplasmasshownbydiscretesectorsofredandgreen cells, themitochondriadidnotreadilyspreadthroughout drial fusion.Interestingly,in10%offusedMfn1mutant Thus, mutantcellshaveseverelyreducedlevelsofmitochon- served even24hafterPEGtreatment(unpublisheddata). Mfn2 mutantcellswithunfusedmitochondriawereob- extensive fusion,and30%showedpartialfusion.Mfn1 fused mitochondriaafter7h(Fig.5,EandF).1%showed 202) offusedMfn2mutantcellsshowedpredominantlyun- treatment, allofthefusedcells( cells. Whenwild-typecellswereexamined7hafterPEG vent synthesisofnewfluorescentmoleculesinthefused plied tofusethecells.Cycloheximidewasincludedpre- mitochondrially targetedGFP,andPEGwastransientlyap- ally targeteddsRedwascoculturedwithacelllineexpressing (PEG) cellfusionassay.Alineexpressingmitochondri- sion, and8%showedpartialfusion.Similarly,69%( fused cell.35%ofcellsshowedextensivemitochondrialfu- red andgreenmitochondriaweredispersedthroughoutthe nantly unfusedmitochondria(Fig.5,BandC)evenwhen Although themutantcellsarecapableofrespiration,we The JournalofCellBiology n

364) ofthefusedcellscontainedpredomi-

Volume 160,Number2,2003 n

200) containedexten- fzo1 yeast, fzo1 n

is compromisedintheabsenceofeitherMfn1orMfn2. respiratory activity,thefunctionofindividualmitochondria cence. Theseresultsindicatethatwhilebulkculturesdisplay their membranepotentialwouldstillcontainEYFPfluores- of EYFP,mitochondriathatsubsequentlyloseorreduce membrane potentialforimport.Becauseofthehighstability tial becausethematrix-targetedEYFPrequiresanintact detected bythisassayinitiallycontainedmembranepoten- (Fig. 6F).Itislikelythatthecompromisedmitochondria dria thatweremarkedbyEYFPbutnotMitoTrackerRed cultures overonethirdofthecellsshowedsomemitochon- ing Mfn1-Myc Mfn1 mutantcultureswereinfected witharetrovirusexpress- that lackedinterconnections(Fig. 7,AandF).However,when cultures ormutant infected withanemptyvirus, on anintactGTPasedomain. InuninfectedMfn1mutant toration ofMfnfunctionand whethersuchrescuedepended Mfn, wetestedwhetherthesedefectscouldberescuedbyres- phological defectsobservedinmutantcellsareduetolossof To unequivocallydemonstratethatthemitochondrialmor- fusion orfission Rescue ofmitochondrialtubulesbymodulation Figure 5. mitochondrial networks consistingoflongtubules (Fig.7,B (D) SectoringeffectinMfn1mutantcell. (C andF)MagnifiedviewsofboxedportionsinBE,respectively. mutant cellsdisplayingpredominantlyunfusedmitochondria. (E) extensive mitochondrialfusion.(BandE)Mfn1(B)Mfn2 mitochondrially targeteddsRedandGFP.(A)Wild-typecellshowing 95% ofthecellsshowedhighly fragmentedmitochondria Mitochondrial fusionassay. 90% oftheinfected cellsshowedextensive

PEG fusionofcellscontaining

on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology such aphenotype (Fig.7F). works, butonly 25%ofMfn2-Myc–infected cellsshowed Mfn1-Myc–infected cellsshowed entirelytubularnet- (Fig. 8F).Incontrast,with Mfn1-deficientcells,91%of works, resultinginrescue 75–80%ofexpressingcells equally effectiveinrestoring extensivemitochondrialnet- Mfn2-deficient cells,bothMfn1-Myc andMfn2-Mycwere reveal differentialactivitiesof thetwohomologues.With mitochondrial fusion.Interestingly,theseexperimentsalso pressed, eachproteiniscapableofactingalonetopromote normal mitochondrialmorphologyinMEFs,whenoverex- that althoughbothMfn1andMfn2arerequiredinvivofor tochondrial tubules(Fig.7,DandF).Theseresultsshow Mfn2 inMfn1-deficientcellswassufficienttorestoremi- Inaddition, overexpressionof phology (Fig.8,DandF). cient cellswassufficientforrescueofmitochondrialmor- single mitofusin.OverexpressionofMfn1inMfn2-defi- rescue themorphologicaldefectsbyoverexpressionof a normal mitochondrialtubulesinMEFs,wewereableto and F).Similarly, drial fusioninaGTPase-dependentmanner. sults showthatbothMfn1andMfn2promotemitochon- structure (Fig.7,CandF,Fig.8,F).Thesere- Mfn2[K109A]) hadgreatlyreducedabilitytorestoretubular rying mutationsintheGTPaseG1motif(Mfn1[K88T]and mitochondria (Fig.8,BandF).Incontrast,constructscar- a Mfn2-expressingretrovirusshowedpredominantlytubular Although bothMfn1andMfn2areclearlyrequiredfor 80% ofMfn2mutantcellsinfectedwith pensated, atleastpartially,by a reductioninfission. tion infusioncausedbylossofMfn1orMfn2canbecom- ential rings.Nevertheless,theseresultsindicatethatareduc- the cellnucleusthatoftengaveappearanceofcircumfer- were generallylessdispersed,withanincreaseindensitynear tached tothickerknobs.Second,themitochondrialnetworks were lessuniform,oftenshowingregionsofthinningat- First, thethicknessoftubulespromotedbyDrp1(K38A) with thenetworksobtainedbyrescueMfn1orMfn2. infected cellsweretubular,theyimperfectcompared respectively. Althoughthemitochondrialnetworksinthese 90% (Fig.7,EandF)50%8,ofthecells, resulted inastrikingrestorationofmitochondrialtubules Mfn2 mutantcellswitharetrovirusexpressingDrp1(K38A) Drp1 (Smirnovaetal.,2001).InfectionofeitherMfn1or mutation (K38A)asinadominant-negativealleleofhuman sion, weconstructedanalleleofmouseDrp1withthesame key roleinmediatingmitochondrialfission.Toinhibitfis- drial fissioncouldalsorestoretubularstructure.Drp1playsa clearly reversible,wetestedwhetherinhibitionofmitochon- to promotemitochondrial fusion.Toexplore thisidea,we chondrial morphology, itispossiblethatthey actinconcert Because bothMfn1andMfn2 areessentialfornormalmito- heterotypic oligomers Mfn1 andMfn2formhomotypic and Because themorphologicaldefectsinmutantcellswere

Essential roleofmitochondrialfusioninmice| appear green. stain poorlywithMitoTrackerRedandthus cells (EandF)asubsetofmitochondria(arrows) In thesemergedimages,notethatinthemutant mitochondria issensitivetomembranepotential. MitoTracker Red,whosesequestrationinto targeted EYFP(green)werestainedwiththedye Mfn2 mutant(F)cellsexpressingmitochondrially potential. Wild-type(D),Mfn1mutant(E),and mitochondria usingdyessensitivetomembrane and Mfn2(C)mutantcells.(D–F)Staining of probe. (BandC)COXIexpressioninMfn1(B) detected bySouthernblotanalysisusinga in mitochondriaofmutantcells. Figure 6. Stochastic lossofmembranepotential

Chenetal.

(A) mtDNAis

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on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology 196 complexes inMfn1-deficient cells. homotypic. Likewise, wealsodetectMfn2-Mfn2 homotypic deficient cells,therebyshowing thatthisinteractionisstrictly Mfn1-Mfn1 homotypiccomplexes areformedinMfn2- cipitation assayinmutantMEFs (Fig.9B).Wefindthat relieve theseconcerns,wealso performedtheimmunopre- sion ofendogenousMfn1and Mfn2intheparentalcells.To detected mightnotbestrictly homotypicduetotheexpres- trol Drp1-Mycisused. tagged proteinisomitted(unpublisheddata)orwhenacon- is foundintheanti-MycimmunoprecipitatewhenMyc- these interactionsarespecificbecausenoHA-taggedprotein Mfn1-HA iscoimmunoprecipitatedwithMfn2-Myc.Allof HA iscoimmunoprecipitatedwithMfn1-Myc;conversely, ond, Mfn1andMfn2formheterotypiccomplexes.Mfn2- Mfn2-HA iscoimmunoprecipitatedwithMfn2-Myc.Sec- is coimmunoprecipitatedwithMfn1-Myc;analogously, Mfn1 andMfn2canformhomotypiccomplexes.Mfn1-HA vealed threetypesofintermolecularinteractions.First,both pressed inwild-typeMEFcells(Fig.9A).Ourresultsre- tested whetherthesetwoproteinsformacomplexwhenex- for eachinfection. morphological classifications.600cellswerescored percentage ofinfectedcellsbelongingtoeachfour results aresummarizedinF,whichdepictsthe most oftheDrp1residesinacytosolicpool.The In E,thesignalsarelargelynonoverlappingbecause immunofluorescence withananti-Mycantibody(green). Red staining,andinfectedcellsareidentifiedby mitochondrial morphologyisrevealedbyMitoTracker negative Drp1(K38A)(E).Inthemergedimages, Mfn1 (B),Mfn1(K88T)(C),Mfn2(D),ordominant- a retrovirusexpressingMycepitope-taggedversionsof mutant cells. Figure 7. It isaformalpossibilitythatthehomotypicinteractions The JournalofCellBiology Rescue ofMfn1-deficientcellsMfn1 Mfn1mutantcells(A)wereinfectedwith

Volume 160,Number2,2003 phoblast giant cells maybeparticularlyvulnerable topertur- of theirhighmetabolic rate,wespeculatethat, invivo,tro- bolically activecells(Edgarand Orr-Weaver,2001).Because endoreplication, aprocessoften associatedwithhighlymeta- Mfn2 isonlyrequiredforselective developmentaltransitions. that despiteitsbroadexpression pattern(unpublisheddata) tent withadefectinmitochondrial fusion.Theseresultsshow tocysts showfragmentationofmitochondrialtubules,consis- cells. TrophoblastcelllinesculturedfromMfn2mutantblas- velopment, mostobviouslyinthepaucityoftrophoblastgiant cient mice,weobserveadramaticdisruptioninplacentalde- ment. Bothlinesofmicedieinmidgestation.ForMfn2-defi- essential roleformitochondrialfusioninvertebratedevelop- Our analysisofMfn1andMfn2mutantmicedemonstratesan logical importanceoftheseprocesseshasremainedunclear. has beenpreviouslydemonstratedinvertebrates,thephysio- Although mitochondrialdynamicsinvolvingfusionandfission in mousedevelopment An essentialroleformitochondrialfusion Discussion Trophoblast giantcellsarepolyploid cellsthatarisefrom

on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology 1999; Sesakiand Jensen,1999;Fekkesetal., 2000;Mozdyet by reducingfusion andfissionsimultaneously (Bleazardetal., networks. Giventhatmitochondrial tubulescanbemaintained and fissionpathwaysincellsthat have“stable”mitochondrial It hasbeenparadoxicalwhyeukaryotes haveinvestedinfusion protecting mitochondrialfunction A modelfortheroleofmitochondrial fusionin iological andexperimentalconditions. of mitochondrialdynamicsinadulttissuesunderbothphys- alleles ofMfn1andMfn2thatshouldfacilitatetheanalysis namics isessential.Thus,wehaveconstructedconditional cesses inwhichthepreciseregulationofmitochondrialdy- wendter etal.,1998;Talosi2000). underlying causemaybeamitochondrialdefect(Widsch- tardation. Geneticandcellularevidencesuggeststhatthe low trophoblastinvasion,oftenresultinginfetalgrowthre- maternal morbidityintheUnitedStates,ismarkedbyshal- tion. Infact,preeclampsia,theleadingcauseoffetaland bations inmitochondrialdynamicsandpresumablyfunc- We anticipatethatthereareotherdevelopmentalpro- vidual, defective mitochondriahavelostmtDNA. mtDNA inmutant cells,wecurrentlydonot know iftheindi- (Nakada etal.,2001a).Although thereisnogrosslossof store localdepletionsandmaintain mitochondrialfunction membrane andmatrixcontents andthereforemayhelptore- intermitochondrial cooperation byallowingexchangeofboth are alwaystransient(Fig.10A). Mitochondrialfusionenables perhaps bylocaldepletionofmetabolic substratesormtDNA, by ensuringthatregionallossesofmembranepotential,caused the dynamicnatureofmitochondriaprotectstheseorganelles of asinglemitochondrialtubule(Loew,1999).Wesuggestthat and transientlossesofmembranepotentialwithinsmallregions to mitochondrialmembranepotentialhasrevealedoccasional potential. Inothercellculturesystems,theuseofdyessensitive nonfunctional mitochondriaasevidencedbylossofmembrane are examinedwefindthatmanycellscontainapercentage of oxygen electrodemeasurements,whenindividualmitochondria show normallevelsofendogenousandcoupledrespirationby question. AlthoughbulkculturesofMfn-deficientfibroblasts maintaining mitochondriainahighlydynamicstate? al., 2000;TieuandNunnari,2000),whatistheadvantageof Our analysisofMfn-deficientcellssuggestsananswertothis

Essential roleofmitochondrialfusioninmice| scored foreachinfection. Fig. 7.TheresultsaresummarizedinF.200cellswere negative Drp1(K38A)(E).Thecellswerestainedasin Mfn2 (B),Mfn2(K109A)(C),Mfn1(D),ordominant- a retrovirusexpressingMycepitope-taggedversionsof mutant cells. Figure 8. Rescue ofMfn2-deficientcellsMfn2 Mfnz mutantcells(A)wereinfectedwith Chenetal. 197

on August 21, 2006 2006 21, August on www.jcb.org from Downloaded The Journal of Cell Biology 198 fission, themitochondrial populationisessentially function- have distinctfunctionsinaddition tofusion. Mfn complexesisinvolvedin fusion,itispossiblethatthey ing mitochondrialtubularshape. Thus,althougheachofthe homotypic complexes,playa moredirectroleinmaintain- Alternatively, itmaybethat Mfn2, andbyextensionMfn2 flect thelossofcytoskeletalinteractions asnotedinFig.4. Mfn1 mutantcells.Thelossoftubularshapemaysimplyre- higher residualfusionactivityinMfn2mutantcellsthan Mfn1. Thelargermitochondrialfragmentsmaybedueto a spheres. Thisphenotypeiseasilydistinguishedfromlossof leads totheformationofbothlargeandsmallmitochondrial drial fusion.ItisalsointerestingtonotethatlossofMfn2 plexes functioninthesameordistincttypesofmitochon- Mfn2-deficient embryosbutnotMfn1-deficientembryos. are mostimportant.Asaresult,thiscelltypeisaffectedin blast giantcells,itappearsthatMfn2homotypicoligomers homotypic complexesarefunctionalforfusion.Intropho- store mitochondrialtubules,clearlydemonstratingthatthe oligomers orMfn2homotypicissufficienttore- mentation. Nevertheless,overexpressionofMfn1homotypic of eitherMfn1orMfn2leadstoseveremitochondrialfrag- plexes arelikelytoplayimportantrolesbecausedisruption (Fig. 10B).Inmousefibroblasts,allthreeoligomericcom- of mitofusinactioncanchangedependingonthecelltype propose thattherelativeimportanceofeachthesemodes typic oligomers,andMfn1-Mfn2heterotypicoligomers.We chondrial fusion–Mfn1homotypicoligomers,Mfn2homo- molecular complexesthatarecapableofpromotingmito- Our resultsshowthatthetwomitofusinsformthreedistinct unclear whytherearetwoseparatemammalianmitofusins. Given theirbroadandoverlappingexpression,ithasbeen Multiple molecularmodesofmitofusinaction mutant cells(indicatedontop)wereusedinananalysissimilartoA. precipitates (top)andtotalcelllysates(bottom)fromMfn1orMfn2 compared withtheimmunoprecipitates.(B)Anti-Mycimmuno- The totalcelllysatesamplescontainonesixthequivalents (bottom) wereanalyzedbyWesternblottingagainsttheHAepitope. on top.Anti-Mycimmunoprecipitates(top)andtotalcelllysates Mfn1 (labeled1),Mfn22),orDrp1D)asindicated cells wereinfectedwithretrovirusesexpressingMyc-orHA-tagged Figure 9. In conclusion, in cellswithcontinualcycles offusionand It remainstobedeterminedwhetherthesethreecom- The JournalofCellBiology Immunoprecipitation ofMfncomplexes.

Volume 160,Number2,2003 (A)Wild-type ments wereamplified byPCR,whichalsointroduceda stopcodonjustbe- Cambridge, MA)as therightarms.Toinsertleft arms, genomicfrag- Gertler andL.Jackson-Grusby,Massachusetts InstituteofTechnology, cloned intothetargetingvectorpPGKneobpAlox2PGKDTA (agiftfromF. SvJ mousegenomiclibrary(Stratagene). Genomicfragmentsweresub- DDBJ underaccessionnos. AY174062 andAY123975. Mfn1 a mousekidneycDNAlibrary,resulting inisolationofcDNAsforboth encodes ahighlyhomologouspolypeptide. ThiscDNAwasusedtoscreen 1997) identifiedamurineEST(IMAGEConsortiumCloneID733269)that Homology searchesusingthe Cloning of thus protectmitochondrialfunction. molecular complexestopromotemitochondrialfusionand lian mitofusins,Mfn1andMfn2,functioninthreedistinct accumulate toaffectthewell-beingofcell.Themamma- the stochasticdifferencesbetweendistinctmitochondriacan lins etal.,2002).Therefore,withoutmitochondrialfusion, vidual mitochondriaarefunctionallydistinctentities(Col- ally homogeneous.However,atanygiventimepointindi- are affectedinMfn2mutantsandnotMfn1mutants. trophoblast giantcellspredominantlyuseactivityIIIbecausethey appear touseallthreeactivities(indicatedbyasterisks).Incontrast, Mfn2 fragmentsmitochondriaandresultsindistinctphenotypes, mutant cellscontainonlyactivityI.SincedisruptionofeitherMfn1or Discussion fordetails.Mfn1mutantcellscontainonlyactivityIII;Mfn2 complexes thatleadtothreeactivities(I,II,III)involvingfusion.See modes ofmitofusinaction.Mitofusinsformhomotypicandheterotypic deficient cells,suchrescueoccursatamuchreducedrate.(B)Three fusion withfunctionalmitochondriaandregainsactivity.InMfn- In wild-typecells,adefectivemitochondrion(shaded)undergoes At alowrate,individualmitochondriastochasticallylosefunction. Figure 10. Materials andmethods Genomic clonesof and Mfn2 Mfn Models. . Thesesequencesareavailablefrom GenBank/EMBL/ cDNAandgenomicconstructs Mfn1 (A) Theprotectiveroleofmitochondrialfusion. and Drosophila Mfn2 wereretrievedfromalambda129/ Fzosequence(HalesandFuller,

MEFs

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The Journal of Cell Biology epitope-tagged cDNAswerereclonedintotheretroviralvectorpCL Myc-HisA vectorcontainingsevenMycepitopetagsattheCOOHterminus. sis. TheDrp1(K38A)insertwassubclonedintoamodifiedpcDNA3.1( placental RNA,andDrp1(K38A)wasconstructedbysite-directedmutagene- ing aCOOH-terminal3 pCL R. Jensen,JohnsHopkinsUniversity,Baltimore,MD)weresubclonedinto GFP anddsRed(fromplasmidspHS1pHS51;giftsfromH.Sesaki used toinfectMEFcultures.Fragmentsencodingmitochondriallytargeted 293T cells.Retroviralstockswereharvested48haftertransfectionand vector pCLEco(agiftfromC.Lois,CaliforniaInstituteofTechnology)into sion vectorswerecotransfectedwiththeecotropicretroviralpackaging gift fromC.Lois,CaliforniaInstituteofTechnology).Theretroviralexpres- Mfn1 directed mutagenesis(Kunkeletal.,1991).TomaketheMfn-HAconstructs, form pMfn-Myc.Mfn1(K88T)andMfn2(K109A)wereconstructedbysite- derived frompMMHb-3 Linkage analysiswasperformedbytheJacksonLaboratoryMappingResource. oratory (C57BL/6JEi laser scanningconfocal microscope(CarlZeissMicroImaging, Inc.) ters anddichroics (q497lp, HQ500lp;Chroma)were used onaZeiss410 with lightmineraloilandimagedin a37 onto chamberedglasscoverslips.Cells withculturemediumwereoverlaid stained cellswerepostfixedin4%PFA. stained with2.5U/mlrhodamine-phalloidin (MolecularProbes).The drial morphology.Tolabelactinfilaments, cellswerefixedin4%PFAand 1986). Neitherretroviralinfectionnor immortalizationaffectedmitochon- from L.Jackson-Grusby,Massachusetts InstituteofTechnology)(Jatetal., were laterinfectedwitharetrovirusexpressingSV40largeTantigen(agift dena, CA)(Okadaetal.,1999).Tofacilitateimmortalization,theMEFs matrix (agiftfromR.Lansford,CaliforniaInstituteofTechnology,Pasa- human cytochromecoxidase,whichdirectsEYFPtothemitochondrial retrovirus expressingEYFPfusedtothepresequencefromsubunitVIII of 150 nMMitoTrackerRedCMXRos(MolecularProbes)orinfectedwith a tamine, penicillin/streptomycin[LifeTechnologies/GIBCOBRL]). MEF media(DME,10%FCS,1 persed byrepeatedpassagethroughaP1000pipettetipandplatedwith MEFs werederivedfrome10.5embryos.Embryosmechanicallydis- MEF andTScelllines the vectorpcDNA3.1( duced immediatelybeforethestopcodon.ThecDNAwassubclonedinto To constructMfn1orMfn2withMycepitopetags,aBamHIsitewasintro- Retroviral andplasmidconstructs scraped offunstainedslides.DNAwasrecoveredandgenotypedbyPCR. detected usingsenseprobes.Forgenotyping,embryonictissuewas RT-PCR usinge11.5placentalRNAastemplate.Nospecificstainingwas ously (Vortkampetal.,1996).Allriboprobetemplateswerederivedfrom for hematoxylin-eosinstainingorinsituhybridizationasdescribedprevi- in atransverseplanewithrespecttotheplacenta.Slideswereprocessed and embeddedintoparaffinblocks.Forstaining,10- maldehyde, dehydratedthroughanethanolseries,treatedwithxylenes, Individual implantationsiteswerefixedovernightat4 In situhybridization (residues 348–579)orMfn2369–598)fusedtoanNH wild-type allelesinasinglereaction. and areverseneomycinprimerwereusedtodetectboththetargeted typing ofoffspring.Aforwardgenomicprimer,areverse bridized withaflankinggenomicprobe.PCRwasusedforroutinegeno- gested withXbaI(Mfn1mutantline)orEcoRI(Mfn2andhy- For Southernblotanalysisoftargetedalleles,genomicDNAsweredi- Confirmation oftargetingevent Excision oftheneomycincassettehadnoeffectonphenotypes. allele wereinjectedintoC57BL/6blastocyststogeneratechimericmice. Southern blotanalysis.Twoindependentlyisolatedclonesforeachtargeted dures (Chesteretal.,1998).Neomycin-resistantcolonieswerescreenedby into lowpassage129/SvEvembryonicstemcellsusingestablishedproce- Each genereplacementvectorwaslinearizedwithSacIIandelectroporated Construction ofknockoutmice otides usedinthisstudyareavailablefromtheauthors. were verifiedbyDNAsequenceanalysis.Thesequencesofalloligonucle- fore theconservedGKSsequenceineachGTPasedomain.Allconstructs Mfn1 residues348–579orMfn2369–598. umn coupledtoamaltose-bindingproteinfusioncontainingeither histidine tag.IgYpurifiedfromchickeneggswasaffinityonacol- To generateretroviralexpressionconstructsforeachoftheabove, For time-lapseconfocalmicroscopy, cellswereplatedatlowdensity For visualizationofmitochondria,theMEFswereeitherstainedwith For Westernblotanalysis,chickenantiserawasgeneratedagainstMfn1 To identifythechromosomallocationsof and W togenerateretroviralexpressionvectors. Mfn2 cDNAsweresubclonedintoapcDNA3.1( SPRET/Ei)F1 )/Myc-His A(Invitrogen).Mycepitopetagcassettes HA tag.Murine Myc weretheninsertedintotheBamHIsiteto nonessentialaminoacids,1mM SPRET/Eibackcrosspanelwasused. Drp1 Mfn1 C chamber.EYFP-optimizedfil- wasamplifiedfrommouse and Mfn2 m sectionswerecut C in4%parafor- , theJacksonLab- ) vectorcontain- 2 -terminal L W (a -glu- )/ 410 laserscanningconfocalmicroscope(CarlZeissMicroImaging,Inc.). were used.CellsimagedwithaPlanNeoFluar63 ary antibodies(JacksonImmunoResearchLaboratoriesandMolecularProbes) Probes) wasused.Fordetection,Cy3-orAlexaFluor488–conjugatedsecond- 9E10 wasused.ForCOX1,mousemonoclonal1D6-E1-A8(Molecular body. ForMycepitope–taggedproteins,themousemonoclonalantibody were blockedwith5%bovinecalfserumandincubatedprimaryanti- nM) andSyto16(100nM;MolecularProbes). (Tanaka etal.,1998a).LivecellswerestainedwithMitoTrackerRed(150 min beforefixationandthenpermeabilizedwithacetoneat some experiments,cellswereincubatedwith150nMMitoTrackerRedfor30 ples wereimmunoblottedwithHA.11(Covance). 9E10 antibodycoupledtoproteinA–Sepharosebeads.Afterwashing,sam- X-100, andaproteaseinhibitorcocktail)immunoprecipitatedwith resuspended inlysisbuffer(150mMNaCl,50Tris,pH8.0,1%Triton ing Myc-andHA-taggedMfn1,Mfn2,Drp1(K38A).Monolayerswere Cell lineswereinfectedwithvariouscombinationsofretrovirusesexpress- Immunoprecipitation probing witharadio-labeledPCRfragmentcontainingthe Southern analysisofmtDNAwasperformedbylinearizingwithXhoIand Analysis ofmtDNA (37 Cells grownonpolylysine-coatedcoverslipswerefixedwithprewarmed Immunofluorescence dium containing30 1500 (Roche).Thecellswerewashedandgrownfor7hor24inme- targeted dsRed.Thenextmorning,cellswerefusedfor60swith50%PEG night on25-mmcoverslipswith40,000cellsexpressingmitochondrially 40,000 cellsexpressingmitochondriallytargetedGFPwereculturedover- PEG fusion Bakeeva, L.E.,S.ChentsovYu,andV.P.Skulachev.1978.Mitochondrialframe- References Accepted: 12December2002 Revised: 12December2002 Submitted: 12November2002 tutes ofHealth(grant1RO1 GM62967-01). in BiomedicalSciences.ThisresearchwassupportedbytheNationalInsti- and recipientofaBurroughsWellcomeFundCareerDevelopmentaward Chan isaBrenscholar,RitaAllenBeckmanYounginvestigator, ology fundedbytheBurroughsWellcomeFundInterfacesprogram.D.C. A.J. EwaldisaparticipantintheInitiativeComputationalMolecularBi- grant NIHGM07616andE.E.GriffinisfundedbyaFergusonfellowship. and E.E.GriffinaresupportedbyaNationalInstitutesofHealthtraining and A.LombesforstimulatingdiscussionsaboutthePEGfusionassay. tance andA.Harringtonforblastocystinjections.WethankDrs.M.Rojo We aregratefultoM.Michelmanforembryonicstemcellcultureassis- We thankDr.PhilipLederforhissupportintheearlystagesofthiswork. croscopic fieldis96 eos 1and3are20-minrecordings;video2isa10-minrecording.Themi- eos 1–3showmitochondrialdynamicsinwild-typeandmutantMEFs.Vid- available athttp://www.jcb.org/cgi/content/full/jcb.200211046/DC1.Vid- (video 1),Mfn1mutant2),andMfn23)MEFsare Confocal time-lapsevideosofEYFP-labeledmitochondriainwild-type Online supplementalmaterial Bakeeva, L.E.,S.ChentsovYu,andV.P. Skulachev.1983.Intermitochondrialcon- Bakeeva, L.E.,Y.S.Chentsov,andV.P.Skulachev.1981.Ontogenesisofmito- and 75 Bleazard, W.,J.M.McCaffery,E.J.King, S.Bale,A.Mozdy,Q.Tieu,J.Nunnari, Bereiter-Hahn, J.,andM.Voth.1994. Dynamics ofmitochondriainlivingcells: TS cellsfrome3.5blastocystswerederivedusingestablishedprotocols H. ChenissupportedbyanAlcottPostdoctoralfellowship.S.A.Detmer C) 3.7%formaldehydeandpermeabilizedinPBS/0.1%TritonX-100.In Acta. work (reticulummitochondriale)inratdiaphragmmuscle. tacts inmyocardiocytes. chondrial reticuluminratdiaphragmmuscle. chondrial fissioninyeast. and J.M.Shaw.1999. Thedynamin-relatedGTPaseDnm1 regulatesmito- Res. Tech. shape changes,dislocations,fusion,and fissionofmitochondria.

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