The Journal of Cell Biology
JCB
Introduction and Lawrence S.B.Goldstein disruption ofneuronal kinesin heavy chain KIF5A Abnormal neurofilament transport causedby targeted powering slowaxonaltransporthavebeencontroversialand axonal transport,theexistenceandidentityofmotorproteins its relativesarethemotorproteinsdrivingfastanterograde transport system. proteins,aremovedbytheslowaxonal rofilament (NF)* axonal transportsystem.Cytoskeletalproteins,suchasneu- mitochondria, andothervesicles,aretransportedbythefast Membranous organelles,suchassynapticvesicleprecursors, transported tothenerveterminalsviaaxonaltransport. axonal proteinsmustbesynthesizedinthecellbodiesand Because theaxonhaslittleornoproteinsynthesismachinery, or moreinlengthtoreachpostsynaptictargetstheperiphery. such asspinalmotorandsensoryneurons,extendaxonsof1m polarity andsizeofthesecells.Inhumans,someneurons, Axonal transportinneuronsisessentialbecauseoftheextreme T 1 the remaininganimalssurvived to3moorlonger. Inyoung mice exhibitedseizuresanddeathataround3wkofage; of KIF5Ainneuronspostnatally. Three fourths ofsuch mutant Cre-recombinase transgene was usedtodirectinactivation mutants dieimmediatelyafterbirth,asynapsin-promoted ventional kinesinheavy chain, KIF5A.BecausenullKIF5A nation togenerate micelacking theneuronal-specific con- Chun-Hong Xia, neuron neurofilament; axonalcaliber;DRG sensory neuronalkinesinheavychainKIF5A; Key words: slow axonaltransport; chain; KLC,kinesinlightNF, neurofilament. root ganglion;E,embryonicday;ES, embryonicstem;KHC,kinesin *Abbreviations usedinthispaper:c-section, caesariansection;DRG,dorsal 9701. E-mail:[email protected] Drive, LaJolla,CA92093-0683.Tel.:(858)534-9702.Fax:534- West Room336,UniversityofCalifornia,SanDiego,9500Gilman Address correspondencetoDr.LawrenceS.B.Goldstein,HHMI/CMM- http://www.jcb.org/cgi/doi/10.1083/jcb.200301026 The Journal ofCellBiology
Department ofCellularandMolecularDepartment Medicine, Howard Hughes Medical Institute, La Jolla, CA92093 3
TheRockefeller University Press, 0021-9525 Ludwig Institute forCancerResearch,
Article Although numerousstudiesdemonstratethatkinesinand
neurofilaments (NFs),weusedhomologousrecombi- motor proteinspower theslow axonaltransport of o testthehypothesis thatfastanterograde molecular
1 Elizabeth A.Roberts, , Volume 161, Number 1,April 14,200355–66 1 /2003/04/55/12 $8.00 4 Department ofMedicine,Department and 1 Lu-Shiun Her, heavy 1 XinranLiu, (110–130 kD)andtwokinesin lightchain(KLC)subunits composed oftwokinesinheavy chain(KHC)subunits 1985; Valeetal.,1985).Nativekinesin-Iisaheterotetramer first identifiedmemberofthekinesinsuperfamily(Brady, intermediate filamentproteinsataslownetrate. such askinesin-Icouldbeinvolvedinthetransportofthese pauses. Thus,itispossiblethatafastaxonaltransportmotor driven byafastmotorproteininterruptedprolonged slow axonaltransportmaybetheresultofrapidmovements the possibility,stillnotrigorouslytested,thatslowrateof (Wang etal.,2000).Togethertheseobservationshaveraised and highlyasynchronousmannerinculturednervecells tagged NF-Mhasbeenshowntomoveinarapid,intermittent, in nonneuralcells(Prahladetal.,1998).Recently,GFP- also reportedtoassociatewithvimentin-containingstructures kD), andNF-H(115kD)(Yabeetal.,1999).Kinesin-Iwas in theslowaxonaltransportofNF-L(61kD),NF-M(90 possible involvementofconventionalkinesin(calledkinesin-I) Yabe etal.,1999).Otherinvitroexperimentssuggestedthe energy move anterogradelyinaxonsthroughamicrotubule-and mysterious. SomeexperimentssuggestthatNFsubunits transport ofatleastonecargo,theNFproteins. plays aroleinthemicrotubule-dependentslow axonal These datasupportthehypothesis thataconventional kinesin in axons,lossoflargecaliberandhindlimbparalysis. accumulation ofNFsubunitsincellbodiesandareduction developed a reductioninsensoryaxoncaliber. Olderanimalsalso cell bodiesofperipheral sensoryneuronsaccompaniedby but NF-H,aswellNF-MandNF-L,accumulatedinthe mutant animals,fastaxonaltransport appearedtobeintact, KLC subunitsareprobablyinvolved incargobindingormod- that interactswiththemicrotubule trackandhydrolyzesATP. (60–70 kD) Kinesin-I wasfirstfoundinsquidaxoplasmandthe 5 Neuroscience, University ofCalifornia,San Diego, -dependent mechanism(KoehnleandBrown,1999; 2 age-dependent sensoryneurondegeneration, an David S. Williams, (Bloom etal.,1988).KHChas amotordomain 2 Department ofPharmacology,Department 2 Don W. Cleveland, 3,4,5 55
The Journal of Cell Biology 56 Results elegans were detectedintypeII(KIF5A bination. Thestrategy (Fig.1A)wastocreate anullmutant We madeaKIF5A deletionmutantbyhomologous recom- KIF5A nullmutantswerelethal species, including Although onlyoneconventionalKHCgeneisfoundinmany and Philp,1999;Rahmanetal.,Kamal2000). ulation ofKHCactivity(BloomandEndow,1995;Goldstein used toprobeKIF5A,KIF5B,andKIF5C; brain homogenatesweremadefromtwolittersofmice,and100 null andconditionalKIF5Amutants inmice. axonal transportofNFs,wehave nowgeneratedandanalyzed function ofKIF5A,andtotesttheroleKIF5Ainslow isoform thatmightbeinvolvedisunclear.Toinvestigatethe and Brown,1999;Yabeetal.,1999),theidentityofKHC to playaroleintheslowaxonaltransportofNFs(Koehnle al., 2002).Althoughconventionalkinesinhasbeensuggested of hereditaryspasticparaplegiainhumans(SPG10;Reid et 2000), andKIF5Amutationshavebeenfoundtocauseaform be importantfortheviabilityofmotorneurons(Kanaietal., KIF5C areunknown,althoughhasbeensuggestedto al., 1997;Xiaet1998).ThefunctionsofKIF5Aand ronal tissues(Navoneetal.,1992;Niclas1994;Meng et both KIF5AandKIF5Cappeartobeexpressedonlyinneu- KIF5C). KIF5Bappearstobeubiquitouslyexpressed,whereas selection. (BandC)SouthernblotanalysesofHindIII-digestedG418-resistantESclonesaftertransfectingtargetingvector.( second stepwasperformedbytransfectingaCreplasmidintorecombinantEScellsisolatedfromthefirststep,followedGan type IIdeletionEScells.ThefirststepwasdonebytransfectinglinearizedtargetingvectorintocellsfollowedG418s Cre transfection.WiththeloxPprobe,onlya6-kbbandwasdetectedintypeIdeletion(KIF5A 3), whereasclones1and4onlyhadthefirsttwoloxPsites.(D)SouthernblotanalysisofHindIII-digestedGancyclovir-resist The correctrecombinantclonesshouldhaveallthreeloxPsites,both8.2-kband2.5-kbbandsbedetectedbytheprobe( 3.5-kb selectioncassette)wasalsodetectedintherecombinantclones.(C)ThepresenceofthreeloxPsitesconfirmedbya probe wasusedtoidentifytherecombinantclones.A4.7-kbwild-typebanddetected,andanadditional8.2kb(withadd The JournalofCellBiology , mammalshavethreeKHCgenes(KIF5A,KIF5B,and
Drosophila melanogaster
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Volume 161,Number1,2003 flox ) EScells.(E)NoKIF5AproteinwasdetectedinnullmutantmicebyWesternblotanalysis.Mouse -tubulin wasusedasaloadingcontrol. and Caenorhabditis g ofproteinwasloadedineachlane.Isoform-specificantibodieswere extracts ofwild-type, heterozygous,andhomozygous KIF5A No visiblestructuraldefectswere observedinanyorgans. able fromtheircontrollittermates bysizeandappearance. The KIF5Ahomozygousmutant pupswereindistinguish- heterozygous (153/309),and homozygous mutant(73/309). Mendelian ratioof1:2:1was observed:wildtype(83/309), tained byc-sectionwerePCR genotyped.Thepredicted C57BL background,atotalof45littersE18.5pupsob- blue andusuallydiedwithin10min.Inamixed129/ removal fromtheuterus).Mutantpupsgraduallyturned not (althoughlikecontrollittermates,theygaspedjustafter veloped anormalbreathingpattern,themutantpupsdid were aliveatE18.5.Whereascontrollittermatesquicklyde- served bynaturalbirth,allnullpupsrecoveredc-section genotyped. Althoughlivenullmutantpupswereneverob- andtheembryonicday18.5(E18.5)pupswere (c-section), the pregnantfemalemicewerekilledbycaesariansection KIF5A nullmutants,heterozygousmiceweremated, probably neonatallethal.Todirectlyobservethebehaviorof mozygous pupswerefound.Thus,KIF5Anullmutantsare KIF5A homozygousmutantssurvived,onlythreedeadho- ters wereanalyzed.Initialgenotypingrevealedthatno tation weremated,and33newbornoffspringfromfourlit- encoded protein.MiceheterozygousfortheKIF5Anullmu- by deletingtwocriticalexons,causingaframeshiftofthe Immunoblots with aKIF5Apolyclonalantibody ofbrain Figure 1. were performedtogeneratetypeIdeletion(nullmutant)and were flankedbyloxP2andloxP3.Twostepsoftransfection loxP sites(loxP1andloxP2),thetwoexonstobedeleted pGK-neo andHSV-tkselectioncassettewasflankedbytwo (A) KIF5Agenetargetingstrategy.Inthevector, Targeted disruptionofthemouseKIF5Agene. null ) EScells,whereas4.7-kband2.5-kbbands election; the B) A5 ant clonesafter loxPprobe. clones 2and cyclovir external ition ofa
The Journal of Cell Biology (KIF5A Figure 2. mates. Electrophysiologyofthesecellsdidnotrevealany difference whencomparedwithcellsfromcontrollitter- from KIF5Anullmutantsdidnotshowanymorphological mates (Fig.2,BandC).Hippocampalneuronscultured appeared tobebiggerthanmotorneuronsofcontrollitter- ies ofspinalcordmotorneuronstheKIF5Amutant tant brain(Fig.2,D–F).Interestingly,nucleiandcellbod- vious pathologicalchangeswereobservedintheKIF5Amu- analysis ofthebrainandspinalcordwasperformed.Noob- mality wasfoundinthemutant(unpublisheddata). muscle structuredevelopednormally.Noconsistentabnor- examine whethertheneuromuscularjunctionregionand was studiedbyimmunostainingandelectronmicroscopyto underlying mechanismisunclear.Thediaphragmmuscle lungs maybethecauseofdeath,althoughnature those ofcontrollittermates(Fig.2A).Thus,unexpanded toxylin-eosin. KIF5Amutantlungsdidnotexpandaswell control littermatesweresectionedandstainedwithhema- pattern ascontrollittermatesdid,lungsfrommutantand died soonafterbirthanddidnotdevelopanormalbreathing amount ofeitherintheKIF5Amutant. and KIF5Cantibodiesshowednosignificantchangeinthe duced KIF5Aasexpected.ReprobingtheblotwithKIF5B the homozygousmice(Fig.1E).Heterozygoushadre- mutant littermatesrevealedthatKIF5Awastotallyabsentin (C) 20 mutant andcontrol littermatesexceptthatthecell bodies ofthemotorneuronswere largerinthemutantspinalcord.Bars: (B hippocampus (E), andcerebellum(F)werestained withcresylviolet.Notethatno obvious differenceswereobserved betweenKIF well expanded.Bar,50 Because KIF5Aisonlyfoundinneurons,histological Because KIF5Anullmutantpupsdeliveredbyc-section null m; (E)100 /KIF5A Histology ofKIF5Anullmutantmice. null ) andcontrol(KIF5A m. m. (B–F)HistologyofKIF5Anull mutant nervoustissues.Paraffinsectionsfromspinalcord(BandC), cortex(D),
WT
/KIF5A
WT
(A)LunghistologyofKIF5Anull mutant. 7- ) littermateswerestainedwithhematoxylin andeosin.Notethatthemutantlungwasnot 1993). BymatingKIF5A course withamaximumaroundday20(Hoescheetal., gene whoseexpressionfollowsabiphasicpostnataltime known tobeaneuron-specific,developmentallyregulated dorsal rootganglion(DRG)andspinalcordfrom To observedirectlyKIF5Aexcisionatthecellularlevel, reflect variableefficiencyofCre-mediatedgeneexcision. The heterogeneityinthelevelofreductionKIF5Amay ranged from6to56%ofthatcontrolbrains(Fig.3A). animals showedthatKIF5Aproteinlevelsinmutantbrains spring wereidentified. KIF5A KIF5A-specific antibody(Fig.3,BandC).Thisstaining old mutantswereexaminedbyimmunostainingwitha moter (Cre Cre recombinaseunderthecontrolofsynapsinIpro- tant, whichwascombinedwithatransgeneencodingthe transport ofNFs,weconstructedaconditionalKIF5Amu- and totestwhetherKIF5Amighthavearoleinslowaxonal To understandbetterthelethalitycausedbylossofKIF5A, and sensoryneurondegeneration Cre-mediated postnatallossofKIF5Acausesseizures KIF5A (unpublisheddata). defect insynaptictransmissioncausedbytheabsenceof Quantitative immunoblotanalysisofasmallsetinitial Neurofilament transportdefectinakinesinmutant| WT ; Cre synapsin synapsin ) (Zhuetal.,2001).Thistransgeneis m lungparaffinsectionsfromKIF5A null mice,KIF5A flox /KIF5A null /KIF5A flox micetoKIF5A flox , D,andF)50 ; Cre Xiaetal. synapsin 5A null 3-wk- off- null m; 57 / The Journal of Cell Biology 58 among 3-wk-oldlittermateswithdifferentgenotypes.NotetheobviouslowbodyweightinKIF5A Note thedecreasedorlackofKIF5Astaininginsomeneurons(arrowheads).Bar,100 with KIF5A-specificantibody.Spinalcordsectionswerealsodoublestainedananti-BIPantibodytovisualizethemotorne mutant bandandcontrolafternormalizingwiththeactinband.(BC)KIF5AexcisionbyCre n (F) AbnormalhindlimbpostureinanolderKIF5A a groupofmice(142total,113controland29mutant)isshownhere. Therateofsurvivalthedifferentgenotypeswasplott KIF5A (KIF5A cord motorneurons(C)ofKIF5A nal cordmotorneurons. excision occurredinmanyDRG sensoryneuronsandspi- had almostnoKIF5Astaining.Weconcludethat ies wasmuchweakercomparedwiththecontrol;somecells that KIF5Astaininginthemutantmotorneuroncellbod- BIP antibody(anendoplasmicreticulummarker)indicated the mutant.Inspinalcord,costainingwithamonoclonal revealed anabsenceofKIF5Ainsomesensoryneurons Figure 3. KIF5A the weightofmutant micewasonly mice wereobviously smallerthantheirlittermates. At3wk, protein levelsinthebrainsofKIF5A (KIF5A thecriticalneonatalstageasdidcontrol littermates vived