The Journal of Cell Biology

JCB

Introduction and Lawrence S.B.Goldstein disruption ofneuronal heavy chain KIF5A Abnormal neurofilament transport causedby targeted powering slowaxonaltransporthavebeencontroversialand axonal transport,theexistenceandidentityofmotorproteins its relativesarethemotorproteinsdrivingfastanterograde transport system. ,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, . 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 - 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 .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., 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

As predicted,KIF5A The JournalofCellBiology 4foreachgroup.*,P flox flox WT ) areshown. null /KIF5A ; Cre Gross analysisofKIF5Aconditionalmutantmice(KIF5A /KIF5A WT synapsin ) littermateswereloaded;actinwasusedasaloadingcontrol.Eachmarkednumberrepresentstheratiobetween flox ). By2–3wkofage,however, mutant , KIF5A

null | 0.01.(E)MostKIF5A

Volume 161,Number1,2003 /KIF5A null flox /KIF5A /KIF5A null /KIF5A flox flox ; Cre ; Cre WT 50% ofthecontrol flox , andKIF5A ; Cre synapsin synapsin null null /KIF5A /KIF5A synapsin mutant.Tissuesectionsfrom micesur-

mice.Equalamountsofbrainhomogenatefrom3-wk-oldmutantandcontrol flox flox

; Cre ; Cre

flox

synapsin

synapsin

/

null

mutantmouse.Two7.5-mo-oldlittermates(controlandKIF5A /KIF5A mutantmicediedaround3wkofage.Postnatalsurvivalcurve

Cre were observed as wellseizuresapparently inducedwhen these miceappeared todieofseizures.Spontaneous seizures (72%) ofthemutantmicedied betweenday15and25; ground), andsurvivalcurves were plotted(Fig.3E).21/29 113 littermatecontrolmice in amixed129/C57BLback- fects orsensorydefects.At3wkofage,KIF5A cult toassesswhethertheseproblemsresultfrommotorde- type miceonarotoroddevice(unpublisheddata).Itisdiffi- sensory test),buttheyfellmuchmorefrequentlythanwild- weight (Fig.3D).Thesemicerespondedtopinch(acrude lowed (29KIF5A although theyfrequentlyexhibited atremor.

In aninitialcohort,142mice from16litterswerefol-

synapsin

flox

; Cre mutantmiceexhibitedarelativelynormalposture, 3-wk-old mutantandcontrollittermateswerestained synapsin m. (D)Acomparisonofbodyweight(mean ). (A)WesternblotanalysistomeasureKIF5A null /KIF5A null synapsin /KIF5A flox transgeneinDRG(B)andspinal ; Cre flox ; Cre synapsin synapsin mutantmiceand mutantgroup, uron cellbodies. ed againstage. null /KIF5A SD) null / flox ; The Journal of Cell Biology showed almostnormalpostureuntil and 8mo).Allmutantmicethatsurvivedto5moofage (the remainingmicewerekilledforexperimentsat5.5,7, mice, onediedat3mo,4andanother5.5mo termates (KIF5A eration). Thisphenotypewas not observedinthecontrollit- merous profilesofmyelindebris (typicalofWalleriandegen- showed strikingdegeneration ofaxons(Fig.4B),withnu- four oldermutantmice(two 5.5- andtwo7.5-mo-oldmice) axons ofthedorsalrootsfrom 3-wk-oldanimals(Fig.4B), tant mice.Whereasnodegenerationwasdetectedinsensory obvious abnormalityin3-wk-,5.5-mo-,or7.5-mo-oldmu- thin sectionsofventralroots(Fig.4A)didnotdetectany 3 wkor7.5moofage(unpublisheddata).Examination animals werestressed.Eachseizureepisodelasted that diedat3wkofage,theremaining recover fromsevereseizures.Distinctthe72%ofmice repetitive seizurewasobserved,andthemiceusuallydidnot hind limbposture afterlossofKIF5A. data areconsistent withanage-dependentonset ofabnormal observed inKIF5A vious morphologicalabnormalityinspinalcordorDRGwas normal hindlimbpostureduringresting. were notcompletelyparalyzed,andtheanimalsadoptedab- limbs behavedabnormallyduringwalking,althoughthey after thattheydevelopedatremorwhenwalking.Thehind 3 mo.Thispoolcontainseightanimals;amongthese In spiteoftheobviousneuromuscularphenotype,noob- flox /KIF5A null /KIF5A WT orKIF5A flox ; Cre synapsin 5 mo(Fig.3F),but null 28% ofmicelived mutantsateither /KIF5A flox 20–30 s; ). These sory axonswerelost,whereasnosignificantloss(P motor axonsandaprofoundloss( 60% (P flected byapreferentiallossoflargecalibersensoryaxons. surement ofaxonaldiameteralsorevealedsensorylossre- ( Cre mutantanimals( were significantlyreducedinthe3-wk-oldKIF5A–synapsin ever, sensoryaxonnumberswithindorsalroots(Fig.5A) fect onthesurvivalofmotorneuronsat3wkage.How- 0.112), indicatingthatlossofKIF5Ahadnosignificantef- mates (Fig.5A)revealednosignificantaxonloss(P of 3-wk-oldKIF5A-depletedmutantsandcontrollitter- the numberofL5lumbarmotoraxonswithinventralroots the lumbarsciaticnerverootswerecounted.Comparisonof fected thesurvivalofmotorand/orsensoryaxons,axons To determinequantitativelywhetherthelossofKIF5Aaf- caused bypostnatallossofKIF5A Loss oflargecaliberaxonsandNFtransportdefects large caliberaxons. Inthedorsalroots(Fig. 5D),profound most substantialforaxons Again, sensoryandmotor(Fig. 5,CandD)axonlosswas was observedforsmallcaliberaxons. mutant group, andtherewasanobviousloss of the largeaxonswereclearly shifted tosmallersizesinthe wild-type andKIF5A-depleted neurons,butthecalibersof showed atypicalbimodaldistribution ofaxonalsizesinboth n

By 5.5moofage,weobservedasmallloss( twocontrolandmutant animals)(Fig.5B). Neurofilament transportdefectinakinesinmutant| 0.002)oflargecaliber( decrease oflargecaliberaxons.Bar,20 degenerating axons(arrowheads)andastriking 5.5-mo-old mutant,therearelargenumbersof degeneration in3-wksensoryaxons,butthe 5.5-mo-old mutantmice.Notethelackof Bar, 50 mutant mice.Notethelackofdegeneration. (A) Ventralrootsof3-wk-oldand5.5-mo-old KIF5A Figure 4. null /KIF5A 86% ofcontrol;P m. (B)Dorsalrootsof3-wk-oldand Sensory axondegenerationin flox 4 ; Cre 3 m. Motoraxonsstill synapsin 36%) ofsensoryaxons

m indiameter)sen-

mutantmice.

0.045).Mea-

Xiaetal.

12%) of

7.5-

m.

0.26)

m 59

The Journal of Cell Biology 60 *, P difference isobservedbetweenthenumberofmutantandcontrolaxonsinventralroot.Thereasignificantlossdorsa hood ( ginning atweeks2–3andcontinuingthroughyoungadult- Zhu etal.,1997,1998).Concomitantwithmyelinationbe- ons isamajordeterminantofaxonalcaliber(forreviewssee axon losswasobservedinsensoryaxonswithcalibers Figure 5. axons shiftsfrom7–7.5 roots oftwomutantandcontrolmiceisshown.Notethatintheventralroot(C),peakdistributiondiame L5 ventralroot(C)anddorsal(D)from5.5-mo-oldmutantcontrolmice.Averageddistributionofaxondiametersfro especially largecaliberaxons.Thelossofventralrootaxonsisnotasprofoundobservedinthedorsalroot.(CandD)Axo and dorsalrootsfrom5.5-mo-oldcontrol(12,control)mutant(34,mutant)mice.Thereisaprofoundlossofdors large ( NF subunitsaccumulated inKIF5A-depleted DRGs,while As expectedifKIF5A wasneededforefficient NFtransport, (comprised primarilyofthecell bodiesofsensoryneurons). sory axons,weusedimmunoblotting ofextractsDRGs reduced NFtransportinto,and accumulationwithin,sen- motor forNFtransportintoaxons. most NF-richaxons,suggestedthatKIF5Amightbeone caliber byKIF5A-deficientaxons,especiallyofthelargest, (e.g., seeFig.4,AandB).Lossofacquisitionthecorrect m), butnolossofsmallcalibersensoryaxons( m, withcompletelossofthelargestsensoryaxons( Many earliereffortsdemonstratedthatNFcontentinax- To testwhetherdiminishedaxonal caliberwascausedby The JournalofCellBiology 0.05(0.045);**,P 3 5 mo),thereisarapidgrowthinaxonalcalibers Loss oflargecaliberaxonsintheKIF5A m diameter)myelinatedaxonsinL5ventralanddorsalrootsfrom3-wk-oldcontrol( m incontrolsto5.5–6 0.01(0.002);

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Volume 161,Number1,2003 t test.(B)Axonnumbersoftotal,small( m inmutants.Alsonotetheobviouslossoflargecaliberaxonsdorsalroot(D). null /KIF5A 1.5 flox m). ; Cre 7 2 synapsin ( pendent ofitsphosphorylationstate,revealedasignificant COOH terminusofNF-H,whichrecognizesNF-Hinde- shown inFig.6A).Apolyclonalantibodyagainstthe (three litterswereexamined;onerepresentativelitter is KIF5A levelswerereducedto20%ofthatinthecontrols DRGs. Noobvious changesintheamounts ofNF-H, NF-M,NF-L,andperipherin wereselectiveforthe NF-H, ron-specific was alsoincreasedinthemutant DRGs,whereastheneu- crease. ThetypeIIIintermediate filamentproteinperipherin phorylated NF-H(SMI-31)revealed aslight(10–15%)de- of NF-H,whereasamonoclonal antibodyspecificforphos- SMI-32, whichisspecificforthedephosphorylatedisoform in NF-Hwasconfirmedwiththemonoclonalantibody increased buttoalowerextent(by20–50%).Theincrease equivalent amount( The amountofNF-LwasincreasedinmutantDRGsbyan mutantmice. 150%) increaseintotalNF-HproteinmutantDRGs. 4 m), andlarge( (A)Countsoftotal,small( III-tubulin isoformwasnot. Increasesin n

4)andmutant( 150%), whileNF-Mamountswere 4 m) myelinatedaxonsinL5ventral n

3 6)mice.Nosignificant m diameter),and ter oflargecaliber nal diametersof l rootaxons. al rootaxons, m theentire The Journal of Cell Biology KIF5A by antibodySMI-32)aswellthe increaseinNF-M,NF-L,andperipherin.(B)Westernblotofbrain andsciaticnervefrom3-w in dephosphorylatedNF-H(revealed bythelowerbandlabeledNF-H,apolyclonalantibodyagainst theCOOHterminusofNF-H, pooled fromoneII/ sciatic nerve.(CandD)Immunostaining ofDRGsensoryneurons7.5-mo-oldKIF5A clear reductionofKIF5Aprotein inthemutant.ThelevelsofNFsandperipherinwerenotsignificantly changedinthemutantb Figure 6. lations oftheseproteinsintheDRGs. and8-mo-oldmutants,accompaniedbyaccumu- 6-wk-old and peripherinwereobservedinthesciaticnervesof neuron degeneration.SignificantreductionsofNF-M,NF-L, nerve andintheDRGwereseen,perhapsbecauseofsensory animal, obviousreductionsinthecontentofNFs ever, hadverypoorKIF5Aexcision.Inthe7-mo-oldmutant teins wasobservedinthesciaticnerve.Thisanimal,how- tant animal,noobviousreductionintheamountofNFpro- protein levelwasincreasedintheDRGof3-mo-oldmu- among theseunusualsurvivinganimals.AlthoughtheNF bly owingtovariationinCre-mediatedKIF5Aexcision mo). Avariablephenotypewasseenintheseanimals,possi- by Cre-inducedrecombination(6wk,3mo,7and8 the rareoldersurvivingmiceinwhichKIF5Awasreduced atic nerve(Fig.6B). NF-L,orperipherinwerefoundinthebrainandsci- NF-M, intense SMI-32staining insomeDRGneurons.Bars, 200 DRG staining.Bottom panel,highermagnification. (D)MutantDRGstaining,higher magnificationinthelowerpanel. Notetheap with KIF5A(green)andSMI-32(recognizes dephosphorylatedNF-H,red)wasperformedonamutant andacontrollittermate.(C)C analyses ofDRGsfromthefirstcohort3-wk-oldKIF5A We alsoexaminedNFlevelsinsensoryneuronsoffour null /KIF5A Accumulation ofNFproteinsinDRGsensoryneuroncellbodies of KIF5A flox ; Cre andoneCre/ synapsin mutantmice.20 littermates;mutantDRGs(M)were pooledfromtwomutantlittermates.Notetheobviousincrease g ofproteinsmutantandcontrol (II/ III-tubulin was null m. /KIF5A flox ; Cre (APP, Rab3,synaptophysin,andKIF5C),usingacombi- the integrityofseveralfastanterogradetransportpathways from fivedifferentlitters.Weusedtheseanimalstoassess mals and123-wk-oldmatchedlittermatecontrolanimals cohort of123-wk-oldKIF5A defect inslowaxonaltransportofNFs,webredasecond controls (Fig.6,CandD). elevated staininginsectionsofDRGfrommutantsbutnot the amountwithinsensoryneuroncellbodies,asjudgedby blotting oftheDRGwasconfirmedtoreflectanincreasein the increasedaccumulationofNF-Hobservedbyimmuno- tant DRGsandsciaticnerves(unpublisheddata).Finally, Rab5) wereunalteredatboth3and6wkofageinthemu- the levelsofotheraxonalproteins(KIF3A,KIF3B,and the DRGs.Thesechangesappearedtobeselectivebecause accompanied byaslightaccumulationoftheseproteinsin modestly reducedinsciaticnervesamplesoftheseanimals, synapsin To testfurtherwhetherKIF5Amutantshaveaspecific Neurofilament transportdefectinakinesinmutant| mutantmice.Inthelittershown,controlDRGs(C)were null null /KIF5A ) littermateswasloadedineach lane. Notethe /KIF5A flox flox ; Cre ; Cre synapsin synapsin mutantmice. null mutantmice.Doublestaining /KIF5A flox (A)Westernblot ; Cre Xiaetal. synapsin rain and k-old parent ontrol and ani- 61 The Journal of Cell Biology 62 binding partner isreduced. thus theamount ofKIF5Cmaybedecreased whenits form heterodimerswithKIF5A (Kanaietal.,2000),and the reducedfunctionofKIF5A. Alternatively,KIF5Cmay able fortransport,moreKIF5C isusedtocompensatefor with thoseofKIF5A,suchthat whenlessKIF5Aisavail- that KIF5Cmaycarryout functions thatareredundant KIF5C reductionsintheDRG, oneobviouspossibilityis further workwillbenecessarytounderstandtheoriginof were comparableinthemutantandcontrol.Although abundance inthesciaticnerveanditstransportbehavior slightly decreasedinthemutantDRGs,althoughits were observed.Interestingly,KIF5Cproteinamountwas the proteinlevelsofAPP,Rab3,andsynaptophysin in mulation intheDRGs,whereasnosignificantchanges KIF5A-deficient miceexhibitedstrikingNFproteinaccu- from thatoftheircontrollittermates.10out12 Rab3, synaptophysin,andKIF5Cwasindistinguishable (Fig. 7),whereastheaxonaltransportprofileofAPP, levels inallofthemutantDRGscomparedwithcontrols fore, weobservedsignificantlyreducedKIF5Aprotein full antibodyset,yieldingareduced antibodies (someofthesefilterscouldnotbeusedforthe lanes ongelsandprobethesemultipletimeswithdifferent ligations ofindividualanimalswasobtainedtoloadsingle ple amountsavailable,sufficientproteinfromsciaticnerve forming nerveligationsonthemseverelylimitedthesam- of theKIF5Aanimalsandtechnicaldifficultyinper- (Kamal etal.,2000).Althoughthesmallsizeandfragility bodies inDRGsandsciaticnerveligationexperiments nation ofproteinaccumulationinsensoryneuroncell The JournalofCellBiology

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Volume 161,Number1,2003 n ; Fig.7C).Asbe- inconsistent sonotallprobescouldbeusedonanimals. possible wereusedforreprobingofeachfilter,butfilterlifetimewas animals (C) Summaryofdatafrom12mutantandlittermatecontrol of APP,Rab3,andsynaptophysinwerenotobviouslychanged. NF-H andNF-LlevelsinthemutantDRGs,whereasproteinamounts lysate fromeachanimalwasloadedintolane.Noteincreased content ofmutantandcontrolanimals.Approximately12 are shown.P,proximalside;D,distalside.(B)AnalysisofDRG by Westernblotting.Datafromtwopairsofmicedifferentlitters proteins ( from eachanimalwerehomogenized,andequalamountsofnerve unligated controlsarefromthecontralateralnerve.Nerves after ligationofsciaticnervesfromcontrolandmutantanimals; as assessedbyWesternblottingofproximalanddistalsegments6h mice. second cohortof3-wk-oldKIF5A Figure 7. ons. panied byareducednumberofthelargestcalibersensoryax- tion experiments.NFaccumulationintheDRGwasaccom- appeared unchangedintheDRGandsciaticnerveliga- onal transportpathways(APP,Rab3,andsynaptophysin) comes fromourobservationthatmarkersofseveralfastax- a resultofspecificdeficitinslowaxonaltransportNFs of DRGsensoryneurons.Evidencethatthisaccumulation is (NF-H, NF-L,andNF-M)peripherininthecellbodies natal removalofKIF5AwasaccumulationNFsubunits normal. Theonlycellularphenotypewedetectedafterpost- normal inappearance,andmostphenotypesassessedwere organismal lethality,mutantcellsandtissuesareremarkably slow axonaltransport. whether eachofthesesystemsevenhasthemachineryfor been 1999; Prahladetal.,2000)models.Thisproposalhas Yabe etal.,1999)andsquidaxoplasm(Galbraith transport ofNFsincellculture(KoehnleandBrown,1999; sin orkinesinfamilymembersareimportantforslowaxonal Previous invitroexperimentsraisedthepossibilitythatkine- Discussion ways, butdoes havearoleinthevivotransport ofatleast major rolein typical kinesin-Ifastaxonal transport path- clusion fromthesefindingsis thatKIF5Adoesnothavea more complexinterpretations arepossible,thesimplestcon- KIF5A playsanyrolein ther workwillberequired to betterunderstandwhether indicative ofdefectivetransport insomeolderanimals;fur- it wasunchangedinyounger animals,butshowedbehavior We haveshownherethatalthoughlossofKIF5Acauses (A)AxonaltransportofAPP,Rab3,synaptophysin,andKIF5C highly controversialwithunresolvedissuesincluding III-tubulin waslessconsistentinitsbehaviorbecause from fivelitters.SYP,synaptophysin.Asmanyantibodiesas 40 Analysis ofDRGaccumulationandaxonaltransportin g/lane) wereloadedontoSDS-PAGEgelsandanalyzed III-tubulin transport.Although null /KIF5A flox ; Cre synapsin mutant g ofDRG The Journal of Cell Biology rons intheKIF5A–synapsin Cremice.However, KIF5Ais sensory neurons aremoreseverelyaffectedthan motorneu- lesser extent,ofmotoraxons. portant roleintheradialgrowth ofsensoryaxonsand,toa which werelostby9wkofage. Thus,NF-Hplaysanim- the growthoflargestcaliber sensoryaxons, (Rao etal.,1998).Knockout ofNF-Happarentlyinhibited ported theconsequenceoflossNF-Hinsensoryaxons of motoraxons(Elderetal.,1998b).Onestudyalsore- study describedamorepronouncedreductioninthecaliber ventral roots(Raoetal.,1998;Zhu1998),thethird slight reductioninthecaliberofmyelinatedaxonsfrom ported onNF-Hnullmice.Twostudiesreportedonly a der etal.,1998a;Jacomy1999).Threelabshavere- large myelinatedaxons(bothmotorandsensoryaxons)(El- duction inNFnumberandreductionofaxonalcaliber al., 1997),andNF-Mknockoutmiceshowedprofoundre- have noNFsandexhibitsevereaxonalhypotrophy(Zhu et ing thecalibersoflargeaxons.NF-Lgeneknockoutmice NFs thathaveprovidedevidencefortheirroleindetermin- ported material. the form(subunit,oligomeric,orpolymeric)oftrans- the KIF5Amutant.Ourdataaresilentonquestionof with thecompromiseintransportofNFsandperipherin nerves ofsomeolderanimals(unpublisheddata),consistent duced levelsofNF-M,NF-L,andperipherininthesciatic after mostrecombinationhasoccurred.Wedidobservere- animals atthisagearemostlikelybeinganalyzednotlong Cre-mediated excisionusing 3,500 totalaxonsinthesciaticnerve).Second,although ined atthistimehavedetectablecaliberdeficits( normally begin.Onlyasubsetofaxonsinmutantsexam- stantial NFdepositionandradialgrowthinaxonalcaliber in transportisobservedat3wkofage,theagewhichsub- dent linesofevidence.First,theonsetapparentdeficit nerve. Thisbehaviorisasexpectedbasedontwoindepen- was notaccompaniedbyobviousreductionsinthesciatic these proteins.ElevationofNFsubunitlevelsintheDRG cell bodyaccumulationisnotcausedbyup-regulationof not significantlychangedinthebrain,suggestingthat at reducedrates.Overall,thelevelsoftheseproteinswere sized atnormalratesbutweremovedoutofthecellbodies simplest explanationisthattheseNFsubunitsweresynthe- mutant DRGs,andthusinsensoryneuroncellbodies.The crease ofNF-H,NF-M,andNF-Lby3wkageinthe Our quantitativeimmunoblotdatashowedasignificantin- A fastmotorinslowaxonaltransport? Eyer andPeterson,1994;Zhuetal.,1997). into axonsisessentialforsuchgrowth(Oharaetal.,1993; quence ofreducedslowtransportNFs,whosemovement for normalpostnatalradialgrowth;presumablyaconse- dence alsorevealsanessentialroleforaconventionalkinesin one componentofslowaxonaltransport,theNFs.Theevi- that activity peaks(thiswouldbeconsistentwiththeobservation sion ismostactiveat3wkofagewhensynapsin-Ipromoter early asE12.5(Zhuetal.,2001),itislikelythatCreexci- One intriguingobservationfrom ourstudyisthatDRG Our dataareconsistentwithgeneknockoutstudiesof 72% ofthesemutantsdieat Cre synapsin hasbeendetectedas 3 wkofage).Hence, 19% of 250/ nesins, KIF5B and KIF5C. Likely candidates includetheotherpairof conventional ki- other motorproteinsalsoparticipate intransportofNFs. however, thatourdatadonot ruleoutthepossibilitythat identity ofamotorthatdrives thesemovements.Itisclear, longed pauses.Ourdatanow provideamissinglink,the may betheresultofrapidmovements interruptedbypro- the suggestionthatslow rateofslowaxonaltransport in culturednervecells(Wangetal.,2000);thedataledto an NFproteintaggedwithGFPhasbeendirectlyobserved 1993; Nixon,1992,1998a,b).Recently,axonaltransportof the motorforrelativelylongerperiods(Laseketal.,1992, est movingNFsrepresentthosethathavedissociatedfrom more timeinassociationwiththemotor,whereasslow- est movingNFswouldrepresentthosespendingrelatively vidual NFswiththeirputativetransportmotor,i.e.,thefast- NFs reflectsdifferentialassociationanddissociationofindi- been proposedthatbroadeningofthetransportedwave been observedinvariouspulse-labelingexperiments.Ithas of radiolabeledNFsubunitsduringcontinuedtransporthas ticipate inslowaxonaltransportofNFs?Abroadenedpeak transport. Howmightafasttransportmotor,kinesin-I,par- transport arenotanindirectresultoflargedefectsinfast pears normalinKIF5AmutantssuggeststhatchangesNF of thisview,ourobservationthatfastaxonaltransportap- ers’, data(Yabeetal.,1999;Wang2000).Insupport rect roleofKIF5AinNFtransportbetterfitsour,andoth- indirect effectontransportofNFsinsensoryneurons,adi- tor neurons(Kanaietal.,2000). other conventionalkinesin,KIF5C,inmaintenanceofmo- gested thepossibilityofapreferentialrequirementforan- 1995; Yangetal.,1996,1999).Anotherrecentstudysug- sumably duetoperturbedaxonalarchitecture(Guoetal., generation ofsensoryneuronsbutnotmotorneurons,pre- orNFsandmicrotubules, resultedinrapidde- filaments tein thatmayformcross-bridgesbetweenNFsandactin et al.,1998).Similarly,deletingBPAG1n/,apro- growth ofthelargestcaliberaxonssensoryneurons(Rao calibers ofmotorneuronsbutapparentlyinhibitedthe rons, lossofNF-Hinmicehadverylittleeffectonaxonal of axonalcaliber)inbothmotorneuronsandsensoryneu- knockout miceshowedprofoundaxonalatrophy(reduction neurons. Forexample,althoughbothNF-LandNF-M have cytoskeletalandtransportdifferencesrelativetomotor mice alsosupportsthenotionthatsensoryneuronsmight sciatic nerve)examined.Evidencefromotherknockout DRG thaninotherneuronaltissues(brain,spinalcord,and considerably higherrelativetoKIF5BandKIF5Cinthe observation (unpublisheddata)thatthelevelsofKIF5Aare transport. Supportforthislatterpossibilitycomesfromour dent uponKIF5Afornormalfunctions,includingNF have aspecializedaxonalarchitecturethatismoredepen- spinal cordmotorneurons.Second,sensoryneuronscould preferentially occursinDRGsensoryneuronsbutnot ble thatKIF5AexcisionbyCrerecombinaseprimarilyor tween motorneuronsandsensoryneurons.First,itispossi- mechanisms couldexplainthephenotypicdifferencebe- sensory neuronsbyimmunolocalization.Twopossible clearly presentinbothspinalcordmotorneuronsandDRG Although itisformallypossiblethatlossofKIF5Ahasan Neurofilament transportdefectinakinesinmutant| Xiaetal. 63 The Journal of Cell Biology 64 earized withNotI and transfectedintoRIEScellsby electroporation. The by WurstandJoyner (1993).TheKIF5A-KO/pfloxplasmid DNAwaslin- The KIF5A-targetedembryonicstem (ES) cellsweregeneratedasdescribed Generating chimericmiceandmouse tailDNAgenotype 129SVJ/Lambda FIX A KIF5Agenomicclone(encodingaminoacids98–547)isolatedfroma Construction oftheKIF5Atargetingvector Materials andmethods volved inthedevelopmentoftheseotherdiseasesaswell. axonal transportresultingfromKHCimpairmentisin- degeneration, itwillbeofinteresttotestwhetherimpaired of ourfindingthatlossKIF5Acanprovokesensoryaxon with diabeticneuropathy(Fernyhoughetal.,1999).Inlight phosphorylation wasobservedinsensoryneuronsofrats chodne, 1996;Yagihashi,1997).Moreover,aberrantNF paranodal demyelination,andlossofmyelinatedaxons(Zo- ropathy inhumans,alsohasperipheralaxonaldegeneration, polyneuropathy, themostcommonformofdiabeticneu- fails inthesedisorders.Intriguingly,symmetricalsensory out tobeanimportantcomponentofthemachinerythat lecular basisofsuchblockageisobscure.KIF5Amayturn by impairedslowaxonaltransportofNFs,althoughthemo- lien, 1999).Itispossiblethatsuchaccumulationsarecaused and Alzheimer’sdisease(JulienMushynski,1998;Ju- cluding amyotrophiclateralsclerosis,Parkinson’sdisease, have beenobservedinmanyneurodegenerativediseases,in- system. ItisalsostrikingthatabnormalNFaccumulations proper axonaltransportforthehealthofhumannervous 2002), underscoringtheimportanceofthisproteinand form ofhereditaryspasticparaplegia(SPG10)(Reidetal., More studiesneedtobedonesortoutthesepossibilities. due toaxonalblockagesecondaryanNFtransportdefect. tial, althoughitispossiblethattheneonataldeathcouldbe port ofother,asyetunidentified,cargoesKIF5Aisessen- NF knockoutmutantsareviable,wepresumethatthetrans- flecting impairedaxonaltransportofKIF5Acargoes.Asall loss ofKIF5Acouldberelatedtonervedamage,perhapsre- the KIF5Anullmutantsuggeststhatdefectscausedby view, theenlargedcellsizeofspinalcordmotorneuronsin (KIF5B andKIF5C)donotprovide.Consistentwiththis reflects aneuronalfunctionthattheothertwoKHCs imals. BecauseKIF5Aisneuronspecific,itlikelythatthis KIF5A wasfoundtobeessentialforviabilityinneonatalan- Are thereotheressentialfunctionsofKIF5A? blunt ligation.An for aminoacid323wasinsertedintotheBamHIsiteofpfloxvector by amino acids239–322(VSKTGAtoTLMFGQ)andthefirsttwonucleotides follows. An construct thetargetingvector.TheKIF5Avectorwasmade as kb AvaIIfragment. HSV-tk selectioncassette,thesecond andthirdloxPsitesflankedthe1.1- sulting vectorhadthreeloxPsites, thefirsttwoflankedpGK-neo, The finishedtargetingvectorwasdesignated asKIF5A-KO/pflox.There- sequence deletionalsoshouldnotcauseanyphenotypicconsequence. ably wouldnotaffecttherecombinationefficiencyandsuchashortintron SpeI–AvaII intronsequencewasdeletedinthetargetingvector,thisprob- right arm/pfloxbybluntligation.Forcloningconvenience,an SpeI fragment,itwassubclonedintotheSalIsiteof1.1-kbAvaII 4.5-kb fragmentwastherightarm.Theleftarman in pSK)wasbluntligatedintotheXhoIsiteof1.1-kbAvaII/pflox,this AvaII–EcoRV andEcoRV–SalIfragments,thenbluntligatedtogether Recent workhasrevealedthatKIF5Aisdefectiveinone The JournalofCellBiology 1.1-kb AvaIIfragmentcontainingtwoexonsencoding ® II mousegenomiclibrary(Stratagene)wasusedto 4.5-kb AvaII–SalIfragment(madebysubcloning

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Volume 161,Number1,2003 3.2-kb XbaI– 80-bp cific antibodies. cross-reacting antiseraandbinding to aKIF5CcolumngetKIF5C-spe- passing throughamixedKIF5Aand KIF5Bcolumntoimmunodeplete (pH 8).TheKIF5C-specificantibodies weregeneratedbyasimilarstrategy; with 0.2Mglycine(pH2.5)andneutralized with1/10volumeof1MTris lowed bythreewasheswith10mMTris.Theantibodiesweretheneluted KIF5A antibodieswerewashedwith10mMTris(pH7.5),0.5MNaClfol- pernatant) wasappliedtoaKIF5Aaffinitycolumn.Theresinswithbound and KIF5Cmixedfusionproteincolumn,theflow-through(unboundsu- erate KIF5A-specificantibodies,therabbitantiserawereappliedtoaKIF5B (pH 7.5),appliedtothecolumns,andincubatedovernightat4 The antiserawereheatedto65 for 1hatRT,washedagain,andlastlywith10mMTris(pH7.5). Hepes, pH6.9,50mMNaCl,andthecolumnswereblockedwith5%BSA The uncoupledproteinswereremovedwithseveralwashesof10mM Biomax-30 unit)to2mlofAffigel-15(Bio-RadLaboratories)for1hatRT. NaCl, and2MureawithaMilliporeUltrafree mg offusionproteins(reconstitutedin10mMHepes,pH6.9,50 form-specific antisera.Theaffinitycolumnswerepreparedbycoupling20 polyclonal seraagainstKIF5AorKIF5C. slices ofeachfusionproteinwereinjectedintothreerabbitstoproduce eluted with500mMimidazole(in6Murea/solutionA).SDS-PAGEgel with 20mMimidazole/6Murea/solutionA,andthefusionproteinswere agarose waswashedwith6Murea/solutionA,followedbyafinalwash min) andincubatedat4 urea-solubilized fraction(byspinat35,000rpminaTi1270rotorfor20 for 1hat4 (15 min,Sorvall647.5rotor),wasresuspendedin6Murea(insolutionA) hibitors) andlysedwithaFrenchpress.Thepellet,after25,000-rpmspin NaPO bacterial pelletwasresuspendedinlysisbuffer([solutionA]50mM IPTG for2h.TheHis-taggedfusionproteinsweremadeasfollows. by inductionoftransformedBL21(DE3)bacteriaatlogphasewith0.5mM generated inpET-23b. sion proteincontainingaminoacidsAVNYDQKStoQARQDLKwasalso 781 (DKTRANEQtoDKREQARE)intopET-23b.AcomparableKIF5Bfu- was generatedbysubcloningaPCRfragmentencodingaminoacids506– DQKSQ toLQLELEKL)intopET-23b.KIF5C–Hisfusionproteinconstruct ing aHincII–HindIIIfragment(encodingaminoacids496–745,VNY- His-tagged fusionproteins.KIF5Aconstructwasmadebysubclon- were subclonedintopET-23b(Novagen,Inc.)togenerateCOOH-terminal DNA fragmentsencodingportionsofthestalkregionKIF5AandKIF5C Preparation ofantibodiesfromKHC–Histagfusionproteins fixed in4%paraformaldehyde/PBS overnightat4 For KIF5Anullmutants, dissectedE18.5micebrainand spinalcordwere Histology ofmousetissues ES cellswereallowedtogrowfor24–36hbefore200 (GIBCO BRL)wasaddedforselection; P2 primers(TGTGGAGGTCAGAGGTCAAGT)willamplifyan GATCAATTCGAG) willproducean GCTGGGAACATA) andthe3 bined toperformtypeIgenotype,the5 and weregenotypedbyPCR.Threeprimers(P1,P2,Pflox)com- founders tobreedF1hybridswithC57BL/6Jfemalemice. C57BL/6J blastocyststoproducechimeras.Malechimeraswereusedas clones D17-37andD17-38typeIIcloneD17-35wereinjectedinto selected, 30clonesweretypeIEScells,4IIcells.Type fection, thesurvivingcloneswerepickedup5dlater.Among48 ES cellsand2 and D17.Crerecombinase(PMCpGK-Hyg)wastransfectedintoD17 nant cloneswiththreeloxPsites;theywerenamedasB2,B6,B18, were isolatedafter GAGGCAAAGA) andP4(GATGGGACAGCAGCAGTGCA)willdetectan wild-type band.FortypeIIgenotype,aprimersetP3(CGTTCTGGAC- for 1.5min,andafinalelongationat72 cycles ofdenaturationat95 which detectsan TTCACCGGCATCAACGT; 3 loxP sequence)typeIIband.Apairofspecificprimers(5 PCR conditionsforallprimersetsweredenaturationat95 370-bp wild-typebandandan Cross absorptionandaffinitypurificationwereusedtogenerateiso- Recombinant His-taggedKIF5AandKIF5Cproteinswereoverexpressed Mouse tailDNAsweremadeasdescribedbyWurstandJoyner(1993) 4 , 300mMNaCl,0.5MgCl C, andthentheNi-NTA-agarose(QIAGEN)wasaddedto M Gancyclovirwasaddedtothemedium3–4daftertrans- 300-bp band,wasusedforCretransgenegenotype. 8 dselection.Fourcloneswerehomologousrecombi- C for60min.Thefusionprotein–boundNi-NTA- C for30s,annealingandelongationat72 loxPprimer(PfloxkpnI,CGGTACCCGGG- primer,CTGCATTACCGGTCGATGCA), C for15min,diluted10-foldin10mMTris 470-bp (withtheadditionof 300-bp mutantfragment,theP1and 2 , 0.01%NP-40,andproteinasein- C. 120 neomycin-resistantESclones primerP1(GATACTCCAAG- ® -4 centrifugalfilterdevice C. KIF5A–synapsin Cre C for2min,30 primer,ACG- g/ml G418 C. Togen- 100 bp 450-bp C The Journal of Cell Biology overnight. 10- overnight at4 1 hatroomtemperature,followedbyincubationinprimaryantibodies cence staining.Sectionswereblocked(5%BSA,0.15%saponininPBS)for confocal imagingsystem. body. AllimageswerecollectedwithaBio-RadLaboratoriesMRC-1000 (Jackson ImmunoResearchLaboratories)wasusedasthesecondaryanti- FITC- orTexasred–conjugatedgoatanti–rabbitanti–mouseIgG and monoclonalAbSMI-32(SternbergerMonoclonalsInc.)at1:1,000. 1:20; monoclonalanti-Grp78(BIP;StressGenBiotechnologies)at1:200; ments duringthecourse ofthiswork.Weespecially thank JoeMarszalek We thankmembers oftheGoldsteinandClevelandlabs forhelpfulcom- by Westernblotanalysis. buffer (50mMTris-HCl,pH8.0,150 mMNaCl,1%NP-40)andexamined ments fromeachindividualanimal werehomogenizedinNP-40lysis dissected fromthecontrolnervein thecomparableregion.Nerveseg- distal) neartheligaturefromeachanimal; twosimilarsizesegmentswere gated; theothernervewasunligatedandservedasacontrol.After6 h, (Hanlon etal.,1997).Inbrief,onesciaticnervefromeachanimalwas li- Sciatic nerveligationexperimentsweredoneaspreviouslydescribed Sciatic nerveligationexperiments postfixed for1–2hat4 with 4%paraformaldehyde/PBS.Tissueswereremovedbydissectionand Mice wereeuthanizedwithcarbondioxideandtranscardiallyperfused Immunostaining KIF3B at1:1,000;rabbitanti– (Chemicon)at1:1,000;rabbitanti-KIF3A at1:1,000;rabbitanti- Zymed Laboratories)at1:1,000;mouseanti–NF-L1:5,000;rabbitanti- terminal regionofNF-Hat1:5,000;mouseanti–NF-M(cloneRMO-44; berger MonoclonalsInc.)at1:5,000;apolyclonalAbagainsttheCOOH- Francisco, CA)at1:100;monoclonalantibodiesSMI-31andSMI-32(Stern- KIF5B-specific Ab(fromR.Vale,UniversityofCaliforniaSanFrancisco, KIF5A, usedat1:100dilution;polyclonalAbspecificforKIF5C1:200; blots. Theantibodiesusedforblottingwereaffinity-purifiedrabbitanti- nescence ECLkit(AmershamBiosciences)wasusedfordevelopingthe ondary antibodies(JacksonImmunoResearchLaboratories).Achemilumi- followed byHRP-conjugatedgoatanti–rabbitIgGoranti–mousesec- with 5%milkandthenprobedprimaryantibodiesinthemilk-TBST, blocked inTBST(0.15MNaCl,10mMTris-HCl,pH8.0,0.1%Tween-20) ferred toPVDFmembrane(Bio-RadLaboratories).Theproteinblotswere ferent genotypeswereloadedon7.5%SDS-PAGE,andthegelstrans- Bradford assay(Bio-RadLaboratories).Equalamountsofproteinsfromdif- centrifuge) toremovelargedebris.Proteinconcentrationsweremeasuredby mogenized tissueswerequicklycentrifuged( 150 mMNaCl,1%NP-40,0.5%sodiumdeoxycholate,0.1%SDS).Ho- Mouse tissueswerehomogenizedinRIPAbuffer(50mMTris-HCl,pH8.0, Quantitative immunoblottinganalysis of equivalentarea. cross-sectional areaofeachaxonwasconvertedtoadiametercircle lens. Allmyelinatedaxonsineachrootweremeasured.Themeasured lumbar rootswerestainedwithtoluidineblueandviewedundera40 system (BioquantR&MBiometrics,Inc.).Inbrief,1- Axonal diametersweredeterminedwithaBioquantNovaimageanalysis Morphometric analysis toluidine blueandexaminedbylightmicroscopy. 812; ElectronMicroscopySciences).Semi-thinsectionswerestainedwith PBS for1h,washed,dehydrated,andembeddedinEponresin(Embed aldehyde inPBSovernightat4 nostaining. Theotherhalfwasfixedin2%paraformaldehyde,2.5%glutar- hyde/PBS, andhalfoftheDRGswereremovedprocessedforimmu- by lightmicroscopy.ImageswerecollectedwithaCCDcamera. 4 fused with4%paraformaldehyde/PBS.Tissueswerepostfixedovernightat mutant miceweresuffocatedwithcarbondioxideandtranscardiallyper- monoclonal anti– GmbH) at1:1,000;monoclonalanti-(Chemicon)1:5,000;anda aptic SystemsGmbH)at1:1,000;rabbitanti-synaptophysin(Synaptic m) werestainedwithhematoxylin-eosin,orcresylviolet,andexamined 0.5 cmofnervesegmentwasdissected frombothsides(proximaland C andthendehydratedembeddedinparaffin.Paraffinsections(7 For DRGroothistology,animalswereperfusedwith4%paraformalde- C. Theantibodiesusedforstainingwererabbitanti-KIF5Aat m cryostatsectionswereprocessedforimmunofluores- -tubulin Ab(cloneDM1A;Sigma-Aldrich)at1:10,000. 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