Syntaxin-1a和granuphilin在胰岛素释放中的作用(2)

Pancreaticisletswereisolatedfromcervicallydislocatedmicebypancreaticductinjectionof500units/mlcollagenasesolution(typeXI;Sigma),followedbydigestionat37°Cfor25minwithmildshaking,andisolatedisletswerepickedupbyhandselectionunderadissectingmicroscope,asdescribed

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elsewhere(16).Proteinextractsfromisletswerepreparedandimmunoblottedasdescribedelsewhere(11).Theimmunoreac-tivebandswerevisualizedusingenhancedchemiluminescentWesternblottingdetectionreagents(GEHealthcare).Chemi-luminescentsignalsonthex-rayfilmwerecapturedwithanimagescannerandquantifiedbyImageJ1.31software.Fortheimmunoprecipitationanalyses,cellextractswerepreparedin1mloflysisbuffer(10mMTris,pH7.5,150mMNaCl,2mMMgCl2,0.5mMEGTA,and0.1%TritonX-100)andwerecen-trifugedat21,880 gfor10min.Thesupernatantswereincu-batedwitheither20 lofratanti-HAaffinitymatrixbeads(RocheDiagnostics)or30 lofmouseanti-FLAGaffinitygel(Sigma),withgentleagitationat4°Cfor1h.

PerifusionAssaysinIsolatedIslets—Isolatedisletswerecul-turedovernightinRPMI1640medium(11mMglucose)sup-plementedwith5%fetalcalfserum,100units/mlpenicillin,and100 g/mlstreptomycin.Thirtyisletswereplacedatthebot-tomofa1-mlsyringethathadbeencuttoavolumeof400 landpluggedwithcotton.TheywereperifusedwithstandardlowglucoseKrebs-Ringerbuffer(15mMHEPES,pH7.4,120mMNaCl,5mMKCl,2mMCaCl2,1mMMgCl2,24mMNaHCO3,0.1%bovineserumalbumin,and2.8mMglucose)ataconstantflowrateof1.0ml/minfor30min.Afterthisstabili-zationperiod,theywerefurtherperifusedwiththesamebufferfor10minfollowedbythebuffercontainingthesecretagogues.Alloftheperifusatesolutionwasequilibratedwith95%airand5%CO2andmaintainedat37°C.Fractionswerecollectedevery1min,andtheinsulinsecretionwasmeasuredusinganAlph-aLISAinsulinkitwithanEnVision2101multilabelreader(PerkinElmerLifeSciences).

ElectronMicroscopicAnalysisofGranuleDistribution—Iso-latedisletswereculturedovernightandthenincubatedinlowglucoseKrebs-Ringerbufferat37°Cfor1h.Theywerefixedbyimmersionwith2%paraformaldehyde,2%glutaraldehyde,0.2%picricacidin0.1Mcacodylatebuffer,pH7.4,for1.5hatroomtemperatureandembeddedinto1%agarose.Theywerethenpostfixed,embeddedinplasticresin,andsectioned.Theultra-thinsections(80nm)wereanalyzedunderanelectronmicro-scope(JEM1010;JEOL,Akishima,Japan)atanaccelerationvoltageof80kV.Micrographswererandomlytakenat 4000magnificationfrom21individual cellsfromthreemiceforeachgenotype.Thedistancefromthegranulecentertotheplasmamembranewasmeasuredasdescribedelsewhere(3).PlasmidConstruction—Full-lengthcDNAsencodingmouseMunc18-1–3wereamplifiedfrommousecDNAsbyPCRusingthefollowingpairsofoligonucleotideswithanEcoRIlinkeroranXhoIlinker:5 -GGGGAATTCATGGCCCCCATTGGCC-TCA-3 and5 -GGGCTCGAGTTAACTGCTTATTTCTTC-ATCTG-3 forMunc18-1;5 -GGGGAATTCATGGCGCCC-TTGGGGCTG-3 and5 -GGGCTCGAGTCAGGGCAGGG-CCACACC-3 forMunc18-2;and5 -GGGGAATTCATG-GCGCCGCCGGTATCG-3 and5 -GGGCTCGAGTTACT-CATCCTTAAAGGAAACTT-3 forMunc18-3.PurifiedPCRproductsweresubclonedintotheEcoRIandXhoIsitesofpcDNA3.1/3 FLAGvector(17)anddesignatedpcDNA3-FLAG-Munc18-1–3.Recombinantadenovirusbear-ingHA-taggedgranuphilincDNAwaspreparedasdescribedelsewhere(10).

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RolesofGranuphilinandSyntaxinsinGranuleDocking

FIGURE1.Proteinexpressioninpancreaticislets.A,proteinextracts(5 g)frompancreaticisletsofcontrolC3H/He,syntaxin-1aknock-out(SynKO),granuphilinknock-out(GrnKO),andgranuphilin/syntaxin-1adoubleknock-out(DKO)micewereelectrophoresedforimmunoblottingwithantibodiestowardtheindicatedproteins.B,proteinexpressionlevelswerequantifiedfromthreeindependentexperimentalpreparations.Thebandintensityofeachproteinwasnormalizedbythatof -actin.Allresultsareprovidedasthemean S.E.*,p 0.05;**,p 0.001versus

C3H/He.

CellCultureandTransfection—Allcellsweremaintainedinahumidifiedincubatorwith5%CO2at37°C.Mouseinsuli-nomaMIN6cellsandHEK293Acellswereculturedinhighglucose(25mM)Dulbecco’smodifiedEagle’smediumsup-plementedwith15%fetalcalfserumand55 M2-mercapto-ethanolandwith10%fetalcalfserum,respectively.ThepcDNA3-FLAG-Munc18-1–3andpcDNA3-HA-granu-philin-avectors(atotalof8 gofplasmids)weretransfectedintoHEK293AcellsusingLipofectamine2000reagent(Invitro-gen).Twodaysaftertransfection,cellswereharvestedandhomogenizedforimmunoprecipitation.

RESULTS

GenerationofGranuphilinandSyntaxin-1aDoubleKnock-outMice—Togeneticallyinvestigatethefunctionalrelation-shipbetweengranuphilinandsyntaxin-1a,wegenerateddou-blemutantmicebycrossinggranuphilinknock-outmicewithsyntaxin-1aknock-outmice.AllthesingleanddoublemutantmicehadaC3H/Hegeneticbackground.Wefirstisolatedpan-creaticisletsandexaminedtheexpressionlevelsofthosetwoproteinsandtheirrelatedmolecules(Fig.1).Thesyntaxin-1aknock-outmiceshoweddecreasedlevelsofgranuphilin-b(82%)butanormallevelofMunc18-1,comparedwiththecon-

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FIGURE2.Invivophenotypesofgranuphilinand/orsyntaxin-1aknock-outmice.A,bodyweight.B,bloodglucoseconcentrationsduringanintra-peritonealglucosetolerancetest.C,plasmainsulinconcentrationsbeforeand30minafteraglucoseload.D,percentageofstartingbloodglucoseconcentrationduringanintraperitonealinsulintolerancetest.Eachmeasure-mentwasperformedinage-matched(9–16-week-old),C3H/He(blue;n 14forA,B,andDandn 8forC),SynKO(red;n 11forA,B,andDandn 9forC),GrnKO(green;n 10forA,B,andDandn 9forC),andDKO(purple;n 11forA,B,andDandn 9forC)mice.Allresultsareprovidedasthemean S.E.Thestatisticalsignificanceofdifferencesbetweenmeanswasassessedbyarepeatedmeasureofanalysisofvariance(Tukey-Kramer’smethod).*,p 0.05;**,p 0.001versusC3H/He.

trolC3H/Hemice.However,thegranuphilinknock-outmiceshowedreducedlevelsofsyntaxin-1a(42%)andMunc18-1(78%),asreportedelsewhere(6).Similarly,doubleknock-outmiceexhibitedareducedlevelofMunc18-1(68%).Wealsoinvestigatedtheexpressionlevelsofothersyntaxinsthatresideontheplasmamembrane(18).Becausesyntaxin-1bisnotpres-entinpancreatic cellsorinsyntaxin-1anull cells(14),weexaminedsyntaxin-2–4.However,incontrast …… 此处隐藏：6647字，全部文档内容请下载后查看。喜欢就下载吧 ……