Syntaxin-1a和granuphilin在胰岛素释放中的作用(3)

MorphometricAnalysesofInsulinGranuleDistributions—Electronmicroscopyhasdemonstratedthatgranuphilinnull cellsexhibitaspecificdeficitofgranuleshavingcenterswithin200nmoftheplasmamembrane(6).Becausethediameterofinsulingranulesis 350nm,thisindicatesthatgranuphilinisessentialforthedirectattachment(docking)ofaninsulingran-uletotheplasmamembrane.Ithasalsobeenreportedthatsyntaxin-1anull cellsshowadrasticallyreducednumberofgranulesatadistanceof 10nmfromtheplasmamembrane(14).Becausegranuphilininteractswithsyntaxin-1a(8),bothmoleculesmaymediategranuledockinginthesamepathway.Ifso,thosesingleanddoublemutant cellsshouldshowquanti-tativelysimilardockingdefects.Totestthishypothesis,allthosemutantisletsaswellasthewild-typeisletswereprocessed

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FIGURE3.Insulinsecretionprofilesinperifusedislets.Isletsisolatedfromage-matched(20–30-week-old),C3H/He(blue;n 4forAandn 9forB),SynKO(red;n 3forAandn 8forB),GrnKO(green;n 4forAandn 8forB),andDKO(purple;n 3forAandn 8forB)micewereperifusedwithstandard2.8mMglucose-containingKrebs-Ringerbufferfor30min.Thereaf-ter,thecollectionofeachfraction(1ml/min)wasstarted,andanappropriatesecretagoguewasappliedat10minafterthecollection.Forglucosestimula-tion(A),isletswereperifusedwiththebuffercontaining16.7mMglucosefor30minfollowedbythestandard2.8mMglucosebufferfor20min.Forstim-ulationbyhighK concentration(B),isletswereperifusedwithbuffercon-taining60mMKClplus65mMNaClfor15minfollowedbythestandardbufferfor10min.

forandexaminedbyelectronmicroscopyunderthesamecon-ditions.Theelectronmicrographswerethenmorphometricallyanalyzedasdescribedelsewhere(3,6).Wecountedallthegran-ulesinthecytoplasmandfoundthattheaveragegranuleden-sitywasunchangedamongwild-typeandthethreekindsofmutantcells(Fig.4A).Whenthosegranuleswerecategorizedaccordingtotheirdistancefromthegranulecentertotheplasmamembrane,at500-nmincrements,therewerenodiffer-encesinthenumberofgranules,exceptforthatofgranuleswithin500nmoftheplasmamembrane(Fig.4B).Thegranuphilinnullanddoublemutantcells,butnotthesyn-taxin-1anullcells,showedasignificantlyreducednumberofgranuleswithin500nm(p 0.05versusC3H/Hecells).Whenthosegranuleswithin500nmwerefurthercategorizedat100-nmintervals,thenumberofgranulesat100–200nmwasspecificallydecreasedingranuphilinnullanddoublemutantcells(Fig.4C;p 0.05versusC3H/Hecells).Bycontrast,syn-taxin-1anullcellsshowednosignificantchangeofgranuledis-tributions,althoughtheytendedtohaveadecreasednumber

of

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RolesofGranuphilinandSyntaxinsinGranuleDocking

FIGURE4.Electronmicroscopicanalysisofthedistributionofinsulingranules.Isletsisolatedfrom24-to28-week-old,C3H/He(blue),SynKO(red),GrnKO(green),andDKO(purple)micewereincubatedat37°Cwith2.8mMglucose-containingbufferfor1handthenfixed.A,averagegranuledensity(granulenumberpercytoplasmicarea).BandC,granuleswhosecenterslocatedwithin7000nm(B)and500nm(C)oftheplasmamembranewerecategorizedinto14and5bins,respectively.Allresultsareprovidedasthemean S.E.Thestatisticalsignificanceofdifferencesbetweenmeanswasassessedbyarepeatedmeasureofanalysisofvariance(Tukey-Kramer’smethod).*,p 0.05.D,electronmicrographsof cellsfromC3H/He,SynKO,GrnKO,andDKOmice.Solidlinesindicatea200-nmdistancefromtheplasmamembrane.NotethatgranuleswhoselimitingmembranesweredirectlyattachedtotheplasmamembranearepresentinSynKOcellsincontrasttoGrnKOandDKOcells.Bar,1 m.

granulesat100–200nm.Importantly,doublemutantcellsshowedadecreaseingranulesat100–200nmevencomparedwithsyntaxin-1anullcells(p 0.05).Ascanbeseenintheactualelectronmicrographs(Fig.4D),syntaxin-1anull cellscontainedasignificantnumberofgranulesdirectlyattachedtotheplasmamembrane.Bycontrast,thesesortofdockedgran-ulesweremissinginboththegranuphilinnullanddoublemutantcells.Thesefindingsclearlydemonstratethatsyn-taxin-1adeficiencydoesnotcauseadistinguishablegranule-dockingdefectofthesortseeningranuphilindeficiency,con-trarytoapreviousreport(14).

GranuphilinInteractswithMultipleSyntaxinsonthePlasmaMembrane—Becausesyntaxin-1anull cellsshowedneitheracleardockingdefectnoradrasticsecretorydefect,wespecu-latedthatothersyntaxinisoformsontheplasmamembraneparticipateinthedockingandfusionofinsulingranulestotheplasmamembrane.Infact,syntaxin-4hasbeenshowntobeinvolvedinglucose-stimulatedinsulinsecretion(19).WeexpressedHA-taggedgranuphilininthe celllineMIN6andinvestigatedwhetheritinteractswiththeendogenoussyntaxinspresentontheplasmamembrane.Asfoundinpancreaticislets(Fig.1),syntaxins1aand-2–4wereallexpressedintheMIN6cells(Fig.5A).Granuphilininteractedwithsyntaxin-2andsyn-taxin-3,inadditiontosyntaxin-1a(8),butnotwithsyntaxin-4,whichisconsistentwiththepreviousfindinginco-immuno-precipitationassayusingCOS-7cells(20).Granuphilinisalso

knowntointeractwithMunc18-1(8,12,21).BecauseMunc18-1specificallybindstosyntaxin-1a,-2,and-3,butnottosyntaxin-4(22),theinteractionofgranuphilinwithsyntaxinsmaybemediatedthroughitsspecificinteractionwithMunc18-1.Toinvestigatethispossibility,wesimulta-neouslyoverexpressedgranuphilinandMunc18isoformsinHEK293Acellsandexaminedthecomplexformation(Fig.5B).GranuphilinselectivelyinteractedwithMunc18-1,butnotwithMunc18-2orMunc18-3,thelatterofwhichisknowntospecif-icallybindtosyntaxin-4(23).ThesefindingssuggestthatgranuphilinmediatesgranuledockingthroughtheinteractionswithcomplexesconsistingofMunc18-1andeithersyntaxin-1a,-2,or-3,andtherefore,syntaxin-1adeficiencycanbecompen-satedforbyusingsyntaxins2and3,whichdoesnotcauseadistinguishablegranule-dockingdefect.

DISCUSSION

Wehaveshownthatalthoughsyntaxin-1anull cellstendtohaveadecreasednumberofinsulingranulesclosetotheplasmamembrane,theyshowonlyaminorgranule-dockingdefect,incontrasttoapreviousreport(14).Thisdiscrepancymayreflectdifferencesinthefixationprotocolforthesamples,thedefini-tionofdockedgranules,orinthegeneticbackgroundofthemice.However,oursimultaneousexaminationofmutantmicewiththesamegeneticbackgroundclearlydemonstratesthatthedockingdefectinsyntaxin-1anul …… 此处隐藏：6077字，全部文档内容请下载后查看。喜欢就下载吧 ……