fromonNovember18,2010 TwoPairsofNeuronsintheCentralBrainControlDrosophilaInnateLightPreferenceZhefengGong,etal.Science330,499(2010);DOI:10.1126/science.1195993 Thiscopyisforyourpersonal,mercialuseonly. Ifyouwishtodistributethisarticletoothers,youcanorderhigh-qualitycopiesforyourcolleagues,clients,orcustomersbyclickinghere.Permissiontorepublishorrepurposearticlesorportionsofarticlescanbeobtainedbyfollowingtheguidelineshere.Thefollowingresourcesrelatedtothisarticleareavailableonlineat(thisinfomationiscurrentasofNovember18,2010):Updatedinformationandservices,includinghigh-resolutionfigures,canbefoundintheonlineversionofthisarticleat:SupportingOnlineMaterialcanbefoundat:AlistofselectedadditionalarticlesontheScienceWebsitesrelatedtothisarticlecanbefoundat:#relatedThisarticlecites24articles,6ofwhichcanbeessedfree:#ref-list-1Thisarticlehasbeencitedby1articleshostedbyHighWirePress;see:#related-urlsThisarticleappearsinthefollowingsubjectcollections:Development/cgi/collection/development Science(printISSN0036-8075;onlineISSN1095-9203)ispublishedweekly,exceptthelastweekinDecember,bytheAmericanAssociationfortheAdvancementofScience,1200NewYorkAvenueNW,Washington,DC20005.Copyright2010bytheAmericanAssociationfortheAdvancementofScience;allrightsreserved.ThetitleScienceisaregisteredtrademarkofAAAS. ReferencesandNotes1.A.Meissner,
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H.Kimura.WethankM.Tachibana,
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T.H.Endo,andS.L.Marjanifortheirinvaluablesuggestions.ThemicroarraydatahavebeendepositedintheGeneExpressionOmnibusandgiventheseriesessionnumberGSE23181. SupportingOnlineMaterial/cgi/content/full/science.1194174/DC1MaterialsandMethodsSOMTextFigs.S1toS8TablesS1toS3References 24June2010;epted2September2010Publishedonline16September2010;10.1126/science.1194174Includethisinformationwhencitingthispaper. DownloadedfromonNovember18,2010 TwoPairsofNeuronsintheCentralBrainControlDrosophilaInnateLightPreference ZhefengGong,1*JiangquLiu,1,2ChaoGuo,1,2YanqiongZhou,1,2YanTeng,3LiLiu1* Appropriatepreferencesforlightordarkconditionscanbecrucialforananimal’ssurvival.Innatelightpreferencesarenotstaticinsomeanimals,includingthefruitflyDrosophilamelanogaster,whichprefersdarknessinthefeedinglarvalstagebutpreferslightinadulthood.Toelucidatetheneuralcircuitunderlyinglightpreference,weexaminedtheneuronsinvolvedinlarvalphototacticbehaviorbyregulatingneuronalfunctions.Modulatingactivityoftwopairsofisomorphicneuronsinthecentralbrainswitchedthelarvallightpreferencebetweenhobicandhilic.Theseneuronswerefoundtobeimmediatelydownstreamofpdf-expressinglateralneurons,whichareinnervatedbylarvalphotoreceptors.Ourresultsrevealedaneuralmechanismthatcouldenabletheadjustmentofanimals’responsestrategiestoenvironmentalstimuliordingtobiologicalneeds. Preferencebetweenlightanddarknessplaysanimportantroleinanimallife(1–4).ThefruitflyDrosophilamelanogasteravoidslightatthefirsttomid-thirdinstarlarvalstage,butthishobicbehavioristhereafterreduced,beforepupation(5–7).Inadditiontothecircadianphotoreceptorcryptochrome(CRY),thelarvalvisualsystemincludestwobilateralgroupsof12photoreceptors(8,9):theBolwig’ans(BO),whichsendoutBolwig’snerves(BNs)toinnervatethepace-makingneurons,thepigment-dispersingfactor(Pdf)–expressinglateralneurons(pdfneurons)inlarvalcentralbrain(6,10).BlockingeitherBOorpdfneuronscauseslarvalblindness,asmeasuredbyphototacticassay(6,11,12). Toinvestigatedownstreamneuronsunderlyinglarvalphototacticbehavior,wescreenedabatchofupto800Gal4lines[obtainedfromDrosophilaicResourceCenter(DGRC), 1StateKeyLaboratoryofBrainandCognitiveScience,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,People’sRepublicofChina.2GraduateUniversityoftheChineseAcademyofSciences,Beijing100039,People’sRepublicofChina.3ProteinScienceCoreFacilityCenter,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,People’sRepublicofChina. *Towhomcorrespondenceshouldbeaddressed.E-mail:zfgong@(
Z.G.);liuli@(
L.L.) Kyoto]inasimplelight-darkchoiceassay(6)usingtheGal4/UASsystemtodriveicexpressionofthetetanustoxinlightchain(TeTxLC;UAS-TNTG),aneuron-specifictoxinthatpreventspresynapticreleaseofsynapticvesicles(13).WhereasmostGal4linesmanifestedhobiaandseveralGal4linesexhibitedlossoflightpreferencewithicTeTxLCexpressionatearlytomid-thirdinstarlarvalstage,oneGal4line,NP394-Gal4,demonstratedapreferenceforlight.ThislineshowedpositivelarvalphototaxiswhenTeTxLCexpressionwasdrivenbyNP394Gal4[Fig.1A,performanceindex(PI)=−0.35T0.07,P<0.001,n=16;fig.S1].Furthermore,temporaryTeTxLCexpressioninNP394-Gal4–labeledneuronswasabletoconferpositivephototaxisatvariouslarvalstages(fig.S2).Thepositivelarvalphototaxiswasreproducedbyicexpressionofamutatedformoftheopenrectifierpotassiumchannel,dORK∆C(Fig.1A,PI=−0.25T0.09,P<0.05,n=16),theoverexpressionofwhichhyperpolarizesneuronsandsubsequentlyinactivatesneuronalfunction(14). Tofindmorephototaxis-positiveGal4linesmonlabelingwiththeNP394-Gal4,werescreenedalltheGal4linesbyoverexpressingdORK∆
C.ThismethodwaschosenbecausetheoverexpressionofTeTxLCledtolethalityordefectsinotioninalargenumberofGal4 lines,meaningthatbehavioralassayscouldnotbeconducted.Twolines,NP423-Gal4andNP867Gal4,manifestedpositivelarvalphototaxiswhenthelabeledneuronswereinhibitedbydORK∆C(Fig.1A,PI=−0.22T0.06forNP423-Gal4>dORK∆CandPI=−0.34T0.07forNP867-Gal4>dORK∆C,n=16forbothlines).Forfurtherconfirmationatthebehaviorallevel,weappliedthetemperature-sensitiveformofDynamin(shits)thatinstantlyinhibitscellendocytosisattherestrictivetemperature(15).InallthreeGal4lines,icexpressionofshitsattherestrictivetemperatureresultedinpositivelarvalphototaxis(Fig.1B,PI=−0.22T0.05forNP394-Gal4>UAS-shits,PI=−0.22T0.05forNP423-Gal4>UAS-shits,andPI=−0.29T0.05forNP867Gal4>UAS-shits,n=16foralllines).Bycontrast,hyperactivationofNP394-Gal4–labeledneuronsbyexpressingthesodiumchannelNaChBac(16)significantlyenhancedlightavoidanceinlatethirdinstarlarvae,whichgenerallyexhibitreducedlightparedwithyoungerlarvae(figs.S3andS4).Thus,weconcludedthatregulationofactivityinneuronslabeledbytheseGal4linescouldswitchlarvalphototaxisbetweennegativeandpositive,suggestingthattheseneuronsmediatethelarvalpreferencebetweenlightanddarkness. Inparallelwithbehavioralscreening,weusedamembrane-tetheredgreenfluorescentprotein(mCD8-GFP)tovisualizetheexpressionpatternsoftheGal4linesatthelarvalstage.Outsidethecentralnervoussystem(CNS),monlabelingwasfoundonlyinthesalivaryglandinallthreeGal4lines(tableS1).InthelarvalCNS,theNP394Gal4expressionpatternwasmostrestrictedofthethreeGal4lines(Fig.1,CtoH,andfig.S5).NP394-Gal4labelingwasmostmarkedintwopairsofmirror-symmetricallyarrangedneuronsinthesupraesophagealganglionfromasearlyasthefirstinstarandthroughoutlarvaldevelopment(fig.S6).Forconvenience,werefertothesecellsasNP394-neurons.IntheothertwoGal4lines,labelingofneuronswithmorphologyandlocationsimilartothoseoftheNP394-neuronswasalsoobserved(Fig.1,CtoH).ToconfirmthatthesameNP394-neuronswerelabeledinallthreeGal4lines,webinatorialGal4labeling.Infliesbinationsoftwo SCIENCEVOL33022OCTOBER2010 499 REPORTS DownloadedfromonNovember18,2010 Fig.1.PositivelarvalphototaxiswasinducedbyinhibitionofneuronslabeledbythreeGal4lines.(A)ExpressionofTeTxLC(UAS-TNTG)anddORKDCwiththeGal4/UASsysteminthethreeGal4linesledtopositivelarval phototaxis,whereastheGal4andUASlinesaloneresultedinnegativephototaxis.(B)ExpressionofshitswiththeGal4/UASsysteminthethree Gal4linesledtopositivelarvalphototaxisatrestrictivetemperature.Early tomid-thirdinstarlarvaewereused.DataarepresentedasthemeanTSEM.*P<0.05,**P<0.01,***P<0.001;n=16forallgroups.(CtoH) ExpressionpatternsofNP394-Gal4(CandF),NP423-Gal4(DandG),andNP867-Gal4(EandH)inthirdinstarlarvalCNS.(IandJ)DoubleGal4expressionpatternsofNP394-Gal4+NP423-Gal4(I)andNP394-Gal4+NP867-Gal4(J)inthirdinstarlarvalCNS.(K)Single-neuronmorphologyoftheNP394-neuronsinNP423-Gal4.BackgroundisFasII-labeledneuraltracts(magenta).mCD8-GFP(green)wasusedtolabeltheneuron.ArrowsindicatethesomaoftheNP394-neuronsin(C)to(J)andtwoarborizationregionsin(K).Scalebars,20mm. Gal4linessuchasNP394-Gal4andNP423Gal4,orNP394-Gal4andNP867-Gal4,thenumberofNP394-neuron–likeneuronsvisualizedwasthesameasthatinthesingleGal4lineofNP394Gal4(Fig.1,IandJ).SuchalackofincrementintheneuronnumbersuggeststhattheNP394neuronswerelabeledbyalltheGal4lines.Thus,theNP394-neuronsappeartobetheonlyneuronslabeledmonintheCNSinallthreeGal4lines. WeproposethattheNP394-neuronsarecrucialforswitchingthelarvallightpreferencebetweendarknessandlight.Toverifythis,weintroducedelav-Gal80,whichcanspecificallyrepressGal4activityinneurons,intoNP394-Gal4fliestopreventGal4expressioninNP394-neurons(17).InparallelwiththerepressionofGal4labelinginNP394-neuronsbyelav-Gal80(Fig.2,AandB),thepositivelarvalphototaxisintheTeTxLC-expressingNP394-Gal4linebecamenegative,possiblybecauseTeTxLCexpressiondrivenbyNP394-Gal4wasexcludedfromtheNP394-neurons(Fig.2C,PI=−0.31T0.07forNP394-Gal4>UAS-TNTG,andPI=0.39T0.09forNP394-Gal4+elav-Gal80>UAS-TNTG,n=16forbothlines).Thus,inhibitionoftheactivity Fig.2.MaskingNP394-neuronsfrominhibitionrestorednegativelarvalphototaxis.(AandB)Introductionofelav-Gal80repressedNP394-Gal4labeling(green)inNP394-neuronsbutleftlabelinginotherregionsintact.Anti-FasIIlabeling(magenta)wasusedasbackground.Scalebars,20mm.(C)Larvalphototaxischangedfrompositivetonegativewhenelav-Gal80wasintroducedintoNP394-Gal4larvaeexpressingTeTxLC.DataarepresentedasthemeanTSEM.***P<0.001;n=16forallgroups. oftheNP394-neuronswasabletoreversethelarvallightpreference. WetheninvestigatedthemorphologyoftheNP394-neurons.ThecontralateralprojectionsofNP394-neuronsfrombothsideswerelargelyoverlapped,makingsingle-neuronmorphologydifficulttoidentify.Hence,theFLP-outtechniquewasappliedtoinvestigatethemorphologyofsingleNP394-neurons(Fig.1Kandfig.S7). Onlyonetypeofsingle-neuronmorphologywasobserved.Thecellbodywaslocatedinthecenterofeachbrainhemisphere.Theneuronprojectedposteriorlyforashortdistanceinmostcases,thenmedially,crossingthemidlinealongthesamepathasthatinthecorrespondingcontralateralNP394-neuronsandturningposteriorlyintheend.Twomajorarborizationregionswereformed:oneclosetotheterminalregionoflateralneurons 500 22OCTOBER2010VOL330SCIENCE DownloadedfromonNovember18,2010 Fig.3.SynapticcontactbetweenNP394-neuronsandpdfneurons.(AandB)PatternsofNP394-neurons(green)andpdfneurons(magenta)inlarvalbrain.(A)Patternsinz-axisprojection.(B)Patternsinasinglescanninglayerof0.3mm.Insetin(B)showstheoverlapbetweenNP394-neuronsandpdfneuronsinanx-zview.Arrowsindicateoverlappingregions.(CtoG)GRASPsignalsvisualizedbetweenpdfneuronsandNP394-neuronslabeledbyNP394-Gal4(CtoE),NP423-Gal4(F),andNP867-Gal4(G).GRASPsignalintheframedregionin(C)isshownathighermagnificationin(D)and(E).Insetsin(F)and(G)showonlytheGRASPsignals.ArrowsindicatetheGRASPsignal.Scalebars,20mm. Fig.4.AblationofpdfneuronsallowsstrongerandfasterresponsetolightstimulationinNP394neurons.(A)ChangeinGCAMP3.0fluorescence(DF/F)uponlightstimulation.Scalebar,10mm.(B)RepresentativeresponseprocessesofNP394-neuronsincontrolandpdf-DTIlarvaeuponlightstimulation.(C)Averagepeakresponseincontrolandpdf-DTIlarvae.(D)Averagetimeforfluorescencechangetoachieve20%(20%DF/F)afterlightstimulationincontrolandpdf-DTIlarvae.DataarepresentedasthemeanTSEM.*P<0.05,***P<0.001;n=6forallgroups. REPORTS labeledbyanantibodyagainstPdf(18),andtheotherclosertothemidline(figs.S7andS8).Thepartofthetractthatcrossedthemidlineappearedtocontainlittlearborization. BecausetheNP394-neuronsappearedtobeinvolvedinlarvallightpreference,wespeculatedthattheymightsomehowreceivevisualinformationfromthelarvalvisualsensorysystem.paredtheNP394-Gal4–labeledNP394-neuronswithPdf-specificantibody–labeledpdfneurons.ThedendritesofNP394-neuronswereclosetotheaxonalterminiofpdfneurons,althoughtheinterfaceareaappearedtobelimited(Fig.3andfig.S8).Consequently,itseemsthattheNP394-neuronsarepostsynaptictopdfneurons.Thus,thepolarityofNP394-neuronswasexamined.Weexpressedboththepresynaptic-specificmarkers,suchasSyt-GFP(19),andpostsynapticmarkerDscamGFP(20),inNP394-neuronsusingNP423-Gal4todeterminethepre-andpostsynapticregionsoftheneurons(fig.S9).Thearborizationregionswerenotlabeledbythepresynaptic-specificmarkerSytGFP,butwerestronglylabeledbythepostsynapticmarkerDscam-GFP.Thus,thearborizationregionofcontactwiththepdfneuronswasmostlikelypostsynaptic. ToverifythesynapticinteractionbetweentheNP394-neuronsandthepdfneurons,weusedtherecentlydevelopedGRASP(greenfluorescentproteinreconstitutionacrosssynapticpartners)technique(21,22).Inthistechnique,theponentsofGFP,GFP1-10andGFP11,aredifferentiallyexpressedintwoneighboringcells,andformfluorescentreconstitutedGFPacrosscellulargapsifthecellsarecloseenoughtoeachother.WeexpressedGFP1-10inNP394-neuronsandGFP11inpdfneurons(23)andobservedsignalsofreconstitutedGFPinregionswherepdfneuronsmaysynapsewithNP394-neurons(Fig.3,CtoG,andfig.S10).TofurtherconfirmthecontactbetweenNP394-neuronsandpdfneurons,weexaminedthemorphologyofNP394-neuronsintheabsenceofpdfneuronstotestwhetherthepresenceofpdfneuronsisrequiredforthenormalmorphologicaldevelopmentofNP394-neurons.WedidnotfindanysubstantialmorphologicalchangeofNP394-neuronsinapdf-DTIlineinwhichpdfneuronsinbrainhemispheresweremissing(fig.S11).Nevertheless,calciumimaginganalysisrevealedthattheNP394-neuronresponsetolightsimulation,asindicatedbyGCAMP3.0fluorescence(24),wasstrongerandfasterintheabsenceofpdfneuronsthanintheirpresence(Fig.4,peakDF/F:0.72T0.10forcontroland1.11T0.12forpdf-DTI;timelatencyfor20%DF/F:30.19T1.44sforcontroland8.03T0.73sforpdf-DTI;moviesS1toS3).Thus,ourresultssuggestthatfunctionalcontactdoesexistbetweentheNP394-neuronsandpdfneurons.Becausepdfneuronsreceivevisualinformationnecessaryforphototacticbehavior,weproposethattheNP394-neuronsmayreceiveinputsignalsfrompdfneuronsforthereversionoflightpreference. AslarvaewithinhibitedNP394-neuronsmanifestedpositivephototaxiswhereasthosewith SCIENCEVOL33022OCTOBER2010 501 REPORTS hyperactivatedNP394-neuronsshowedpromotedlightavoidance,weproposethatNP394-neuronactivityispositivelycorrelatedwithlarvallightavoidanceability.Twopossiblescenarioscouldbeoperatinginthissituation.First,theactivityofNP394-neuronsitselfcontrolsthelarvalphototaxisbyanunknownmechanism.Second,theNP394-neuronsactivatethepathwaythatmediatesavoidanceoflightwhereasotherunidentifiedneuronsactivatethepathwaythatunderliesavoidanceofdarkness,aswasshownforthemechanismsunderlyingodor-taxisinadultDrosophila(25). ReferencesandNotes1.J.N.Crawley,What'sWrongWithMyMouse?
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Y.Jan,andR.Jiaoforflystocksandconstructs;andC.WangandX.Haoforfacilitysetup.WethanktheDGRC(Kyoto)andtheBloomingtonflystockcenterfortheflystocks,andDevelopmentalStudiesHybridomaBankforantibodies.WealsothankH.Gongfortechnicalassistance,andJ.FlemingandR.Wolfforments.ThisworkwassupportedbytheNationalNaturalSciencesFoundationofChina[grant30770682(
Z.G.)andgrants30621004and30625022(
L.L.)],the‘973Program’[grants2005CB522804and2009CB918702(
L.L.)],andtheKnowledgeInnovationProgramoftheChineseAcademyofSciences[grantKSCX2-YW-R-247(
L.L.)]. SupportingOnlineMaterial/cgi/content/full/330/6003/499/DC1MaterialsandMethodsFigs.S1toS13TableS1ReferencesMoviesS1toS3 3August2010;epted8September201010.1126/science.1195993 DownloadedfromonNovember18,2010 FastVesicleFusioninLivingCellsRequiresatLeast ThreeSNAREComplexes RalfMohrmann,1,2*HeidideWit,3MatthijsVerhage,3ErwinNeher,1JakobB.Sørensen1,4,5* ExocytosisrequiresformationofSNARE[solubleN-ethylmaleimide–sensitivefactorattachmentprotein(SNAP)receptor]plexesbetweenvesicleandtargetmembranes.Recentassessmentsinreducedmodelsystemshaveproduceddivergentestimatesofthenumberofplexesneededforfusion.Here,weusedatitrationapproachtoanswerthisquestioninintact,culturedchromaffincells.Simultaneousexpressionofwild-typeSNAP-25andamutantunabletosupportexocytosisprogressivelyalteredfusionicsandfusion-poreopening,indicatingthatbothproteinsassembleintoheteromericplexes.Expressingdifferentwild-type:mutantratiosrevealedathird-powerrelationforfast(synchronous)fusionandanear-linearrelationforoverallrelease.Thus,fastfusiontypicallyobservedinsynapsesandneurosecretorycellsrequiresatleastthreefunctionalplexes,whereasslowerreleasemighturwithplexes.Heterogeneityinplexnumbermayexplainheterogeneityinvesicularreleaseprobability. TheSNARE[solubleN-ethylmaleimide–sensitivefactorattachmentprotein(SNAP)receptor]plexformedbetweentwofusingmembranesisattheheartofthemolecularmachinerythatmediatesexocytosis
(1).Itisacoiledbundleoffourparallelahelicesprovidedby 1DepartmentofMembraneBiophysics,Max-PlanckInstituteforBiophysicalChemistry,Göttingen,Germany.2DepartmentofPhysiology,UniversityofSaarland,Homburg,Germany.3CenterforNeurogenomicsandCognitiveResearch,DepartmentofFunctionalGenomics,VrijeUniversiteit(VU)AmsterdamandVUMedicalCenter,Amsterdam,Netherlands.4DepartmentofNeuroscienceandPharmacology,FacultyofHealthSciences,UniversityofCopenhagen,Copenhagen,Denmark.5LundbeckFoundationCenterforBiomembranesinNanomedicine,UniversityofCopenhagen,Copenhagen,Denmark. *Towhomcorrespondenceshouldbeaddressed.E-mail:Ralf.Mohrmann@uks.eu(
R.M.);jakobbs@sund.ku.dk(
J.B.S.) threeSNAREproteins:SNAP-25(synaptosomeassociatedproteinof25kD),synaptobrevin-2,andsyntaxin-1
(2).plexformationproceedsfromtheN-totheC-terminalendinadiscontinuousprocessthatinvolvespartiallyassembledintermediates.AssemblyofthemostCterminalthreetofourinteractionlayerscoincideswithmembranemerger
(3).Thoughithasbeenunclearwhetherassemblyofoneplexgeneratessufficientenergytoinitiatevesiclefusion(4–7),itwasrecentlyreportedthatliposomescanfusewiththehelpofasingleplex,albeitwithlowspeed
(8).Otherstudieshaveconcludedthat5to11plexesmightbeinvolvedinfastermodesoffusion(9–13). Tostudythedependenceoffastvesiclefusiononhigher-orderplexesinintactcells, weusedanexceptionallyinhibitorySNAP-25mutationinatitrationassaythatallowedustorelateexocytosistotherelativeexpressionlevelsofmutantandwild-type(WT)proteininagivencell.Ourmutantharboredtwoalaninesubstitutions[Met71→Ala71(M71A)andIle92→Ala92(I192A)]intheinteractionlayer+5,facingtheinsideofplex
(2).IfincorporatedintheSNAP-25Aisoform,thispletelyfailstoreconstituteexocytosisinSnap-25–/–adrenalchromaffincells(14).Here,weintroducedthemutationintoSNAP-25B,becausethisisoformsupportstwotothreetimesmorefast-phasesecretion(15).SNAP-25BWT(SN25B)ormutantprotein(denotedSN25BL5**)wereN-terminallyfusedtoenhancedgreenfluorescentprotein(EGFP)ormCherry(mCh),allowingforthequantitativeanalysisofexpressionlevelsandproteinlocalization. UsingtheSemlikiForestvirus(SFV)expressionsystem,wecharacterizedmCh-taggedSN25BL5**andmCh-SN25BexpressedseparatelyinSNAP25–deficientchromaffincells(16).Bothproteinswerelocalizedtotheplasmamembraneandexpressedtosimilarlevels(Fig.1,EandF).Secretionwasassayedbymembranecapacitancemeasurementsandamperometryafterflashphotoreleaseofcagedcalcium.ExpressionofmCh-SN25BL5**suppressedsecretion(totalcapacitancechange:12T3fFafter5s;n=28cells)(Fig.1,AtoE)paredwithmCh-SN25B–infectedcells(510T54fF;n=36;P<0.0001)andevenSNAP-25–deficientcells(39T5fF;n=35;P<0.0001;Student’sttest).ThismadeSN25BL5**anattractiveinhibitorforatitrationexperiment.SeverallinesofevidenceindicatethatinhibitionprobablyarisesfrominterferencewithaverylatestepofexocytosisassociatedwiththeC-terminalassemblyoftheplex:(i)SN25L5**formsstableplexes 502 22OCTOBER2010VOL330SCIENCE