Related ESCRT-III Subunit CHMP2B

19 Pages·2015·5.19 MB·English

TheJournalofNeuroscience,February18,2015•35(7):3155–3173•3155
Cellular/Molecular
Regulation of Postsynaptic Function by the Dementia-
Related ESCRT-III Subunit CHMP2B
XRomainChassefeyre,1,2*Jose´Martínez-Herna´ndez,1,2*FedericaBertaso,3,4,5NathalieBouquier,3,4,5Be´atriceBlot,1,2
MarineLaporte,1,2SandrineFraboulet,1,2YohannCoute´,6,7AnnyDevoy,8AdrianM.Isaacs,8KarinPernet-Gallay,1,2
Re´mySadoul,1,2XLaurentFagni,3,4,5andYvesGoldberg1,2,9
1InstitutNationaldelaSant´eetdelaRechercheM´edicale(INSERM),Unit´e836,F-38042Grenoble,France,2Universit´eGrenobleAlpes,GrenobleInstitut
desNeurosciences(GIN),F-38042Grenoble,France,3CNRS,UMR-5203,InstitutdeG´enomiqueFonctionnelle,F-34094Montpellier,France,4Universit´es
deMontpellier1&2,UMR-5203,F-34094Montpellier,France,5INSERM,Unit´e661,F-34094Montpellier,France,6INSERM,Unit´e1038,F-38054Grenoble,
France,7Commissariata`l’EnergieAtomique(CEA),InstitutdeRecherchesenTechnologiesetSciencespourleVivant(iRTSV),LaboratoiredeBiologiea`
GrandeEchelle,F-38054Grenoble,France,8DepartmentofNeurodegenerativeDisease,UniversityCollegeLondonInstituteofNeurology,LondonWC1N
3BG,UnitedKingdom,and9CEA,iRTSV,GroupePhysiopathologieduCytosquelette(GPC),F-38054Grenoble,France
The charged multivesicular body proteins (Chmp1–7) are an evolutionarily conserved family of cytosolic proteins that transiently
assemblesintohelicalpolymersthatchangethecurvatureofcellularmembranedomains.MutationsinhumanCHMP2Bcausefronto-
temporaldementia,suggestingthatthisproteinmaynormallycontrolsomeneuron-specificprocess.Here,weexaminedthefunction,
localization,andinteractionsofneuronalChmp2b.Theproteinwashighlyexpressedinmousebrainandcouldbereadilydetectedin
neuronaldendritesandspines.DepletionofendogenousChmp2breduceddendriticbranchingofculturedhippocampalneurons,de-
creasedexcitatorysynapsedensityinvitroandinvivo,andabolishedactivity-inducedspineenlargementandsynapticpotentiation.To
understandthesynapticeffectsofChmp2b,wedetermineditsultrastructuraldistributionbyquantitativeimmuno-electronmicroscopy
anditsbiochemicalinteractionsbycoimmunoprecipitationandmassspectrometry.Inthehippocampusinsitu,asubsetofneuronal
Chmp2bwasshowntoconcentratebeneaththeperisynapticmembraneofdendriticspines.Insynaptoneurosomelysates,Chmp2bwas
stablyboundtoalargecomplexcontainingothermembersoftheChmpfamily,aswellaspostsynapticscaffolds.Thesupramolecular
Chmpassemblydetectedherecorrespondstoastableformoftheendosomalsortingcomplexrequiredfortransport-III(ESCRT-III),a
ubiquitouscytoplasmicproteincomplexknowntoplayacentralroleinremodelingoflipidmembranes.WeconcludethatChmp2b-
containingESCRT-IIIcomplexesarealsopresentatdendriticspines,wheretheyregulatesynapticplasticity.Weproposethatsynaptic
ESCRT-IIIfilamentsmayfunctionasanovelelementofthesubmembranecytoskeletonofspines.
Keywords: ESCRTfilaments;frontotemporaldementia;postsynapticscaffold;spinoskeleton;structuralplasticity
Introduction cellular membrane remodeling during inward vesiculation of
The endosomal sorting complex required for transport-III multivesicularendosomes,mitoticcellabscission,andviralbud-
(ESCRT-III) is a ubiquitous protein complex that is crucial to ding(forreview,seeHenneetal.,2013;McCulloughetal.,2013).
The core subunits of ESCRT-III are a family of evolutionarily
conservedproteins,calledchargedmultivesicularbodyproteins
ReceivedFeb.10,2014;revisedDec.23,2014;acceptedJan.6,2015.
(Chmp1-7). Dominant mutations in subunit CHMP2B cause
Authorcontributions:R.S.,L.F.,andY.G.designedresearch;R.C.,J.M.-H.,F.B.,N.B.,B.B.,M.L.,S.F.,Y.C.,A.D.,
frontotemporaldementia(FTD;Skibinskietal.,2005;Isaacset
A.M.I.,K.P.-G.,andY.G.performedresearch;Y.C.andA.M.I.contributedunpublishedreagents/analytictools;R.C.,
J.M.-H.,F.B.,N.B.,Y.C.,K.P.-G.,R.S.,L.F.,andY.G.analyzeddata;R.C.,J.M.-H.,A.M.I.,R.S.,L.F.,andY.G.wrotethe al., 2011), raising the question whether some neuron-specific
paper. processiscontrolledbythisprotein.Chmp2bandotherESCRT-
ThisworkwassupportedbyagrantfromtheAgenceNationaledelaRecherche(ANR),MALZprogram, IIIsubunitsexistinthecytosolinamonomeric,autoinhibited
France.R.C.andM.L.weresupportedbytheMiniste`redel’EnseignementSupe´rieuretdelaRecherche,
state.Afterreleaseofintramolecularinhibition,individualsub-
France.J.M.-H.andN.B.weresupportedbyANR.A.M.I.wassupportedbytheUnitedKingdomMedical
ResearchCouncil.WethankY.SaoudiforhelpwithmicroscopyandN.Jaafari,A.Petiot,andW.Weissenhorn unitspolymerizeintocircularorhelicalfilaments(20–200nm
forcriticallyreadingthismanuscript. wide,subjecttosubunitspecies)thatadheretolipidmembranes
*R.C.andJ.M.-H.contributedequallytothiswork. andchangetheircurvature(Shimetal.,2007;Hansonetal.,2008;
Theauthorsdeclarenocompetingfinancialinterests.
Lataetal.,2008;Bajoreketal.,2009;Henneetal.,2012;Buchk-
ThisarticleisfreelyavailableonlinethroughtheJNeurosciAuthorOpenChoiceoption.
ovichetal.,2013).
CorrespondenceshouldbeaddressedtoeitherYvesGoldbergorRémySadoul,,InstitutdesNeurosciences,UJF
Site Sante´, Chemin Ferrini, 38042 Grenoble cedex 9, France. E-mail: [email protected] or
[email protected].
R.Chassefeyre’spresentaddress:TheScrippsResearchInstitute,LaJolla,CA92037. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense
DOI:10.1523/JNEUROSCI.0586-14.2015 (http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse,distributionandreproductioninany
Copyright©2015Chassefeyreetal. mediumprovidedthattheoriginalworkisproperlyattributed.
3156•J.Neurosci.,February18,2015•35(7):3155–3173 Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses
Table1.Plasmidsusedinthisstudy
Plasmidname Cloningtechnique Cloningsites Feature Tags Source
pIRES2-EGFP Startingvector Clontech
pIRES2-EGFP-CHMP2BWT In-Fusion(Clontech) XhoI,XhoI RNAires. C-terminalFLAG Thisstudy
pBI-CMV3 Startingvector Clontech
pBI-CMV3-CHMP2BWT In-Fusion(Clontech) BamHI,HindIII RNAires. Thisstudy
pBI-CMV3-CHMP2BR19/22/26A In-Fusion(Clontech) BamHI,HindIII RNAires. Thisstudy
pBI-CMV3-CHMP2BL4D/F5D In-Fusion(Clontech) BamHI,HindIII RNAires. Thisstudy
pBI-CMV3-CHMP2BL207D/L210D In-Fusion(Clontech) BamHI,HindIII RNAires. Thisstudy
pSuper-mCherry Startingvector Bellyetal.,2010
pSuper-mCherryshRNAvsCHMP2B Standardligation HindIII,BglII Bellyetal.,2010
pSuper-mCherryshRNAvsGFP Standardligation HindIII,BglII Thisstudy
pCDNA3.1-CHMP2BWT Topo(Invitrogen) C-terminalHA Thisstudy
pCDNA3.1-CHMP2BR19/22/26A Topo(Invitrogen) C-terminalHA Thisstudy
pCDNA3.1-CHMP4B Topo(Invitrogen) C-terminalFLAG Bodonetal.,2011
Thecloningtechniqueusedtoconstructeachplasmidand,ifapplicable,theassociatedkitsupplierareindicated.RNAires.IndicatestheencodedmRNAisresistanttoRNAibytheChmp2bshRNA.
We have previously shown that Chmp2b preferentially po- pSuper-mCherry. cDNAs encoding wild-type (WT) or mutant ver-
lymerizesundertheplasmamembraneand,whenoverexpressed, sionsoftheRNAi-resistantChmp2b(Bellyetal.,2010)wereclonedin
organizesintohelicesthatdeformpatchesofthecellsurfaceinto thepBI-CMV3vector(Clontech),yieldingpBI-CMV3-Chmp2bWT,
long protrusions (Bodon et al., 2011). Holomeric ESCRT-III pBI-CMV3-Chmp2bR19/22/26A, and pBI-CMV3-Chmp2bL4D/F5D. In
consistsofheteropolymericChmpfilamentsassembledatsitesof thesevectors,Chmp2biscoexpressedwithfluorescentproteinZsGreen
from a bidirectional promoter. cDNA encoding wild-type, RNAi-
endosomalorplasmamembranedeformation,afterrecruitmentby
resistantChmp2b(carryingasingleC-terminalFlagtag;Bodonetal.,
the upstream-acting complexes ESCRT-I and -II and/or by the
2011)wasinsertedupstreamoftheIRESsequenceofthepIRES2-EGFP
Chmp4b-bindingproteinAlix.ESCRT-IIIassembliesatmembranes
vector(Clontech),yieldingpIRES2-EGFP-Chmp2b(usedinFig.6B).In
arethoughttobetransient,beingdissociatedafterinteractionwith
thisvector,Chmp2biscoexpressedwithEGFPfromabicistronictran-
the depolymerizing ATPase Vps4. This depolymerization step is
script.Allconstructswereverifiedbysequencing(BeckmanCoulter).
coupledtoscissionofmembranenecksmodeledbyESCRT-IIIfila- Antibodies.SeeTable2fordetailsofantibodiesanddilutions.
ments(Henneetal.,2013). Animals.AnimalswerehandledandkilledinconformitywithEuro-
FTD-associatedCHMP2Bmutantsarederegulatedandform peanlawandinternalregulationsofINSERM.PregnantOncinsFrance
aberrantpolymericstructuresintransfectedcells(Skibinskietal., soucheA(OFA;asubstrainofSpragueDawley)rats(CharlesRiver)were
2005).StrongexpressionofsuchaCHMP2Bmutantincultured usedforprimaryneuroncultures,and2-month-oldOFAratsofboth
corticalneuronsisrapidlylethal(Leeetal.,2007),andtransgenic sexeswereusedforbiochemicalanalysisofbraintissue.Theratswere
miceexpressingthesamemutantthroughouttheirbraindisplay deeplyanesthetizedbyisofluraneinhalationbeforedecapitation,quickly
followedbycesareansectionorbraindissection.Three-month-oldmale
progressive neurodegenerative changes (Ghazi-Noori et al.,
C57BL/6miceand1.3-month-oldfemaleChmp2bKOmice(herecalled
2012).Theseeffectshavebeenlinkedtodefectsinautophagicand
KD mice since knock-out is incomplete;Ghazi-Noori et al., 2012) or
endosomaldegradationpathways(Filimonenkoetal.,2007;Lee
femaleWTlittermates(onC57BL/6background)wereusedforimmu-
etal.,2007;Urwinetal.,2010).However,thesephenotypesarise
nohistochemistry and electron microscopy (EM) studies. Mice were
fromatoxicgainratherthanlossoffunctioninthemutantallele.
anesthetizedbyintraperitonealinjectionof0.1mlofsodiumpentobar-
Indeed, degeneration was not seen in knock-out mice severely bital(5.6%w/v;CEVASante´Animale)andintracardiallyperfusedwith
(thoughnotcompletely)deficientforChmp2b(Ghazi-Nooriet phosphate-buffered 0.9% NaCl (PBS), followed by 0.1 M phosphate-
al., 2012). Furthermore, Chmp2b depletion did not affect sur- buffered4%formaldehyde,pH7.4,supplementedwith0.05%glutaral-
vivalofculturedneurons(Leeetal.,2007;Bellyetal.,2010)but dehyde(Sigma).Thebrainswerecarefullyremovedandpostfixedfor4h
significantlyimpairedthemorphologicalmaturationofdendritic inthesamefixative,and60-(cid:2)m-thickcoronalbrainsectionswerecut
spines(Bellyetal.,2010).This,togetherwiththefactthatsynap- withaVibratome(VT1000S;Leica).
togenesis correlates with Chmp2b gene transcription in some Cellcultureandtransfection.Primaryculturesofhippocampalneurons
neuronalpopulations(Xuetal.,2012),ledustohypothesizethat werepreparedasdescribedpreviously(Bellyetal.,2010).Briefly,hip-
pocampiweredissectedfromE18ratembryos,treatedwithtrypsin,and
normalChmp2bandESCRT-IIImayfunctionatsynapses.
disruptedbyseventoeightcyclesofaspirationandejectionthrougha
Here,weshowthatChmp2bisrequiredformaintainingthe
micropipettetip.Dissociatedhippocampalcellswereseededatadensity
complexityofdendritictreesaswellasthestability,efficacy,and
of1.5(cid:2)104/cm2in35mmdishesoncoverslipsprecoatedfor4hwith50
plasticityofexcitatorysynapses.Wealsoshowthattheprotein (cid:2)g/mlpoly-D-lysine(Sigma),inneuronalculturemedium(Neurobasal
preferentiallylocalizesbeneaththeplasmamembraneofdendritic
mediumcontainingB27supplement,penicillin/streptomycin,and0.5
spinesandisboundtostableESCRT-IIIcomplexesassociatedwith mML-glutamine;Invitrogen)supplementedwith10%heat-inactivated
thepostsynapticcytoskeleton,suggestingthatESCRT-IIIpolymers horseserum(Invitrogen).Fourteen-mililimeter-wideroundcoverslips
cooperatewithpostsynapticscaffoldsandactinfilamentstoshape wereusedforimmunostaining,and30-mm-widecoverslipswereused
dendriticspines. for live imaging. Neurons were maintained in water-saturated 95%
air/5%CO at37°C.Theseedingmediumwasreplacedafter20hwith
MaterialsandMethods serum-free2neuronalculturemedium.
Plasmids.SeeTable1fordetailsofallconstructs.ThepSuper-mCherry Neuronsweretransfectedusingacalciumphosphatemethodopti-
vector was described by Belly et al. (2010) and expresses the inserted mizedforhighlymatureneuronalcultures(Goetzeetal.,2004;Jiangand
shRNAtogetherwithmCherry.TheChmp2b-specificshRNA(Leeetal., Chen, 2006). For cotransfection experiments, for each dish, 1 (cid:2)g of
2007;Bellyetal.,2010)andtheGFP-specificshRNA[previouslyshown pSuper-mCherry-based plasmid was mixed with 2 (cid:2)g of pBI-CMV3-
byAlvarezetal.(2006)tohavenoeffectonneuronalmorphologyor basedorpIRES2-EGFP-basedplasmid.Fivemicrolitersof250mMCaCl2
electrical properties] have been described and were inserted into were added to 45 (cid:2)l of water containing 3 (cid:2)g of DNA, mixed, and
Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses J.Neurosci.,February18,2015•35(7):3155–3173•3157
Table2.Antibodiesusedinthisstudy
Dilutions
Antibody Species Company(reference) IF WB IHC/EM
Anti-Chmp2b Rabbitpolyclonal Abcam(ab33174) 500 10,000 1000
Anti-Chmp2b Mousemonoclonal Covalab 100 2000
Anti-Chmp4b Rabbitpolyclonal Abcam(ab105767) 500 2000
Anti-Chmp4b Mousemonoclonal Covalab 100 1000
Anti-Alix Rabbitpolyclonal Covalab(ab0204) 6000
Anti-PSD95 Mousemonoclonal NeuroMab(73-028) 200 4000
Anti-GluN1 Mousemonoclonal SynapticSystems(114011) 200 4000
Anti-Synaptophysin Mousemonoclonal MerckMillipore(MAB5258) 10,000
Anti-HA Mousemonoclonal CellSignaling(2367) 200 5000
Anti-HA Rabbitpolyclonal Sigma-Aldrich(H6908) 5000
Anti-Flag Mousemonoclonal Sigma-Aldrich(F3165) 1000 10,000
Anti-Flag Rabbitpolyclonal Sigma-Aldrich(F7425) 1000 5000
Anti-Actin Mousemonoclonal Millipore(MAB1501R) 20,000
Anti-Tubulin Mousemonoclonal GiftfromA.Andrieux’slaboratory 20,000
Anti-PanMAGUK Mousemonoclonal NeuroMab(75-029) 4000
Anti-GluN2B Mousemonoclonal NeuroMab(75-097) 4000
Anti-Glu2 Mousemonoclonal Millipore(MABN71) 5000
Anti-ShankIII Rabbitpolyclonal SantaCruzBiotechnology(30193) 1000
Anti-Homer Mousemonoclonal SantaCruzBiotechnology(17842) 2000
Anti-MouseHRP Goatpolyclonal JacksonImmunoResearch(115-035-166) 20,000
Anti-RabbitHRP Goatpolyclonal JacksonImmunoResearch(115-035-044) 20,000
Anti-MouseAlexaFluor488 Goatpolyclonal Invitrogen(A-11029) 1500
Anti-MouseAlexaFluor594 Goatpolyclonal Invitrogen(A-11032) 1500
Anti-MouseCy5 Goatpolyclonal JacksonImmunoResearch(115-175-146) 1000
Anti-RabbitAlexaFluor488 Goatpolyclonal Invitrogen(A-11034) 1500
Anti-RabbitAlexaFluor594 Goatpolyclonal Invitrogen(A-11037) 1500
Anti-RabbitCy5 Goatpolyclonal JacksonImmunoResearch(111-175-144) 1000
Theantibodydilutionsweusedareindicatedforeachapplication.IF,Immunofluorescence;WB,Westernimmunoblotting;IHC,immunohistochemistry.
immediatelyaddeddropwiseto50(cid:2)lof2(cid:2)N,N-Bis(2-hydroxyethyl)- a humidified chamber with primary antibodies diluted in a blocking
2-aminoethanesulfonicacid(BES)-bufferedsalinesolution,pH7.1.The solution.AfterwashinginPBS,cellswereincubatedfor1hwithsecond-
mixturewasgentlystirredbytappingthetubeandbubblingaironce,left aryantibodiesconjugatedtoAlexaFluor488,AlexaFluor594,orCya-
toincubatefor10min,andaddedtocoverslipsin1mloftransfection nine5(Cy5),dilutedintheblockingsolution.Coverslipswererinsedand
medium[Eagle’sMEM(Invitrogen)including2.2%NaHCO andsup- mountedinMowiol.
3
plementedwithB27,0.05mMglutamine,and20mMglucose].After1hin Golgistaining.Thedendriticspinesofhippocampalneuronsinsitu
theincubator,thecoverslipsweretransferredtodishescontaining2mlof werevisualizedbytheGolgiimpregnationtechnique.Forthis,weused
transfection medium (without B27) preincubated for 30 min in 10% theFDRapidGolgiStainkit(FDNeuroTechnologies).Hippocampaltis-
CO .Thedisheswerequicklyreturnedto5%CO andleftfor5–10min sueblocks(2.5–3mm)wereimmersedinequalvolumesofsolutionsA
2 2
beforereturningthecoverslipstotheiroriginal(conditioned)neuronal andBfor7dandimpregnatedwithsolutionCfor48hat4°C.Then,the
culture medium. For immunofluorescence of exogenously expressed brainswerecutwithavibratomeinto150-(cid:2)m-thicksectionsembedded
proteins,neuronswerefixed16–20haftertransfection.ForRNAiexper- inagarose,washedtwiceindouble-distilledwater,andincubatedfor10
iments,fixationorliveimagingtookplace72haftertransfection. mininamixtureofonepartofsolutionD,onepartofsolutionE,andtwo
HEK-293TandHeLacellsweregrowninDMEM(Invitrogen)supple- partsofdouble-distilledwater.Sectionswerewashedtwice,dehydrated
mentedwith10%FBSand2mML-glutamine(Sigma).Cellsweretrans- withincreasingethanol,andmountedwithepoxyresin(Fluka)between
fectedafter24hofcultureusingjetPEI(Polyplus)forHeLacellsand twocoverslips.Theslideswereanalyzedbyinvestigatorswithoutknowl-
calciumphosphateforHEKcells.Cellswerefixedorlysed36hafter edgeofgenotype.Ineachofthreeanimalspergenotype,ninesecondary
transfection. ortertiarypyramidalneurondendriteswererandomlyselectedinthe
Immunohistochemistry. Brain sections were incubated in 10% goat CA1stratumradiatum.Selectioncriteriawere(1)dendritesdisplayed
preimmuneserum(GPi)dilutedin50mMTrisbuffer,pH7.4,containing completeGolgiimpregnation,(2)thecelltypewasclearlyidentifiable,
0.9%NaCl[Tris-bufferedsaline(TBS)],with0.2%TritonX-100,for1h. and(3)theminimumlengthofthedendriteswas50(cid:2)mfromthesoma.
Sectionswerethenincubatedovernightat4°Cinaprimaryantibodyata Stacksofbright-fieldimageswith0.3(cid:2)mspacingwereacquiredwitha
finalproteinconcentrationof1–2(cid:2)g/ml,whichhadbeendilutedinTBS Zeiss Axioskop 50 microscope with 63(cid:2) oil objective (NA 1.4; Plan-
containing1%GPi.AfterseveralwashesinTBS,thesectionswerefurther Aprochromat)coupledtoaCCDcamera(CoolSnapES;RoperScien-
incubatedfor2hinbiotinylatedgoatanti-rabbitIgG(SigmaImmuno tific)operatedbyMetaviewsoftware(MolecularDevices).Imageswere
Chemicals)diluted1:100inTBScontaining1%GPi.Theywerethen analyzedwithImageJ.Thenumberofspinesofeachbasictypeperunit
transferred into avidin–biotin–peroxidase complex (ABC kit; Vector dendriticlengthwasmeasured.
Laboratories)diluted1:100for2hatroomtemperature.Boundperoxi- Fluorescencemicroscopy.Lightmicroscopyimageswereacquiredwith
daseenzymeactivitywasrevealedusingDAB(0.05%inTB,pH7.4)as a Zeiss LSM 710 inverted confocal microscope, using a 63(cid:2) Plan-
thechromogenand0.01%H O asthesubstrate.Finally,sectionswere Apochromatoil-immersionobjective(NA1.4;Zeiss)or(forShollanal-
2 2
air-driedandcoverslipped. ysis)a40(cid:2)oilobjective(NA1.3;Zeiss).Theinstrumentwascontrolled
Immunofluorescentstaining.Culturedneuronswerefixedfor20minat byZensoftwareandincludesacousto-opticalfiltersforattenuatinglaser
roomtemperatureinphosphate-buffered4%paraformaldehydesupple- power,scanningmirrorswithadjustablespeed,andresolutionofemitted
mented with 4% sucrose. After three washes in PBS containing 0.1% lightbyadiffractiongratingandbeamguidesforsettingspectralcollec-
glycine,cellswerepermeabilizedinPBScontaining0.25%TritonX-100 tionwindows.Laserlinesat488,561,and633nmwereusedforexciting
for10minat4°CandrinsedwithPBS.Coverslipswereblockedfor1hin fluorescenceofEGFP,ZsGreen,orAlexaFluor488;mCherryorTexas
PBScontaining5%GPiandincubatedfor1–2hatroomtemperaturein Red;andCyanine5,respectively.Formulticolorimaging,collectionwin-
3158•J.Neurosci.,February18,2015•35(7):3155–3173 Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses
dowswereadjustedbasedonemissionspectratominimizecross-channel r
contamination. Pinhole size was usually set to generate 0.9-(cid:2)m-thick rN(cid:3)1(cid:4)r (cid:5)1r ,
opticalslices((cid:2)1Airyunit)atallwavelengths.Forfixedsamples,laser 1 2
powerwasadjustedto0.5–1.25mWinallchannels,andthesignalwas wherer istheradialdistancefromthemembranetotheparticleandr is
1 2
averagedoverfourscansanddigitizedonan8bitgrayscale.Intheexper- theradialdistanceoftheparticlefromthegeometriccenterofthespine.
imentsshowninFigure2,BandC,Z-stackswereacquiredwith0.09(cid:2) Tocorrecttheradialdistributionfordistance-dependentincreaseinbin
0.09(cid:2)0.2(cid:2)m3voxelsizeandprocessedbyablinddeconvolutionalgo- area,particledensitiesalongtheradialcoordinateweredividedbynor-
rithm(AutoQuant;MediaCybernetics)toimproveresolutionandre- malizedradialdistance,thusgivingincreasedweighttoparticledensity
move shot noise. The processed images were slightly contrasted with close to the spine center. Three-dimensional reconstruction of spine
ImageJforclarity. headsfromserialsectiondigitalimageswasdoneusingReconstruct
ToevaluatetheprobabilityofrandomoverlapbetweenChmp2band software(JohnC.Fiala,DepartmentofBiology,BostonUniversity,
postsynapticdensitity-95(PSD-95)clusters,adendriticregionwasse- MA). For comparisons between wild-type and Chmp2b-deficient
lectedinawaytoexcludeasmuchaspossibleemptypixels.Pearson’s mice,acquisitionofmicroscopyimagesandmorphometricquantifi-
correlationcoefficient(r)betweenChmp2bandPSD-95intensityvalues cationwereperformedwithoutknowledgeofgenotype.
wascalculatedfortheactualimage,andtheprobabilityofobtainingthe PurificationofsynaptoneurosomesandPSDfractionation.Subcellular
observedcoefficientwascalculatedbasedon1000randomizedimages fractionationwasperformedaccordingtoCarlinetal.(1980)withminor
similartotheoneshownusingtheImageJplug-inJACOP(Bolteand modifications.Sampleswerekeptat4°Cduringallprocedures.Inbrief,
Cordelie`res,2006). oneratforebrainwashomogenizedin13mlofice-coldisotoniclysis
ForShollanalysis,images(1024(cid:2)1024pixels;350(cid:2)350(cid:2)m;four buffer(0.32Msucrose,4mMHEPES,pH7.4,proteaseinhibitors[EDTA-
opticalsectionsinz)weretakenfromneuronscoexpressingZsGreenand freeComplete;RocheMolecular)]usingaTeflon-glasshomogenizer(15
mCherry,withinthesamefluorescencerangeforallneurons(40neurons strokes).Thehomogenatewascentrifugedat1400(cid:2)gfor10minto
pertransfectioncondition,threeindependentcultures).Shollanalysis removenuclei.Thepostnuclearsupernatant(S1)wasspundownat4°C
wasperformedonmaximumintensityprojectionsofmCherryfluores- for10minat13,800(cid:2)g,yieldingcrudesynaptosomes(pelletP2)and
cenceusingtheShollAnalysispluginofImageJ. crudecytosol(supernatantS2).TheP2fractionwashomogenizedinlysis
Forpostsynapticspinecountinginculturedneurons,transfectedneu- bufferandlayeredontodiscontinuoussucrosedensitygradients(made
ronswerestainedbyimmunofluorescencewithanti-PSD-95antibody of3mleachof0.85,1.0,and1.2Msucroselayers).Thegradientswere
and Cy5-conjugated secondary antibody. Triply labeled neurons centrifugedat82,500(cid:2)g(27,000rpminaBeckmanSW41rotor)for120
(mCherry-ZsGreen-Cy5)wereimagedinallthreechannelsasabove.For min,andthepurifiedsynaptoneurosomefraction(SYN)wascollectedat
each neuron, 10–20 optical sections were acquired (voxel size, 0.1 (cid:2) the1.0–1.2Minterface.Thesynaptoneurosomeswereresuspendedin5
0.1(cid:2)0.2(cid:2)m3).ImageswereanalyzedwithImageJ.Postsynapticspines voloflysisbufferandspundownfor20minat150,000(cid:2)g.Thepelleted
weremanuallyidentifiedinsuccessivesectionsasspine-likedendritic synaptoneurosomeswereresuspendedinTritonbuffer[50mMHEPES,
protrusions(visualizedbymCherryfluorescence)carryingPSD-95clus- pH7.4,0.5%TritonX-100,proteaseinhibitors(EDTA-freeComplete;
ters.Spineswerecountedalong(cid:3)540(cid:4)220(cid:2)m(mean(cid:4)SD)dendritic RocheMolecular),2mMEDTA],stirred30mininthecoldroom,and
lengthperneuron.Similarresultswereobtainedwithtwoindependent centrifugedat32,000(cid:2)gfor20mintoobtainthePSDIpellet.PSDIwas
cultures,allowingustopoolthedata.DualimmunostainingofmCherry- resuspendedin50mMHEPES,pH7.4.One-halfofthesuspensionwas
transfectedneuronswithanti-PSD-95andanti-synapsinantibodiesver- solubilizedagainbyadding1volof1%TritonX-100andcentrifugedat
ifiedthatinneuronsatthisstage,virtuallyallPSD-95clusterslocatedon 200,000(cid:2)gfor20min,yieldingpelletPSDII.Theotherhalfwassolubi-
spineswerecolocalizedwiththepresynapticmarkersynapsin.Imagesof lizedbyadding1volof6%Sarkosylandcentrifugedat200,000(cid:2)gfor
neuronsusedtostudyRNAieffectswereacquiredandanalyzedbyinves- 1h,yieldingPSDIII.
tigatorsblindtothetransfectedconstructs. Gelfiltrationofsynaptoneurosomeextract.Size-exclusionchromatog-
Electron microscopy. Free-floating brain sections were incubated in raphy (gel filtration) was used for separation of Chmp2b-containing
10%GPidilutedinTBSfor1hatroomtemperaturetoblocknonspecific molecularassembliesthathadbeensolubilizedbymilddetergentextrac-
staining.Sectionswerethenincubatedovernightat4°CinTBScontain- tionfromgradient-purifiedsynaptoneurosomes.Chromatographywas
ing1%GPiandaprimaryantibodyatafinalproteinconcentrationof performedat4°Cona120mlHiPrepSephacrylS-300HRcolumncou-
1–2(cid:2)g/ml.Thesectionswerethenlabeledeitherbyimmunogoldorby pledtoanAktaFPLCsystem(GEHeathcare).Thecolumnwasequili-
immunoperoxidase.Forimmunoperoxidaselabeling,wefollowedthe brated,andseparationwasachievedin25mMTris-HCl,pH7.4,150mM
immunohistochemistryprocedure,asdescribedabove(startingfromthe NaCl,0.5%TritonX-100,10mMNaF,10mM(cid:6)-glycerophosphateand
secondaryantibodystep).Forimmunogoldlabeling,sectionswereincu- proteaseinhibitors(Roche)ataflowrateof0.5ml/min.Synaptoneuro-
batedfor3hwithgoatanti-rabbitIgGcoupledto1.4nmgoldbeads somes freshly prepared from one brain were resuspended in 3 ml of
(Nanoprobes) diluted 1:300 in TBS containing 1% GPi. After several deoxycholate (DOC) buffer [10 mM Tris-HCl, pH 9.0, 1% sodium
washesinPBS,thesectionswerepostfixedin1%glutaraldehydediluted deoxycholate,150mMNaCl,proteaseinhibitors(Complete;RocheMo-
in the same buffer for 10 min. They were washed in double-distilled lecular),andphosphataseinhibitors(P0044;Sigma-Aldrich)].Thesus-
water,followedbysilverenhancementofthegoldparticleswithaHQ pension was stirred by rotation at 4°C for 30 min and centrifuged at
Silverkit(Nanoprobes). 100,000(cid:2)gfor20min.Thesupernatantwasfiltered(poresize,0.2(cid:2)m;
Theperoxidase-reactedsectionsandthegold–silver-labeledsections Millipore),and2.5mlwasloadedonthecolumn.Fractions(2ml)were
wereprocessedforEM.Thisincludedtreatmentwithosmiumtetroxide collected,andtheelutionprofilewasanalyzedbyimmunoblottingand
(1% in 0.1 M PB), block staining with uranyl acetate, dehydration bymeasurementoftotalproteinconcentrationwithBCAproteinassay
throughagradedseriesofethanolsolutions,andflatembeddingonglass (Pierce).Theprofilewascalibratedbymeasuringtheelutionvolumeof
slidesinDurcupan(Fluka)resin.Regionsofinterestwerecutat70nmon proteinstandardsinthesameconditions(MWGF1000;Sigma-Aldrich).
anultramicrotome(ReichertUltracutE;Leica)andcollectedonone-slot Immunoprecipitation.ForimmunoprecipitationofthebrainChmp2b
coppergrids.Stainingwasperformedondropsof1%aqueousuranyl complex,2mg(totalprotein)ofgradient-purifiedsynaptoneurosomes
acetate,followedbyReynolds’sleadcitrate.EMimageswereacquiredin wereresuspendedin1mlofDOCbufferandincubatedfor30minat4°C.
a JEOL-1200 electron microscope with a digital camera (Veleta, SIS; After centrifugation at 100,000 (cid:2) g for 20 min, the supernatant was
Olympus) and analyzed with ImageJ. Measurements of gold particle collectedanddilutedwith3volofimmunoprecipitationbuffer(50mM
countsalongtheradialandtangentialcoordinatesofthespinecytoplasm Tris-HCl,pH7.4,150mMNaCl,and1%TritonX-100).Thesynapto-
were acquired and normalized as described by Ra´cz and Weinberg somallysatewasincubatedfor2.5hat4°Cwith15(cid:2)gofantibodythat
(2008).Briefly,normalizedradialposition,r (thefractionofthedis- had been covalently coupled to superparamagnetic protein A beads
N
tancefromthecentertotheplasmamembrane),wascomputedaccord- (Dynabeads, 10001D; Invitrogen) by cross-linking with dimethyl-
ingtothefollowingequation: pimelimidate(Sigma)accordingtoHarlowandLane(1988).Afterex-
Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses J.Neurosci.,February18,2015•35(7):3155–3173•3159
Figure1. Chmp2bexpressioninthemousebrain.A,Anadultmalemousewasdissected,andequalamountsoftotalproteinfromeachtissuewereanalyzedbySDS-PAGEandimmunoblotting
withtheindicatedantibodies.ThearrowheadindicatestheChmp4b-specificband.(cid:7)-Tubulinwasusedastheloadingcontrol.B,Mousebrainsatincreasingdevelopmentalageswereanalyzedby
immunoblottingwiththeindicatedantibodies.PSD-95wasusedasamarkerofsynaptogenesis.P,Postnatalage(days);1M,1month.C,Equalproteinamountsfromtheindicatedregionsofanadult
mousebrainwereanalyzedbyimmunoblottingasabove.D,Frontal(left)orsagittal(right)sectionsfromadultmousebrainwerestainedbyimmunoperoxidasewithanti-Chmp2bantibody.Cx,
Cortex;cc,corpuscallosum;Hpc,hippocampus;Hb,habenula;Ce,centralamygdala;OB,olfactorybulb;CB,cerebellum.E,Left,ImmunostainedCA1layerinafrontalsectionofthehippocampus.Note
thestrongstainingofpyramidalcellbodiesandtheweakerbutdefiniteimmunoreactivityoftheneuropil.SO,Stratumoriens;Pyr,stratumpyramidale;SR,stratumradiatum.Right,Higher
magnificationoftheboxedregion.Immunoreactivityisobservedinapicaldendrites(arrowheads)andinterneurons(arrows).F,SpecificityofChmp2bimmunostaining.Asacontrol,weusedbrain
sectionsfromChmp2bKOmice(Ghazi-Noorietal.,2012),herecalledChmp2bKDbecauseofthepresenceofresidualChmp2bproteininthesemice(Fig.7G,H).Chmp2bimmunoreactivitywas
revealedbyimmunoperoxidaseinafrontalsectionofthebrainofa5-week-oldwild-type(Wt)mouse(left)orinanequivalentsectionofasiblingKDmouse(right).NotethatdevelopmentofDAB
stainingwasperformedidenticallyforthetwosections,butforalongertimethaninD,whichthereforeappearslighterthanF(Wt).
tensivewashinginbuffercontaining50mMTris-HCl,pH7.4,0.25% Immunoblotting.CellsortissuelysateswereresuspendedinLaemmli
sodiumdeoxycholate,and0.75%TritonX-100,togetherwithincreasing bufferandresolvedbySDS-PAGEin8–12%polyacrylamidegels.Pro-
concentrationsofNaCl(150–500mM),boundproteinswereelutedby teinswereelectrotransferredontoPVDFmembranes.Membraneswere
boiling in Laemmli buffer, resolved by SDS-PAGE, and blotted onto blockedfor30mininTBScontaining0.1%Tween20and5%drymilk
PVDFmembranes(Millipore).Forsamplesfirstresolvedbygelfiltra- andincubatedfor1hwithprimaryantibodiesdilutedintheblocking
tion,fractionsofinterestwerepooledandimmunoprecipitatedunder solution.AftersixwashesinTBS–Tween,themembraneswerefurther
thesameconditions. incubatedfor30minwithsecondaryantibodiescoupledtoHRP,washed
Forimmunoprecipitationoftaggedproteins,transfectedHEK-293T sixmoretimesasbefore,andincubatedwithluminescence-generating
werewashedwithPBSandhomogenizedinisotoniclysisbuffer,andthe HRPsubstrate,andboundantibodieswererevealedbyluminographyon
lysatewascentrifugedat1000(cid:2)gfor10min.Thepostnuclearsuper- film.
natantwascentrifugedat50,000(cid:2)gfor1h,andthepelletedmem- Massspectrometryanalysis.ADOCextractfromgradient-purifiedsyn-
braneswereresuspendedinDOCbuffer.Immunoprecipitationwas aptoneurosomeswasfractionatedbygelfiltration,andthehigh-molecular-
performedwithanti-tagantibodyimmobilizedonproteinADyna- weight (HMW) fractions were pooled and immunoprecipitated with
beadsasabove. controloranti-Chmp2bantibody.Twoother,independentlyprepared
3160•J.Neurosci.,February18,2015•35(7):3155–3173 Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses
Figure2. Chmp2bformsclustersindendrites.A,Culturedmousecorticalneuronswerelysedatvariousstagesofmaturation,andequalamountsoflysateproteinwereanalyzedbyimmuno-
blottingwiththeindicatedantibodies.PSD-95wasusedasamarkerforsynaptogenesis.B,HippocampalneuronsweretransfectedwithemptypSuper-mCherryvector,fixedat21DIV,andstained
bydualimmunofluorescencewithanti-Chmp2bandanti-PSD-95antibodies.Left,Punctatestainingofadendriticsegmentinatransfectedneuron(singleconfocalplane).Stainingofnontransfected
cellsisalsovisible.Merge,Overlap(yellow)betweenChmp2bandPSD-95puncta,indendriticshaftsandspines,visualizedwithmCherry(whitecontour).Thenumberedarrowheadsindicatespines
withcolocalizedpuncta.Right,Enlargedviewsofthesamespines.Scalebar,20(cid:2)m.C,Top,Dendriticsegmentdoublestainedbyanti-Chmp2bandanti-PSD-95.Bottom,Sameafterrandomization
ofChmp2bstainingpattern.Thecorrelationcoefficient(r)betweenactualChmp2bandPSD-95stainingintensitiesissignificantlyhigherthanthemeanrvaluegeneratedbyrandomizedChmp2b
patterns(p(cid:6)0.0001,ztest).D,SpecificityofChmp2bimmunofluorescence.HippocampalneuronsweretransfectedwithpSuper-mCherryexpressingChmp2b-specificshRNA,stainedby
immunofluorescencewithanti-Chmp2b,andimagedbyconfocalmicroscopy.Left,Representativetransfectedneuron,visualizedbymCherry.Middle,Chmp2bimmunostainingofthesamefield.
Arrowheadsindicatethepositionofthetransfectedcelldendritesandsoma.N,Neighboring,nontransfectedneuron.Right,Immunostaining(redhotpseudocolor)ismostlyabsentfromthe
transfectedneuron(outlinedinwhite),thoughclearlydetectableinthenontransfectedcell(N).Scalebar,10(cid:2)m.
synaptoneurosomeextractsweredirectlyimmunoprecipitatedwiththe ona300(cid:2)m(cid:2)5mmPepMapC18precolumnandseparatedona75
sameantibodies.Thethreepairsofimmunoprecipitatesweredigested, (cid:2)m(cid:2)250mmC18column(PepMap;Dionex).Thenano-LCmethod
and the resulting peptides were analyzed by liquid chromatography- consistedofa120mingradientataflowrateof300nl/min,rangingfrom
tandemmassspectrometry(LC-MS/MS)asdescribedfurtherbelow.In 5to37%acetronitrilein0.1%formicacidfor114minbeforereaching
eachofthethreeexperiments,peptidesderivedfromcontrolandanti- 72%forthelast6min.MSandMS/MSdatawereacquiredusingXcalibur
Chmp2bimmunoprecipitateswereanalyzedinconsecutiveLC-MS/MS (ThermoFisherScientific).Sprayvoltageandheatedcapillarywere,re-
runsperformedonthesameday. spectively,setat1.4kVand200°C.Surveyfull-scanMSspectra(m/z(cid:5)
Forproteindigestion,proteinsinLaemmlibufferwerestacked(2mm) 400–1600)wereacquiredintheOrbitrapwitharesolutionof60,000after
on SDS-PAGE gels (4–12% NuPAGE gels; Invitrogen) before being accumulationof106ions(maximumfillingtime,500ms).The20most
stainedwithCoomassieblueR-250(Bio-Rad).Thegelbandwasmanu- intenseionsfromthepreviewsurveyscandeliveredbytheOrbitrapwere
allyexcisedandcutinpiecesbeforebeingwashedbysixsuccessiveincu- fragmentedbycollision-induceddissociation(collisionenergy,35%)in
bations of 15 min in 25 mM NH4HCO3 and in 25 mM NH4HCO3 theLTQafteraccumulationof104ions(maximumfillingtime,100ms).
containing50%(v/v)acetonitrile.Gelpieceswerethendehydratedwith Forbioinformaticanalyses,datawereprocessedautomaticallyusing
100%acetonitrileandincubatedfor45minat53°Cwith10mMDTTin MascotDaemonsoftware(version2.3.2;MatrixScience).Concomitant
25mMNH4HCO3andfor35mininthedarkwith55mMiodoacetamide searchesagainsttheUniprotdatabase(August2012version,Rattustax-
in25mMNH4HCO3.Alkylationwasstoppedbyadding10mMDTTin25 onomy,42,469sequences),theclassicalcontaminantsdatabase(67se-
mM NH4HCO3 and mixing for 10 min. Gel pieces were then washed quences,homemade),andthecorrespondingreverseddatabaseswere
againbyincubationin25mMNH4HCO3beforedehydrationwith100% performedusingMascot(version2.4).ESI-TRAPwaschosenasthein-
acetonitrile.Atotalof0.15(cid:2)gofmodifiedtrypsin(sequencinggrade; strumentandtrypsin/Pastheenzyme,andtwomissedcleavagewere
Promega)in25mMNH4HCO3wasaddedtothedehydratedgelpieces allowed.Precursorandfragmentmasserrortoleranceswereset,respec-
foranovernightincubationat37°C.Peptideswerethenextractedfrom tively,at10ppmand0.6Da.Peptidemodificationsallowedduringthe
gelpiecesinthree15minsequentialextractionstepsin30(cid:2)lof50% searchwerecarbamidomethyl(C,fixes)acetyl(N-ter,variable),oxida-
acetonitrile;30(cid:2)lof5%formicacid;and,finally,30(cid:2)lof100%aceto- tion(M,variable),anddeamidation(NQ,variable).TheIRMasoftware
nitrile.Thepooledsupernatantswerethendriedundervacuum. (version 1.30.4) was used to filter the results: conservation of rank 1
Fornano-LC-MS/MSanalyses,thedriedextractedpeptideswerere- peptides,peptideidentificationFalseDiscoveryRate(cid:6)1%(ascalculated
suspendedin5%acetonitrileand0.1%trifluoroaceticacidandanalyzed byusingthereversedatabasestrategy),andminimumofonespecific
by on-line nano-LC-MS/MS [Ultimate 3000 (Dionex) and LTQ- peptideperidentifiedproteingroup.Thefilteredresultswereuploaded
OrbitrapVelospro(ThermoFisherScientific)].Peptidesweresampled intoarelationalmassspectrometryidentificationdatabase(MSIdb),and
Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses J.Neurosci.,February18,2015•35(7):3155–3173•3161
Figure3. Chmp2bregulatesdendriticmorphologyinanESCRT-III-dependentmanner.A,Hippocampalneuronswerecotransfectedat10DIVwiththeindicatedmixturesofpSuper-basedand
rescue(pBI-based)plasmids.pSupervectorsexpressedmCherrytogetherwiththeindicatedshRNA[GFPshRNA(shGFP);Chmp2bshRNA(shChmp2borsh2b)].RescueplasmidsexpressedZsGreen,
aloneortogetherwiththeindicatedversionofRNAi-resistantChmp2b(2B*).Imagesofdoublytransfectedneuronsat14DIVwereacquiredbyconfocalmicroscopy.Depictedisthemorphologyof
representativeneuronsineachcondition,asvisualizedbymCherryfluorescence(negativecontrast,maximumintensityprojections).B,Shollanalysisofdendriticarborizationinneuronstransfected
asinA.Foreachplasmidmix,thegraphshowsmeannumbersofdendriticbranchesasafunctionofradialdistance(n(cid:5)40neuronspercondition).Chmp2bknockdownstronglyreducedthe
distance-dependentincreaseinbranchnumber(***p(cid:5)0.00014comparedwithcontrolcurve,Kolmogorov–SmirnovtestwithBonferroni’scorrection).Expressionofwild-typeChmp2b*restored
thecontrolphenotype(p(cid:5)1).Chmp4b-binding-defective(Chmp2b*R19/22/26A)andmembrane-binding-defective(Chmp2b*L4D/F5D)mutantsofChmp2bwereunabletofullyrestorethecontrol
phenotype(**p(cid:5)0.004andp(cid:5)0.006,respectively).C,Hippocampalneuronsweretriplycotransfectedat10DIVwithHA-taggedChmp2bWT,Flag-taggedChmp4b,andmCherryplasmids(1(cid:2)g
ofeachplasmid).Thecellswerefixed24hlaterandstainedbydualimmunofluorescencewithanti-HAandanti-Flagantibodies.Confocalmicroscopyimages(z-projections)ofdendriticfluorescence
areshownforarepresentativeneuron.Chmp2bwasrecruitedtoChmp4bpuncta,someofwhichwerefoundatdendriticbranchpoints(arrowheads)oratthe(Figurelegendcontinues.)
3162•J.Neurosci.,February18,2015•35(7):3155–3173 Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses
ahomemadetool(hEIDI;Milbradtetal.,2014)wasusedforthecompi- tolight.mCherryfluorescencewasexcitedwith75–150(cid:2)Wnominal
lation,grouping,andcomparisonoftheproteingroupsfromthediffer- laser power at 561 nm. The pinhole diameter was set to 1 Airy unit.
entsamples.hEIDIprovidesmodulestohelpexploringvalidatedsearch Z-stacksof20–30opticalsections(voxelsize,0.11(cid:2)0.11(cid:2)0.2(cid:2)m3)
resultsthathavebeencompiledintoaMSIdb.Theusercreatescontexts, perdendriticsegmentwereacquiredat15minintervals.Signalperpixel
whichrepresentsetsofidentifications,andrunspeptidesandproteins wasaveragedovertwoscans.Afteracquiringbaselineimages,anextra-
groupingalgorithmstoclusterpeptidesidentifyingafamilyofproteinsin cellularsolutioncontaining200(cid:2)Mglycine(warmedto37°Cbyin-line
thesecontexts.Thegroupingalgorithmwillfirstidentifyidenticalpep- heating)wasdelivered.Stimulationwasstoppedafter4minbyperfusing
tidesbasedontheirmassandsequence.Groupsofproteinsmatchingthe awarmglycine-freeextracellularsolution.Onlymodestbleachingwas
samesetofpeptidesarethencreatedasisdoneintheIRMatoolboxfora observedduringtheexperiment.Cellsshowingsignsofdamage(pearling
single identification. This is typically useful to consider results at the orblebbing)werediscarded.
samplelevelinsteadofthereplicatelevel.Tocompareseveralcontexts, Tomeasurechangesinspinevolume,imagestacksweresubjected
hEIDIalignseachproteingroupfromeachcontexttobecompared.This to blind (adaptive) deconvolution with AutoQuant as above. The
alignmentisbasedonproteinandpeptidesetsimilaritymetrics. deconvolvedimages(scaledto8bit)wereusedasinputfor3Dreconstruc-
TobeconsideredasspecificallyassociatedwiththeChmp2bimmu- tion of dendritic morphology by the software package NeuronStudio
noprecipitate,theproteinsidentifiedbythisanalysiswerefilteredac- (http://www.mssm.edu/cnic/tools.html),whichallocatesvoxelsto spines
cordingtothefollowinginclusioncriteria:twoormoreuniquepeptides throughaspecificalgorithm(Rodriguezetal.,2006,2008).Individual
(specificspectralcounts);twoormoredistinctproteinfragments[spec- spineslabeledbythesoftwareandthatwereclearlyidentifiableatboth
tralcounts(SC)];andratioofSCinspecifictocontrolimmunoprecipi- initialandfinaltimepointsweremanuallyselected.Thevolumeofthe
tate(SC2B/SCcontrol)(cid:8)5. spinesbeforeandafterstimulationwasestimatedbycountingspinevox-
Electrophysiologicalrecordingsinculturedneurons.Mature[18–21din els.Thefractionalincreaseinspinevolumewascalculatedforeachspine
vitro(DIV)]hippocampalneuronsexpressingthetransfectionmarker asfollows:
ZsGreenwereusedforwhole-cellpatch-clamprecordingatroomtem-
perature(20–24°C).Theneuronswerecontinuouslyperfusedwithan (cid:9)V V (cid:4)V
1ex0trmacMelHluElaPrEsSo,lu1t0iomnMcognlutacionsien,g01.540(cid:2)MmMtetNroadCol,to2xminM,5C0a(cid:2)CMl2,b3icmucMuKlliCnle, V (cid:3) fVi i,
methiodide,and1(cid:2)Mstrychnine,adjustedtopH7.4withNaOHandto withV thevolumeat5minbeforethestartofthestimulationandV the
330mOsmwithNa-acetate.ToinduceLTP,glycine(200(cid:2)Minextracel- volumei at45minaftertheendofthestimulation.Themedianvaluefsof
lularsolution)wasperfusedfor3minontotheneurons(Luetal.,2001). (cid:9)V/Vwerecalculatedforeachneuron.Differencesbetweenculturesdid
For the experiments in which NMDA current amplitudes were mea- notsignificantlycontributetotheobservedeffects(two-wayANOVA,
sured,NMDA(100(cid:2)M)wasaddedtothissolutionandperfusedwitha p(cid:5)0.015forfactorplasmid,p(cid:5)0.93forfactorculture,p(cid:5)0.56for
fast-exchange perfusion system. Recording pipettes were pulled from interaction). Therefore, the results from seven independent cultures
borosilicate glass and had a resistance of 3–5 (cid:7)M when filled with a werepooled.
solution containing (in mM) 115 CsMeSO3, 20 CsCl, 10 HEPES, 0.6 Statisticalanalysis.Thestatisticalsignificanceofdifferencesbetween
EGTA,4Na2-ATP,0.4Na-GTP,10Na-phosphocreatine,and2.5MgCl2, samplemeansorsampledistributionswascalculatedasdescribedinthe
adjustedtopH7.2withCsOHandto300mOsmwithCs-acetate.Cur- figurelegends.Thenormalityofsampleswassystematicallycheckedby
rentswererecordedfromaholdingpotentialof(cid:8)60mVusinganAxon theShapiro-Wilkstest,andnonparametrictestswereusedwhereappro-
200Bamplifier(MolecularDevices).Cellcapacitanceandseriesresis- priate.StatisticaltestswereperformedwithR(RCoreTeam,2013).Error
tancecompensationwereappliedelectronically.Signalswerefilteredat1 barsindicateSEMinallfigures.
kHz,digitizedat5kHz,andrecordedusingthepClampsoftware.Min-
iatureEPSCs(mEPSCs)andNMDA-evokedcurrentswereanalyzedus-
Results
ingClampFit(MolecularDevices).
ApoolofChmp2blocalizesindendriticshaftsandspines
Liveimaging.Imageacquisitionandanalysiswereperformedblindto
transfected plasmids. For live imaging during chemical LTP, neurons ToaddressthephysiologicalfunctionofChmp2binneurons,we
weretransferredtoaperfusionchamber(Pocon),washed,andkeptinan firstevaluatedtherelativelevelofChmp2bproteininthebrain
extracellularsolution(125mMNaCl,2.5mMKCl,2mMCaCl2,1mM comparedwiththatinotherorgans.Mousetissueextractswere
MgCl2,5mMHEPES,33mMglucose,50(cid:2)Mbicuculline,and0.5(cid:2)MTTX, analyzedbyWesternimmunoblottingwithananti-Chmp2ban-
1(cid:2)Mstrychnine,pH7.4)at37°Conaheatedstage.Neuronsthatsimul- tibody.Thesameblotswerealsoprobedwithanantibodyraised
taneouslyexpressedGFPandmCherry(withinthesamefluorescence againstthecoreESCRT-IIIsubunitChmp4b.Forbothproteins,
rangethroughoutexperiments)andhadahealthyappearancewereiden-
expression was strikingly higher in the brain than in the other
tifiedbyepifluorescence,anddendriticsegmentswereselectedsothat
organs, except for testes (Fig. 1A). Analysis of mouse brain at
30–100spinescouldbeimagedperneuronwhileminimizingexposure
variousstagesofdevelopmentindicatedthatChmp2bexpression
increasedduringthefirstpostnatalweek(Fig.1B).BothChmp2b
4 andChmp4bweredetectedinextractsfromvariousregionsof
(Figurelegendcontinued.) baseofspine-lookingprotrusions(asterisk).Scalebar,10(cid:2)m.D, theadultbrain,withthehighestChmp2blevelsfoundinolfac-
N2AcellsweretransfectedwiththeindicatedcombinationsofRNAi-inducingplasmidsand torybulbandcerebralcortex(Fig.1C).
rescueplasmids.Thecellswerelysedandanalyzedbyimmunoblottingwithanti-Chmp2ban- To define the topographical pattern of Chmp2b protein
tibody.E,HeLacellswerecotransfectedwithHA-taggedChmp2bWTorChmp2bR19/22/26Ato- expression, we performed immunohistochemistry on mouse
getherwithChmp4b-Flag.Cellswerestainedbydualimmunofluorescencewithanti-HAand brainsections(Fig.1D).Chmp2bimmunoreactivitywaspar-
anti-Flagantibodiesandimagedbyconfocalmicroscopy.Insets,Highmagnificationofthe ticularlystronginhabenula,olfactorybulb,deeplayersofthe
boxedregions.Scalebars,20(cid:2)m.ColocalizationofChmp2bandChmp4bwas80%for neocortex, amygdala, and hippocampus. This pattern gener-
Chmp2bWTand12%forChmp2bR19/22/26A(quantifiedasthepercentageofChmp2b-HA-
ally coincided with in situ hybridization data from the Allen
positivepixelsoverlappingChmp4b-Flagpuncta).F,HEK-293TcellsweretransfectedwithFlag-
Brain Atlas (http://mouse.brain-map.org/gene/show/44784).
taggedChmp4bplasmid,aloneortogetherwithHA-taggedChmp2bWTorChmp2bR19/22/26A.
In higher-magnificationviews,immunostainingwasevidentin
Anti-HAimmunoprecipitatespreparedfromthetransfectedcellswereanalyzedbyimmuno-
thesomatodendriticcompartmentofnumerousneurons(e.g.,in
blottingwithanti-Flagoranti-HAantibodies.G,HEK-293TcellsweretransfectedwithFlag-
taggedChmp2bWTorChmp2bL4D/F5D.Thetransfectedcellswerehomogenized,themembrane theCA1regionofthehippocampus;Fig.1E).Specificityoflabel-
(P)andcytosolic(S)fractionswereseparatedbycentrifugation,andequivalentamountsofeach ingwasconfirmedbyshowingreducedstaininginthebrainof
fractionwereanalyzedbyimmunoblottingwithanti-Flagantibody. Chmp2b-deficient(KD)mice(Fig.1F).
Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses J.Neurosci.,February18,2015•35(7):3155–3173•3163
Figure4. Chmp2bisinvolvedinthemaintenanceofexcitatorysynapsesinculturedneurons.A,Hippocampalneuronsweretransfectedat16DIVwithpSuper-mCherryencodingcontrol(GFP)
shRNA.Theneuronswerefixedat18DIV,stainedbydualimmunofluorescencewithanti-PSD-95andanti-synapsinIantibodies,anddendriteswereimagedbyconfocalmicroscopy.Postsynaptic
spines(arrowheads)wereidentifiedasdendriticprotrusions(top)containingPSD-95puncta(middle).NearlyallofthespinescolocalizedwiththepresynapticmarkerSynapsin(bottom).mCherry
fluorescencewasusedasamasktofacilitatevisualizationofpunctabelongingtothetransfectedneuron.Scalebar,15(cid:2)m.B,Neuronsweretransfectedat16DIVwiththesameplasmid
combinationsasinFigure3A.Imagesofdendriticmorphologywereacquired,andpostsynapticspineswereidentifiedasinAandcounted.Arepresentativefieldisshownforeachcondition.Scale
bar,5(cid:2)m.C,MeandensityofpostsynapticspinesalongthedendritesofneuronstransfectedasinB.Comparedwiththecontrol(GFP)shRNA,theChmp2b-depletingshRNAinducedalargedrop
inspinedensity(***p(cid:5)2.4(cid:2)10(cid:8)11,two-sidedWilcoxontestwithHolmcorrection).Coexpressingwild-typeChmp2b*completelyrestoreddensity(p(cid:5)1vscontrol,***p(cid:5)6.3(cid:2)10(cid:8)14vs
Chmp2bshRNAalone).CoexpressingtheChmp4bbinding-defectivemutantChmp2b*R19/22/26Adidnotrescuespinedensityatall(***p(cid:5)3.8(cid:2)10(cid:8)6vsChmp2b*WT,p(cid:5)0.14vsChmp2bshRNA
alone);neitherdidthemembranebinding-defectivemutantChmp2b*L4D/F5D(***p(cid:5)2.9(cid:2)10(cid:8)7vsChmp2bWT,p(cid:5)1vsChmp2bshRNAalone).Neuronspercondition:control,n(cid:5)34;Chmp2b
shRNA,n(cid:5)33;Chmp2b*WT,n(cid:5)30;Chmp2b*R19/22/26A,n(cid:5)16;Chmp2b*L4D/F5D,n(cid:5)14.D–F,Hippocampalneuronsweretransfectedat18DIVwiththeindicatedplasmids.Transfected
neuronswereidentifiedbyfluorescenceat21DIV,andtheirminiatureEPSCsweremeasuredbypatch-clamprecordingsinwhole-cellconfiguration.D,Sampletracesfromneuronstransfectedas
indicated.E,HistogramofmeanmEPSCamplitudesinneuronstransfectedasindicated(n(cid:5)5cells,(cid:10)100minispercellineachcondition).TheamplitudeofmEPSCswasmarkedlyreducedin
Chmp2b-deficientneurons(**p(cid:5)0.005,two-sidedttest).F,HistogramofmeanmEPSCfrequenciesinneuronstransfectedasindicated.FrequencieswerelowerinChmp2b-deprivedneurons
(*p(cid:5)0.012,two-sidedttest).G,Neuronstransfectedasabovewerestimulatedat21DIVbybath-appliedNMDA(control,n(cid:5)6cells;Chmp2bshRNA,n(cid:5)5cells).ThedensityofNMDA-evoked
currents(calculatedascurrentnormalizedbycapacitance)wasstronglydecreasedinChmp2b-deprivedneurons(*p(cid:5)0.031,two-sidedttest).
3164•J.Neurosci.,February18,2015•35(7):3155–3173 Chassefeyre,Martínez-Herna´ndezetal.•ESCRT-IIIFunctionatSynapses
Figure5. Chmp2bisinvolvedinthemaintenanceofexcitatorysynapsesinvivo.A,Brainsectionsfrom5-week-oldChmp2bknockdownmice(KD)orsiblingcontrols(Wildtype)werestainedby
theGolgiimpregnationtechnique.Staineddendriticsegmentswerevisualizedbybright-fieldmicroscopyofthestratumradiatum.Shownarerepresentativeimagesofdendriticshaftsandspines
inbothgenotypes.B,Spineswereclassifiedintothin,stubby,ormushroomtypes,andtheirdensityperunitdendriticlengthwasmeasuredforeachtype(n(cid:5)4animals,36dendritespergenotype).
Left,HistogramshowingthemeandensityofeachspinetypeinKDandWTmice.ThetotalspinedensitywassignificantlylowerinKDmicethanincontrols(*p(cid:5)0.039,two-sidedttest).Right,
Histogramshowingthemeanproportionofthethreespinetypes.ThepercentageofmushroomspineswassignificantlyreducedintheKDmice(*p(cid:5)0.02).C,Representativeelectronmicrographs
oftheCA1stratumradiatumfromWtandKDsiblings.Presynapticprofilesarehighlightedinblue,andpostsynapticspines(s)areinbrown.D,Quantificationofsynapsedensityintheelectron
micrographs.WT:n(cid:5)3animals,851axo-spinoussynapsesfrom30micrographs;KD:n(cid:5)3animals,627axo-spinoussynapsesfrom30micrographs.Thedensityofpostsynapticspinesperunit
surfacewassignificantlylowerintheKD(***p(cid:5)0.0008).
Chmp2bwasalsoreadilydetectableinprimaryculturesofrat ogy. To assess the effect of Chmp2b depletion on dendrite
neurons. Consistent with the developmental profile shown in branching,neuronsweretransfectedat10DIVandimagedat14
Figure 1B, immunoblotting analysis of cultured hippocampal DIV by confocal microscopy. As shown in Figure 3, A and B,
(Belly et al., 2010) or cortical (Fig. 2A) neurons showed that dendriticbranchingwasreducedinChmp2b-depletedneurons
endogenousChmp2bexpressionsteadilyincreasedduringneuronal comparedwithneuronstransfectedwithcontrolshRNA.Coex-
maturation,reachingaplateauafter9DIV,thusoverlappingthe pression of an RNAi-resistant version of the Chmp2b mRNA
periodofsynapseformation.Asalreadyobservedinnon-neuronal togetherwiththeshRNAalmostfullyrescueddendriticcomplex-
cells,immunofluorescentstainingofmaturehippocampalneurons ity.ThesedataindicatethatChmp2bisrequiredformaintaining
revealed Chmp2b immunoreactivity throughout the entire cyto- theintegrityofdendritictrees.
plasm.However,focusingondendrites, we noted that Chmp2b Innon-neuronalsettings,Chmp2proteinsareknowntoin-
staining formed numerous small clusters, many of which were teractwithChmp4forformationofbiologicallyactiveESCRT-III
found at the base, neck, and head of dendritic spines. Within (Morita et al., 2011). Consistent with such an interaction, we
spine heads, Chmp2b clusters were often juxtaposed to PSDs observedthatwhencoexpressedinneuronsat10DIV,epitope-
labeled with anti-PSD-95 antibody (Fig. 2B). The overlap of taggedChmp2bandChmp4bcolocalizedintopunctadistributed
Chmp2b with PSD-95 puncta was significantly more frequent along the dendrites (Fig. 3C). Chmp2b/Chmp4b puncta were
thanexpectedbyrandomsuperposition(Fig.2C).Thespecificity repeatedly found at dendritic branch points or at the base of
of synaptic staining with the anti-Chmp2b antibody was con- dendritic protrusions. We then tested whether the effect of
firmedbyextinctionwithawellvalidatedshRNAconstruct(Belly Chmp2bondendritebranchingdependedonitsabilitytobindto
etal.,2010;Fig.2D). endogenous Chmp4b. For this, we designed a Chmp2b point
mutant deficient for binding to Chmp4b. Morita et al. (2011)
Chmp2bregulatesthestabilityofdendriticbranches showedthatbindingofChmp4btothecloseChmp2bhomolog
Aftertheseobservations,weusedculturedhippocampalneurons Chmp2a depends on three arginine residues in the Chmp2a N
toexaminewhetherChmp2bplayedaroleinthemaintenanceof terminus. Based on sequence alignment with Chmp2a, we re-
dendritesandsynapses.Forthis,neuronsweretransfectedwith placedarginineresidues19,22,and26ofChmp2bbyalanine.We
thesameChmp2b-depletingshRNAasabove.Controltransfec- demonstratedthat,unlikeChmp2bWT,Chmp2bR19/22/26Afailed
tionswereperformedwithaGFP-specificshRNAwithnoeffect tocolocalizewithChmp4binthecellcytoplasmandtocoimmu-
on neurons (Alvarez et al., 2006). The shRNA vectors also ex- noprecipitate with Chmp4b in cell lysates (Fig. 3D–F). Thus,
pressedmCherry,allowingvisualizationofthecellularmorphol- Chmp2bR19/22/26AisunabletointeractwithChmp4b.