The Journal of Cell Biology
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Florence Niedergang, in macrophages membrane delivery duringphagocytosis ADP ribosylationfactor6isactivated andcontrols a sharpandtransient activation inmacrophageswhen phago- tion factor6(ARF6),asmallGTP-bindingprotein,undergoes phagocytosis. Here,wereportthatendogenous ADP ribosyla- to play animportantroleinpseudopodextensionduring some. Membrane delivery frominternalpoolsisconsidered extension ofmembrane protrusionstoformaclosedphago- to takeuprelativelylargeparticles( Phagocytosis isthemechanismofinternalizationusedbycells cytosis was initiatedviareceptorsfortheFcportionof 1 that actinpolymerizationprovidesthedrivingforceallows pseudopods thatengulftheparticle.Itisgenerallyconsidered kinases,andeventuallyleadstotheformationof rosine FcRsignalinginvolvesty- Greenberg andGrinstein,2002). for receptorstheFcportionofimmunoglobulins(FcRs;* is followedbyacascadeofactivationthatbestdescribed present onthephagocytesurface.Theclusteringofreceptors exposed ontheparticlesurfacebyspecificreceptorsorlectins 1999). Phagocytosisistriggeredbytherecognitionofligands cell debris,anddiverseparticulates(AderemUnderhill, dritic cells,useittointernalizeanddegrademicroorganisms, macrophages,polymorphonucleargranulocytes,andden- like cellular compartmentorphagosome.Professionalphagocytes, Introduction Key words: VAMP3/cellubrevin; ARF6;recycling; endocytosis;actin membrane protein;WT,wildtype. SRBC, sheepredbloodcell;Tf,transferrin; VAMP,vesicle-associated ear–containing ARF-bindingprotein; PI3K,phosphatidylinositol3-kinase; receptor fortheFcportionofimmunoglobulins; GGA,Golgi-localized *Abbreviations usedinthispaper: ARF, ADPribosylationfactor;FcR, 33-1-42-34-63-77. E-mail:[email protected] d’Ulm, F-75248Pariscedex05,France.Tel.:33-1-42-34-63-67.Fax: eton DynamicsLaboratory,UMR144CNRS,InstitutCurie,26rue Address correspondenceto http://www.jcb.org/cgi/doi/10.1083/jcb.200210069 The Journal ofCellBiology
Membrane andCytoskeleton Dynamics Group and Scientifique, Institut Curie, F-75248Paris Cedex05,France
TheRockefeller University Press, 0021-9525
Article surface, which leads toactinpolymerizationandthe their interaction withspecific receptorsonthecell ngulfment ofparticlesby phagocytes isinducedby
, Volume 161, Number 6,June 23, 20031143–1150 Florence Niedergang,MembraneandCyto 1 Emma Colucci-Guyon, /2003/06/1143/8 $8.00 0.5 m) intoanintra- 2 1 Electron Microscopy Group, UMR144Centre National delaRecherche Thierry Dubois, Thierry skel- in phagocytosisbecauseboth aconstitutivelyactiveand et al., 1996).Inaddition,ARF6wasshowntoplayarole actin-rich membraneprotrusions andruffles(Radhakrishna 2002). ARF6hasalsobeenimplicated intheformationof 1998; RadhakrishnaandDonaldson, 1997;Vitaleetal., cytosis of receptor-mediated endocytosis,endosomalrecycling,andexo- binding proteins.Itisinvolvedinmembranetraffickingduring 6 (ARF6)belongstotheARFfamilyofRas-relatedGTP- delivery islargelyunknown. merization, theactivationcascadeleadingtofocalmembrane However, incontrasttoextensiveknowledgeaboutactinpoly- be deliveredatthesiteofphagocytosis(Bajnoetal.,2000). membrane protein3(VAMP3)markerhavebeenshownto particular, endocyticvesiclesbearingthevesicle-associated nism forsurfaceloss(Boothetal.,2001;Aderem,2002).In compartments hasbeenproposedasacompensatorymecha- Nelson, 1998).Exocytosisofmembranesfrominternal during phagocytosis(Hackametal.,1998;Holevinskyand macrophages, yet,themacrophagesurfaceratherincreases gosomes shouldresultinadecreasethenetsurfaceof Machesky, 2001). engulfing pseudopods(Castellanoetal.,2001;Mayand the plasmamembranetobelocallyelongatedform membrane recycling duringphagocytosis. microscopy. We concludethat ARF6 isamajorregulatorof lation ofintracellular vesicles, asobserved by electron in anearlyblockade inpseudopodextensionandaccumu- whereas actinpolymerizationwas unimpaired. This resulted 3 in thefocaldelivery ofvesicle-associated membrane protein phagocytosis. Expressionof ARF6-T27N leadtoareduction ARF6 (T27Nmutation)dramatically affectedFcR-mediated immu The smallGTP-bindingproteinADPribosylationfactor Simultaneous internalizationofmultipleparticlesinpha- endosomalrecycling membranes atphagocytosis sites, noglobulins (FcRs). A dominant-negative mutantof 1 Graça Raposo, secretory granules(D’Souza-Schoreyetal.,1995, 2 andPhilippe Chavrier 1 1143 The Journal of Cell Biology 1144 Results take inRAW264.7 cells.TheDIII vents clathrincoatassembly, inhibitedtransferrin(Tf)up- 2000), expressionoftheDIII domainofEps15,whichpre- 2000). Aspreviouslyshownin fibroblasts(Benmerahetal., and istargetedtoclathrin-coated pits(Benmerahetal., docytosis withamutantofEps15, aproteinthatbindsAP2 To testthis,wespecifically blocked clathrin-mediateden- an inhibitionofendocytosisinthetransfectedmacrophages. sis bydominant-negativeARF6couldbetheconsequenceof al., 1999;Palacioset2001),theinhibitionofphagocyto- some celltypes(D’Souza-Schoreyetal.,1995;Altschuler et Because ARF6controlsclathrin-mediatedendocytosisin impair theefficiencyofphagocytosis Inhibition ofreceptor-mediatedendocytosisdoesnot red bloodcells(IgG-SRBCs)for60minat37 mutant) ARF6wereincubatedwithIgG-opsonizedsheep expressing wild-type(WT)anddominant-negative(T27N Murine macrophagesfromtheRAW264.7linetransiently FcR-mediated phagocytosis ARF6 istransientlyactivatedduring from recyclingcompartmentstotheformingphagosome. rophages, andplaysacriticalroleinthedeliveryofmembrane siently duringFcR-mediatedphagocytosisinmurinemac- endogenous ARF6becomesactivatedveryearlyandtran- in phagocytosisremainslargelyunknown.Here,weshowthat (Zhang etal.,1998).However,theprecisefunctionofARF6 dominant-negative formoftheproteininhibitedphagocytosis FcR-mediated phagocytosisinRAW264.7cells. Therefore, RacisactivatedbeforeARF6andCdc42during contact ofmacrophageswithparticlesandthendecreased. served forARF6,theactivationofRacpeaked1minafter kinetics ofactivationCdc42wascomparabletothatob- monitored underthesameconditions(Fig.1D).While crucial forFcR-mediatedphagocytosis,theiractivationwas amount ofproteins(Fig.1C).BecauseRacandCdc42are after precipitationwerenotduetovariationsinthetotal quots ofthetotallysatesindicatedthatvariationsobserved when GSTalonewasaddedinthepull-downassay,andali- min (Fig.1C).NoprecipitationofARF6waseverdetected 10 min,anddecreasinglatertoreturnbasallevelafter30 rophages attimesasshort1min,reachingamaximum The activationofARF6wasobservedinRAW264.7mac- ARF-binding protein3(GGA3;SantyandCasanova,2001). the ARF-bindingdomainofGolgi-localized detected byaGSTpull-downassaybasedonfusedto dogenous ARF6duringphagocytosis.GTP-loadedwas process. Wehavenowmonitoredtheactivationlevelofen- and pointedtoanessentialroleofARF6inthephagocytic fect (Fig.1B). dent phagocytosis,whereasARF6-WThadvirtuallynoef- ARF6-T27N stronglydecreasedtheefficiencyofFcR-depen- not affectedbyeitherconstruct.Incontrast,expressionof the associationofopsonizedparticlestomacrophageswas ficiency ofphagocytosiswasassessed.AsshowninFig.1A, These resultsconfirmedpreviousdata(Zhangetal.,1998) The JournalofCellBiology
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Volume 161,Number6,2003 2 constructthat does ear–containing C, andtheef- endocytosis isnotsufficienttoinhibitphagocytosis. results clearlydemonstratethatblockingreceptor-mediated fected phagocytosisofIgG-SRBCs(Fig.2,BandC).These did notinhibitendocytosis(Fig.2A).Neitherconstructaf- not containtheAP2-bindingsitesandwasusedasacontrol been involved in membranerecyclingfrom theendocytic endosomal vesicles(D’Souza-Schorey etal.,1998)andhas Because ARF6hasbeenshown tocolocalizewithVAMP3in exocytosed atthesiteofphagocytosis (Bajnoetal.,2000). Endocytic vesiclespositivefor VAMP3arerecruitedand at thesiteofphagocytosis recruitment (butnotactin polymerization) Dominant-negative ARF6inhibitsmembrane GST-GGA3 incubated withIgG-SRBCfor60minat37 macrophages transientlyexpressingARF6-WTorARF6-T27Nwere Figure 1. (bottom). Dataarerepresentativeoffourexperiments. was performedwithanti-Racantibodies(top),thenanti-Cdc42 lysates werepreparedandincubatedwithGST-CRIB.Westernblot RAW264.7 macrophageswereactivatedasdescribedinC,and (D) KineticsofactivationRacandCdc42duringphagocytosis. anti-ARF6 antibodies.Dataarerepresentativeofthreeexperiments. shows aliquotsoftotallysates.Westernblotwasperformedwith various timesat37 incubated withmedium(control)for10minorIgG-SRBC activated duringphagocytosis.RAW264.7macrophageswere experiments isplotted.NT,nontransfected.(C)ARF6transiently Materials andmethods.Themean association (A)orphagocytosis(B)wascalculatedasindicatedin constructs weredetectedbyimmunofluorescence.Theefficiencyof antibodies. Afterpermeabilization,theexpressedHA-taggedARF6 and externalSRBCswerestainedwithCy3-coupledanti–rabbitIgG ARF6isactivatedduringphagocytosis. 1–226 (top)orGSTalone(middle).Thebottompanel C. Lysateswerepreparedandincubatedwith SEMofthreeindependent C. Thecellswerefixed, RAW264.7 The Journal of Cell Biology phatidylinositol 3-kinase(PI3K)wasshown toblockphago- SRBCs (Fig.3F,arrowhead; Fig.3I).Inhibitionofphos- strong reductionintherecruitment ofGFP-VAMP3around pared withcontrols,whereas onlyARF6-T27Nleadtoa resulted inasmallreduction of F-actinaccumulationcom- (Fig. 3I).Theexpressionof ARF6-WTandARF6-T27N and G)werescored10minafter theadditionofIgG-SRBCs (Fig. 3,BandF)polymerizedactinfilaments C RAW264.7 macrophages.AccumulationsofGFP-VAMP3 recruitment ofVAMP3atthesitephagocytosisin effect oftheexpressionARF6-WTorARF6-T27Non scent phagosomes.Totestthishypothesis,weanalyzedthe plicated intherecyclinganddeliveryofmembranestona- and Donaldson,1997),wereasonedthatARF6maybeim- compartment (D’Souza-Schoreyetal.,1995;Radhakrishna Eps15DIII (bottom)wereincubatedfor30minat37 GFP-Eps15DIII mediated phagocytosis. Figure 2. three independentexperiments. Materials andmethods.Datapresentedarethemean of nontransfected(NT)controlcells,asdescribedin Fig. 1and GFP-Eps15DIII werecomparedwiththeefficiencyofphagocytosis phagocytic (C)efficiencyofcellsexpressingGFP-Eps15DIII endocytosis doesnotblockphagocytosis.Association(B)and cells. Bar,10 right, Texasred-Tf.TheasterisksindicateGFP-positivetransfected by confocalmicroscopy.Amedialopticalsectionisshown.Left,GFP; containing 60nMTexasred-Tf.Cellswerethenfixedandanalyzed An Eps15mutantinhibitsTfendocytosisbutnotFcR- m. (BandC)Inhibitionofreceptor-mediated 2 (top)orthedominant-negativeconstructGFP- RAW264.7cellstransientlyexpressing
C inmedium SEMof 2 or ARF6 controlsmembranerecyclingduringphagocytosis| ARF6 duringphagocytosis. rather thanactinpolymerization,isunderthecontrolof I). Together,theseresultsindicatethatmembranedelivery, the resultsobtainedbyscoringundermicroscope(Fig.3 any ofthetimepointsanalyzed(Fig.3K),whichconfirms nant-negative ARF6resultedinasmalldecreaseF-actinat al., 2002).ComparedwithARF6-WT,expressionofdomi- 2.5–10 min,anddecreased(unpublisheddata;Coppolinoet (by 20–50%,dependingontheexperiments),peakedat amount ofF-actinincreasedoncontactwiththeparticles time pointsduringphagocytosis.Asreportedearlier,the in ARF6-WT–orARF6-T27N–expressingcellsatdifferent Next, wequantifiedbyflowcytometrytheamountofF-actin cytosed particle,withlittle(ifany)effectonactinassembly. an essentialroleinmembranerecruitmentaroundthephago- was increased(Fig.3J).TheseresultsshowthatARF6plays ment ofGFP-VAMP3,whereasthepresenceF-actincups cells incubatedwithwortmanninshowedareducedrecruit- hibit PI3K.ComparedwithDMSO-treatedcontrolcells, VAMP3 andF-actinincellstreatedwithwortmannintoin- data). Therefore,wealsoanalyzedaccumulationsofGFP- cytosis (Arakietal.,1996;Cox1999;unpublished they wereobservedin cells thatwereincontactwithatleastoneSRBC,whereas vesicles werepresentin compared withcellsexpressingARF6-WT(Fig.4B).Large of largeelectronlucentvesicleswasobservedinthesecellsas expressing cellsattheultrastructurallevel.Anaccumulation rophages. Then,weexaminedphagocytosingARF6-T27N– able tomodulatetheconstitutiverecyclingofTfinmac- pressing ARF6-T27N(Fig.4A).ThisshowsthatARF6is By contrast,theeffluxofTfwasclearlyreducedincellsex- 4 A),asreportedearlierinmacrophages(Coxetal.,2000). or incellsexpressingGFPalone,Tfrecyclingwasrapid(Fig. al., 1983;Bajnoet2000).IncontrolGFP-negativecells VAMP3-positive recyclingcompartment(Dautry-Varsatet tively endocytosedandrecycledwithitsreceptorviaa pressing ARF6-T27NandGFP(Fig.4A).Tfisconstitu- and pseudopodextension Dominant-negative ARF6inhibitsmembranerecycling Alexa we followedbyflowcytometrythekineticsofrecycling To characterizetheroleofARF6inmembranerecruitment, membrane ruffles, andallparticleshadbeen internalized WT–expressing cellswerespread andshowedextensive around theparticle(Fig.5,A andB).After60min,ARF6- or ARF6-T27N,andmembrane extensionswereobserved with thesurfaceofmacrophages expressingARF6-WT phagocytosis. After10min,opsonized SRBCswereassociated phages wereanalyzedbyscanning EMatdifferenttimesof affected bydominant-negativeARF6,transfectedmacro- livery ofmembranestothesitephagocytosis. pathway followedbytheTfreceptors,aswellwithde- indicate thatmutatedARF6interfereswiththerecycling membranes (Fig.4B,arrowheads).Alltogether,theseresults served inARF6-T27Ncellssometimescontainedinternal cells boundtoSRBCs.Theenlargedcompartmentsob- To getfurtherinsightintothestageofphagocytosisthatis ® 633–labeledTfinRAW264.7macrophagescoex- 20% oftheARF6-WT–expressing 80% oftheARF6-T27N–positive Niedergangetal. 1145 The Journal of Cell Biology 1146 or ARF6-T27N,wereincubatedwithIgG-SRBCat37 Flow cytometryquantificationofpolymerizedactinduringphagocytosis(K).RAW264.7cellspositiveforGFP,coexpressedwithA or DMSO-treatedcells(J).Dataarethemean cells (I)orforwortmannin-treated(J),andexpressedasapercentageoftheindexesobtainedcontrolnontransfected (open bars)andpolymerizedactin(filledwerescoredasdescribedinMaterialsmethodsforARF6-WT–ARF6-T27N–ex independent experimentsperformed. expressing cellswasthenexpressedasapercentageofthevaluesobtainedforARF6-WT–expressingcells.Dataaremean fore, theinhibition ofphagocytosisbythe dominant-nega- 1995; Altschuler etal.,1999;Palacios 2001).There- endocytosis indifferentsystems (D’Souza-Schoreyetal., low pseudopodextension. membrane fromtheendocytic recyclingcompartmenttoal- phages, andthatGTP-bound ARF6controlsthedeliveryof vated earlyduringFcR-mediatedphagocytosisinmacro- In thispaper,wehaveshownthatendogenousARF6isacti- the phagosome. ARF6-T27N, beforetheformationandcompleteclosureof phagocytosis wasblockedatanearlystageincellsexpressing head; Fig.5,EandF).Theseobservationsconfirmedthat or pedestal-likestructureswereformed(Fig.5D,arrow- the macrophagesurface.Onlysmallextensionsofmembrane pseudopods didnotextendallaroundtheparticlesboundto expressing ARF6-T27N(Fig.5,D–F).Inthesecells,the (Fig. 5C).Incontrast,SRBCswerestillextracellularoncells Discussion phalloidin. ThelevelsoffluorescencewerethenmeasuredbyFACS ARF6 wasshowntocontrolclathrin-coated pits-mediated The JournalofCellBiology
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SEMofatleastfourindependentexperimentsperformedwithtwodifferentstableclones.
C and,atdifferenttimepoints,fixed,permeabilized,andstainedwithAlexa ® cells werethenpermeabilizedandstainedwithAlexa incubated withCy5-IgG-SRBC(DandH)for10minat37 to expressARF6-WT-HA(A–D)orARF6-T27N-HA(E–H)andwere stablyexpressing GFP-VAMP3(BandF)weretransientlytransfected cells the polymerizationofactin)atsitephagocytosis. Figure 3. One sectionisshown.Bar,10 by conventionalepifluorescencemicroscopyanddeconvolution. anti–rat IgG(AandE)torevealHA-taggedARF6.Cellswereanalyzed (C andG)todetectF-actin,anti-HAfollowedbyCy3-labeled onatleast5,000positivecells.TheF-actincontentofARF6-T27N– et al.,1998).However, ourdatademonstrate thatARF6con- formation inmacrophages expressingARF6-T27N (Zhang namics duringphagocytosis,based onthedefaultinactincup scission oftheclosedvesicle). tension aroundthephagosome, actinpolymerization,or fer fromclathrin-coatedpit formation (i.e.,membraneex- and amphiphysininphagocytosis(Tseetal.,2003)thatdif- whichisinfavorofspecificroles fordynamin phagocytosis, receptor-mediated endocytosisisnotsufficienttoinhibit 2000; Perryetal.,1999).Ourdatashownowthatblocking tosis, werealsoinvolvedinphagocytosis(Goldetal.,1999, IIm, whicharekeyregulatorsofreceptor-mediatedendocy- associated proteins,aswelldynamin2andamphiphysin tosis hadbeensuggestedearlierbecauseclathrin-coatedpit- a linkbetweenphagocytosisandreceptor-mediatedendocy- tosis hadnoeffectonFcR-dependentphagocytosis.Indeed, as themostspecifictooltoblockclathrin-mediatedendocy- mutant ofEps15(Benmerahetal.,2000)thatisconsidered of endocytosis.Wecouldruleoutthispossibilitybecausea tive ARF6couldhaveresultedfromamoregeneralblockade A previousreportproposeda roleforARF6inactindy- ARF6-T27N inhibitstherecruitmentofVAMP3(butnot m. AccumulationsofGFP-VAMP3 ® (NT)cells(I) 350-phalloidin RAW264.7 SEMoffour C. The RF6-WT ® 633– pressing The Journal of Cell Biology T27N–expressing cellswhiletherewasadefault inVAMP3 was stilldetected aroundthenascentphagosomes inARF6- rather thanactinpolymerization. Indeed,polymerizedactin trols membranerecruitment atthesiteofphagocytosis, Alexa alone werescraped,pelleted,andresuspendedinthepresenceof Macrophages transientlycoexpressingARF6-T27NandGFPor Figure 4. the mean GFP(closedtriangles)weregatedandanalyzed.Theplotrepresentsfor cells expressingGFPalone(closedcircles),aswellnegative positive forGFPcoexpressedwithARF6-T27N(closedsquares), by flowcytometryasdescribedinMaterialsandmethods.Cells (sorting andanalysis).Bars,2 arrowheads). Dataarerepresentative oftwoindependentexperiments cells oftencontainedinternalmembranes(bottomrightpanel, The largeintracellularcompartmentspresentinARF6-T27N–positive ofcharacteristicenlargedcompartmentsin30macrophages.number intracellular compartments,asassessedbydirectcountingofthe areas. CellsexpressingARF6-T27Nexhibitedenlargedelectronlucent fortransmissionEM.SRBCsappearaslargeelectron-denseprocessed and incubatedwithIgG-SRBCfor20minat37 or ARF6-T27N(right)withGFPweresortedbyflowcytometry RAW264.7 macrophagestransientlycoexpressingARF6-WT(left) expressing ARF6-T27Naccumulatelargeintracellularvesicles. ® 633-Tffor45minat37 Dominant-negative ARF6inhibitsmembranerecycling. SEMofthreeindependentexperiments.(B)Macrophages C. TheeffluxofTfwasthenfollowed m. C, thenfixedand ARF6 controlsmembranerecyclingduringphagocytosis|
sitol 4-phosphate5-kinase ing viatwoeffectors,phospholipase Dandphosphatidylino- phagocytic signal.ARF6could controlmembraneremodel- ate andhowrecyclingmaybe regulatedandpolarizedbya phagocytosis. ItisstillunclearhowARF6andRab11cooper- endocytic membranestothemacrophagesurfaceduring control ofTfrecyclingandtherecruitmentTf/VAMP3 ARF6 andRab11GTP-bindingproteinsparticipateinthe and toblockphagocytosis(Coxetal.,2000).Thus,both ARF6, aRab11mutantwasreportedtoinhibitTfrecycling of recruitedmembranesduringphagocytosis.Similarlyto lipase A2,maybeneededfortheselectivedockingorfusion 1997). Therefore,arachidonicacid,theproductofphospho- specifically tositesofparticleattachment(Lennartzetal., phospholipase A2leadtoamassiveaccumulationofvesicles (D’Souza-Schorey etal.,1995).Bycontrast,theinhibitionof a phagocytictrigger,aspreviouslyshowninfibroblasts stitutive recyclingofTfinRAW264.7cellstheabsence chesky, 2001).Inaddition,ARF6alsoregulatesthecon- control ofRac/Cdc42(Castellanoetal.,2001;MayandMa- cytosis, whereasactinpolymerizationwouldbeunderthe ARF6 controlspolarizedmembranerecyclingduringphago- membrane accumulation.Thus,ourresultssuggestthat T27N incubatedwithIgG-SRBCfor60minat37 Scanning electronmicrographsofmacrophagesexpressingARF6- Arrowhead pointstoanabortivephagosome.Bar,10 60 min(CandD)at37 T27N (BandD)wereincubatedwithIgG-SRBCfor10min(AB)or particles. Figure 5. polymerization (Hondaetal.,1999;Vitale al.,2002). tively, andare known tomodulatevesicular traffic andactin acid andphosphatidylinositol (4,5)bisphosphate,respec- RAW264.7cellsexpressingARF6-WT(AandC)orARF6- ARF6-T27N inhibitsmembraneextensionaroundthe C, thenfixedandprocessedforscanning , whichproducephosphatidic Niedergangetal. C. Bar,1 m. (EandF) m. EM. 1147 The Journal of Cell Biology 1148 Materials andmethods plasmid encoding the GST-fusedRac-interactivebinding domainofPAK1 Dr. T.Galli(INSERM U536,InstitutduFer-à-Moulin, Paris,France).The Cochin, Paris,France).Theplasmid encodingGFP-VAMP3wasagiftof vided byDr.A.Benmerah(INSERM U567-CNRSUMR8104,Institut the nextyears. these relatedcellularfunctionswillbeamajorchallengefor characterization ofthemolecularbasisARF6functionin the plasmamembrane(TurnerandBrown,2001).The pend onpolarizedinsertionofmembraneatspecificsites tility, whicharecontrolledbyARF6,alsothoughttode- fusion withtheformingphagosome.Cellspreadingandmo- tionality forvesiclemovement,budding,docking,or ARF6 couldactatdifferentstagesoftheprocess,i.e.,direc- nated. Tocontrolmembranedeliveryduringphagocytosis, how exocytosisofthesedifferentcompartmentsiscoordi- rived antigens.Thequestionalsoremainsopenwhetherand ingested particles,andeventually,tothepresentationofde- and maturationprocessesthatleadtothedegradationof ments havedistinctfunctionsinthephagosomeformation 2002; Tapperetal.,2002).Itisnotclearifthesecompart- 2001; Aderem,2002;Allenetal.,Gagnon cruited tonascentphagosomes(Muller-Taubenbergeretal., lysosomes andtheERhaverecentlybeenshowntobere- contribute topseudopodformationinphagocytes.Indeed, FcR duringphagocytosis. rien etal.,2002)thatcouldbeactivatedafterengagementof 2000), includingARNOandEFA6familymembers(Der- ARF6 (ChavrierandGoud,1999;JacksonCasanova, sected. Severalexchangefactorshavebeendescribedfor activation ofARF6duringphagocytosisstillhastobedis- tosis (Pateletal.,2002).Thesignalingcascadeleadingtothe been describedrecentlyforRacandCdc42duringphagocy- Furthermore, concertedbutparallelactivationshavealso etal.,1997;PalaciosandD’Souza-Schorey,2003). Schorey umented duringscatteringofepithelialcells(D’Souza- that distinctactivationprofilesforRacandARF6weredoc- spreading (Zhangetal.,1999).However,ithastobenoted corroborating previouslypublishedresultsonmacrophage cells mayindicatethatRacisupstreamofARF6andCdc42, that RacisactivatedbeforeARF6andCdc42inRAW264.7 allow membraneextension.Furthermore,theobservation signaling pathwayscouldintersectandcooperateinorderto kateswarlu etal.,1998),suggestthatARF6-andPI3K- change factorforARF6,dependsonPI3Kactivity(Ven- withthefactthatactivationofARNO,anex- together formation (Coxetal.,1999;thispaper).Alltheseresults, cles wereseverelyinhibited,leadingtoabortivephagosome whereas membranedeliveryandextensionaroundtheparti- as actinassemblyatthecontactpointswerenotaffected, cases, particlerecognitionatthemacrophagesurfaceaswell macrophages (Arakietal.,1996;Cox1999).Inboth ARF6 resemblesthatobservedwhenPI3Kisinhibitedin control construct(pEGFPC2Eps15DIII ing thedominantinhibitorydomain ofEps15(pEGFPC2Eps15DIII)ora previously (D’Souza-Schoreyetal., 1998). Theexpressionvectorsencod- The ARF6-WT–andARF6-T27N–expressing plasmidshavebeendescribed Plasmids andreagents There aremultiplesourcesofintracellularmembranethat The phagocyticdefectinducedbydominant-negative The JournalofCellBiology
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Volume 161,Number6,2003 2; Benmerahetal.,2000)werepro- (Molecular Probes,Inc.);Cy3-labeledF(ab’) gang etal.,2000). nofluorescence (Niedergangetal.,1995)orelectronmicroscopy(Nieder- points, thecellswerewashedandfixedusingstandardprotocolsforimmu- 100-mm plateofcellsgrowntosubconfluenceand20 fected byelectroporationwiththeOptimixkit(EquiBio).Routinely,one The solutionwasadjustedto3.6 were collectedfromthepelletandwashedthreetimesinAlsever’sbuffer. tosis wassynchronizedbycentrifugationfor2minat400 on thecellsgrowncoverslips(SRBC:macrophageratioof10).Phagocy- medium(serum-free completeRPMI1640)anddistributed phagocytosis al., 2000).SRBCsopsonizedwithIgGwereresuspendedinprewarmed used foreachtransfection(20 Hepes, 1mMsodiumpyruvate,and50 RPMI 1640–glutamaxsupplementedwith10%FCS(Biomedia)and10mM RAW264.7 macrophagesweregrownincompletemedium,consistingof Cell culture,Tfuptake,andtransfection against 20mMTris(pH7.4),2EDTA,100NaCl, 2001), exceptthatitwaspurifiedonGSTbeadcolumns,directlydialyzed and GST-CRIBwerepreparedasdescribedpreviously(Duboisetal., antibodies andAlexa ter twowashes,cellswereincubated withCy2-orCy3-coupledanti–rat cells. Subsequentstepswereperformed atRTinpermeabilizingbuffer.Af- the permeabilizingbuffer(PBS-FCS/0.05% saponin)todetecttransfected twice inPBS-FCSandincubatedfor45minwiththeanti-HAantibodies in beling andappearsswolleninphasecontrast.Cellswerethenwashed detect externalIgG-SRBC.InternalizedIgG-SRBCisprotectedfromthisla- anti–rabbit IgGantibodiesin2%FCS/PBS(PBS-FCS)for45minatRT to Cells werefixedin4%PFA-PBSfor45minat4 Immunofluorescence Biosciences; blood:ficoll 6) andcentrifugedonaficollgradient(FicollPaquePlus™,Amersham sodium citrate,114mMglucose,72NaCl,and2.6citricacid,pH France).ThebloodwasdilutedtwotimesinAlsever’sbuffer(23mM Josas, tion Animale,InstitutNationaldelaRechercheAgronomique,Jouy-en- SRBCs werepurifiedfromsheepblood(UnitéCommuned’Expérimenta- Preparation ofSRBCandphagocytosisassays with 50mMNH Tf, Alexa GST-GGA3 London, UK)ThepGEX4T1(AmershamBiosciences)vectorencoding (GST-CRIB) wasobtainedfromDr.A.Hall(UniversityCollegeLondon, were asfollows:Texasred–labeledhumanTf;Alexa antibodies werehomemade.Fluorochrome-labeledorreagents T. Azuma(HarvardMedicalSchool,Boston,MA),andrabbitanti-Cdc42 tal, Boston,MA),monoclonalanti-Rac1antibodieswereprovidedbyDr. ARF6 pAbswereagiftfromDr.V.W.Hsu(BrighamandWoman’sHospi- Kahn, EmoryUniversitySchoolofMedicine,Atlanta,GA). isoform) subclonedintopcDNA3.1(Bomanetal.,2000;agiftofDr.R.A. captoethanol, and10%glycerol,aliquoted,storedat cultivated inthepresenceof1mg/mlG418completemedium. Vantage™ cellsorter(BectonDickinson),clonedbylimitingdilution,and clones expressingGFP-VAMP3,transfectedcellsweresortedonaFACS- coverslips. Efficiencyoftransfectionwas10–40%.Togeneratestable plete culturemediumandplatedin100-mmdishescontaining12-mm Pulser II;Bio-RadLaboratories),thenimmediatelyresuspendedincom- labeled F(ab’) labeled signal wasacquired separately.Serialopticalsections wererecordedat (model SP2;Leica) equippedwithArandHe/Nelasers. Eachfluorescent Tris, pH8.Thesampleswereexamined underaconfocalmicroscope scope slidesin100mg/mlMowiol, 25%(vol/vol)glycerol,and100mM three timesinthesamebufferandtwice inPBS,andmountedonmicro- 37 GIBCO BRL).60nMTexasred-Tfwasinternalizedintocellsfor30minat Link™ Cy™5labelingkit(AmershamBiosciences).GST-fusedGGA3 anti-SRBC IgG(ICNBiomedicals)werecoupledtoCy5withtheFluoro- HRP-coupled anti–rabbitoranti–ratIgG(Sigma-Aldrich).Purifiedrabbit cuvettes (EquiBio)at240V,950 encoding GFPforcotransfections).Thecellswereelectroporatedin0.4-cm ics wasstartedbyincubatingtheplatesat37 conserved at4 Phagocytosis wasperformedessentiallyasdescribedpreviously(Patelet The anti-HAmAbs(clone3F10)werepurchasedfromRoche,theanti- C asdescribedpreviously(Niedergangetal.,1995).Cellsweretrans- ® 633–labeledphalloidin,andAlexa
1–226
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80 C. g plasmid -mer- 1–226 2 The Journal of Cell Biology fast iterativeconstrainedPSF-basedalgorithm. by Westernblotting. reading ofthemanuscript. samples ofsheepblood. WearegratefultoDr.Andrés Alcoverforcritical on phagocytosisexperiments;andJean-François Alkombreforcollecting Danielle Rouillardforcellsorting;Dr. EmmanuelleCaronforinitialadvice convolution; Dr.MichèleGrasset forassistancewithscanningEM; in developingandperformingwide-field fluorescencemicroscopyandde- We thankDrs.Jean-BaptisteSibarita andVincentFraisierfortheirexpertise tion inamicrofuge.Afterincubationat37 medium (SRBC:macrophageratioof10),andsynchronizedbycentrifuga- sion, initiatedbytheadditionofIgG-SRBCinprewarmedphagocytosis pended inphagocytosismedium.Phagocytosiswasperformedsuspen- each pointofthephagocytosiskinetics.Cellswerescrapedandresus- 6–8 millionofRAW264.7cellsgrownatsubconfluencewereusedfor Pull-down assayanddeterminationoftheactivationARF6 system (BectonDickinson),measuringtheGFPandAlexa FCS onice.ThecellswerethenpelletedandanalyzedaFACSCalibur™ the indicatedtimes,aliquotsofcellsweretakenanddilutedinPBS/2% dium, andresuspendedinprewarmedcompleteRPMI1640medium.At 633-Tf. Thecellswerethenplacedonice,washedincoldendocytosisme- and incubatedfor45minat37 rum-free completeRPMI1640mediumsupplementedwith1mg/mlBSA), Transfected cellswerescraped,washedonceinendocytosismedium(se- Tf recycling counted thenumberofcell-associated(bound nontransfected cellsandexpressedasapercentageofcontrolcells.Wealso tained fortransfectedcellswasdividedbytheindexobtainedcontrol phagocytosed SRBCspercell)wascalculated(usually2–5).Theindexob- sen onthecoverslips,andphagocyticindex(i.e.,meannumberof The numberofinternalizedSRBCswascountedin50cellsrandomlycho- Quantitative analysisofphagocytosis ner (HiScan;Eurocore)andprocessedwithAdobePhotoshop (CM120 Twin;Philips)at60kV.Thenegativeswerescannedwithascan- methanol for5minandviewedphotographedwithamicroscope embedded macrophageswerecounterstainedwith2%uranylacetatein ating voltageof17kV.FortransmissionEM,ultrathinsectionsplastic- Microscopie Electronique,UniversitéParis6,Paris,France)atanacceler- with amicroscope(modelJSM840A,JEOL;CentreInterUniversitairede buffer (Niedergangetal.,2000).ForscanningEM,sampleswereexamined medium atRTandfixedin2.5%glutaraldehyde0.066Mcacodylate ods, exceptthatatdifferenttimepoints,cellswerewashedinphagocytosis Phagocytosis wasperformedasdescribedearlierinMaterialsandmeth- EM 0.3- age ofthevaluemeasuredatt cells. Thevaluesobtainedateachtimepointwereexpressedasapercent- Alexa cences. Atleast10,000GFP-positivecellswereacquired,andthemean (25 2002). Precipitationwasperformedinthepresenceof0.5%BSAwithGST cells werelysedoniceinlysisbufferasdescribedpreviously(Pateletal., enlarged compartmentsin30macrophages. under theelectronmicroscopebycountingnumberofcharacteristic ware. Relativequantitationofintracellularvesicleswasperformeddirectly volution modulefromMetaMorph and deconvolutionwasperformedbythenewthree-dimensionaldecon- Scientific). TheZ-positioningwasaccomplishedbyapiezo-electricmotor, equipped withacooledinterlinedCCDcamera(CoolSNAPHQ™;Roper were examinedunderaninvertedmicroscope(modelDMIRE2;Leica) try asdescribedpreviously(Coppolinoetal.,2002). percentage ofthelatter.Alternatively,cellswereanalyzedbyflowcytome- the indexobtainedforcontrolnontransfectedcellsandwasexpressedasa tions percell).TheindexobtainedforARF6-expressingcellswasdividedby and calculatedanaccumulationindex(i.e.,themeannumberofaccumula- of GFP-VAMP3andF-actinin50cellsrandomlychosenonthecoverslips, actin andmembrane-phagocyticcupprofiles,wescoredtheaccumulations and expresseditaspercentageofcontrolnontransfectedcells.Toquantitate culated theassociationindex(meannumberofassociatedSRBCspercell), were centrifugedat13,000 g), GST-GGA3 m intervalswitha100 ® 633fluorescencewascalculatedforGFP-negativeand-positive 1–226 (50 g for15s,thesupernatantwasremoved,and g), orGST-CRIB(50 , 1.4NAobjective.Alternatively,thecells 0. C inthepresenceof15 ® (UniversalImagingCorp.),usingthe C fordifferenttimepoints,cells internalized)SRBCs,cal- g) at4 C andrevealed ® 633fluores- g/ml Alexa ® 5.5soft- ARF6 controlsmembranerecyclingduringphagocytosis| ® Gagnon, E.,S.Duclos,C.Rondeau,E. Chevet,P.H.Cameron,O.Steele-Mor- Dubois, T.,P.Kerai,E.Zemlickova,S.Howell,T.R.Jackson,K.Venkateswarlu, Gold, E.S.,D.M.Underhill, N.S.Morrissette,J.Guo,M.A. McNiven,andA.Ad- Derrien, V.,C.Couillault,M.Franco,S.Martineau,P.Montcourrier,R.Houl- Dautry-Varsat, A.,A.Ciechanover,andH.F.Lodish.1983.pHtherecycling D’Souza-Schorey, C.,E.vanDonselaar,V.W.Hsu,C.Yang,P.D.Stahl,andP.J. D’Souza-Schorey, C.,R.L.Boshans,M.MacDonough,P.D.Stahl,andL.Van D’Souza-Schorey, C.,G.Li,M.I.Colombo,andP.D.Stahl.1995.Aregulatory Cox, D.,D.J.Lee,B.M.Dale,J.Calafat,andS.Greenberg.2000.ARab11-con- Cox, D.,C.C.Tseng,G.Bjekic,andS.Greenberg.1999.Arequirementforphosphati- Coppolino, M.G.,R.Dierckman,J.Loijens,R.F.Collins,M.Pouladi,Jongstra- Chavrier, P.,andB.Goud.1999.TheroleofARFRabGTPasesinmembrane Boman, A.L.,C.Zhang,X.Zhu,andR.A.Kahn.2000.AfamilyofADP-ribosyla- Benmerah, A.,V.Poupon,N.Cerf-Bensussan,andA.Dautry-Varsat.2000.Map- Castellano, F.,P.Chavrier,andE.Caron.2001.Actindynamicsduringphagocy- Booth, J.W.,W.S.Trimble,andS.Grinstein.2001.Membranedynamicsin Araki, N.,M.T.Johnson,andJ.A.Swanson.1996.Aroleforphosphoinositide Bajno, L.,X.-R.Peng,A.D.Schreiber,H.-P.Moore,W.S.Trimble,andS.Grin- Altschuler, Y.,S.-H.Liu,L.Katz,K.Tang,S.Hardy,F.Brodsky,G.Apodaca,and Allen, L.A.,C.Yang,andJ.E.Pessin.2002.Rateextentofphagocytosisin post-doctoral fellowshipfromAssociationpourlaRecherchesurleCancer. tionale contreleCancer(EquipeLabelisée).T.Duboisistherecipientofa en MicrobiologieetMaladiesInfectieusesParasitaires,andtheLigueNa- grants fromtheInstitutCurie,ProgrammedeRechercheFondamentale Aderem, A.,andD.M.Underhill.1999.Mechanismsofphagocytosisinmacro- Aderem, A.2002.Howtoeatsomethingbiggerthanyourhead. 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