Edgar BMCBiology (2016) 14:46 DOI10.1186/s12915-016-0268-z
Q&A:Whatareexosomes,exactly?
JamesR.Edgar
Abstract
Exosomesareextracellularvesiclesfirstdescribedas such30yearsagoandsinceimplicatedincell–cell communicationandthetransmissionofdiseasestates, andexploredasameansofdrugdiscovery.Yet fundamentalquestionsabouttheirbiologyremain unanswered.HereIexplorewhatexosomesare, highlightthedifficultiesinstudyingthemandexplain thecurrentdefinitionandsomeoftheoutstanding issuesinexosomebiology.
Whatisthecurrentdefinitionofanexosome?
Thatisaverygoodquestion.Sincetheoriginaldescriptionofexosomesover30yearsago,thetermhasbeen looselyusedforvariousformsofextracellularvesicle, muddyingthefieldandcontributingtothescepticism withwhichtheresearchhassometimesbeenmet.Exosomesarebestdefinedasextracellularvesiclesthatare releasedfromcellsuponfusionofanintermediateendocyticcompartment,themultivesicularbody(MVB),with theplasmamembrane.Thisliberatesintraluminalvesicles(ILVs)intotheextracellularmilieuandthevesicles therebyreleasedarewhatweknowasexosomes(Fig.1).
Thereareothertypesofmicrovesicle,includingapoptoticbodiesandectosomes,whicharederivedfromcells undergoingapoptosisandplasmamembraneshedding, respectively.Althoughapoptoticbodies,ectosomesand exosomesareallroughlythesamesize(typically40–100nm)andallalsocontain ‘gulps’ ofcytosol,theyare differentspeciesofvesiclesandunderstandingdifferencesbetweenthemisofparamountimportancebuthas toooftenbeenoverlooked.
Howwereexosomesfirstrecognizedasdistinct entities?
Thepresenceofmembranousvesiclesoutsidecellswas firstrecognized50yearsago,althoughthesewereoriginallyassumedtobewasteproductsreleasedviashedding
Correspondence: je333@cam.ac.uk CambridgeInstituteforMedicalResearch,UniversityofCambridge,Hills Road,CambridgeCB20XY,UK
oftheplasmamembrane.Therecognitionofwhatwe nowcallexosomesdidn’tcomeuntil1983,fromstudies onthelossoftransferrinduringthematurationof reticulocytesintoerythrocytes[1].Thesestudiesshowed, byfollowingtransferrin-goldconjugatesthroughthe endocyticsystem,thatILVsgeneratedinMVBscanbe releasedtotheextracellularspacethroughfusionwith theplasmamembrane[2],althoughitwasnotuntil 1987thattheterm ‘ exosome ’ wascoinedforthem[3].
Eventhen,however,theseextracellularvesicleswere largelyignored,forgottenor,again,dismissedasameans ofcellularwastedisposal.Itisonlyinthepastdecade thatinterestinexosomeshasexploded,withanearly tenfoldincreaseinpublicationsinasmanyyears(115in 2006,1010in2015).
Whythisexplosionofinterest?
Foratleastthreereasons.First,theyarethoughttoprovideameansofintercellularcommunicationandof transmissionofmacromoleculesbetweencells.Second, inthepastdecade,exosomeshavebeenattributedroles inthespreadofproteins,lipids,mRNA,miRNAand DNAandascontributingfactorsinthedevelopmentof severaldiseases.Andthird,theyhavebeenproposedto beusefulvectorsfordrugsbecausetheyarecomposed ofcellmembranes,ratherthansyntheticpolymers,and assucharebettertoleratedbythehost.Infact,someof theearliestexosomeresearchindicatedthattheycan carrytheMHC–peptidecomplexesthatarerecognized byTlymphocytes[4]andthatsecretionofsuchexosomescouldpromoteantitumourimmuneresponsesin miceinvivo[5].Exosometherapiesarenowbeingexploredinanti-cancerclinicaltrialsandrecentreports claimtaxol-filledexosomescanbeusedtotreatcancers inmiceat50-foldlowerdosesthanconventionaltreatments,withtheadditionalbenefitthatexosomesdonot invokeanimmuneresponse[6].
Yetdespite20yearsofresearch,theverybasicsofexosomebiologyareintheirinfancyandweknowlittleof theparttheyplayinnormalcellularphysiology.
©2016Edgar. OpenAccess ThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0International License(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninany medium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreative Commonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http:// creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Fig.1. Exosomescorrespondtointraluminalvesiclesof multivesicularbodies.Atransmissionelectronmicrographofan Epstein–Barrvirus-transformedBcelldisplayingnewlyexpelled exosomesattheplasmamembrane.Multivesicularbodies(MVB)can beseenwhichcandelivercontenttolysosomesfordegradationor canfusewiththecellsurfacetoreleaseintraluminalvesiclesas exosomes,indicatedbythe arrows atthetopofthepicture
Sodoweknowhowtheyaregenerated?
Yesandno.WedoknowthattheyaremadeasILVs; butfirstofall,notallILVsfinishupasexosomes,and second,themechanismoftheirgenerationinendosomes isnotfullyunderstood.Mostconventionalmembrane buddingprocessesdeformmembranefromanorganelle intothecytoplasmbutinILVformationthemembrane budsawayfromthecytoplasmandintotheendosome. Thisunconventionalbuddingprocessisnotlimitedto ILVgenerationbutalsotakesplaceduringenveloped virusbuddingfromthecytosolandduringcytokinesis [7],anditrequiresspecialisedmachinery.
ILVs(andthusexosomes)canbegeneratedatthe endosomallimitingmembranebyatleasttwomechanisms,oneofwhichdependsontheESCRTmachinery (ESCRTstandsforendosomalsortingcomplexesrequiredfortransport)whereastheotherisESCRTindependent(Fig.2).
TheESCRTmachineryconsistsofasetofcytosolic proteincomplexesthatarerecruitedtoendosomesby membraneproteinsthathavebeentagged,usuallywith ubiquitinontheircytosolicdomains.Theubiquitintag isrecognizedbythefirstoftheESCRTcomplexes, ESCRT-0,whichisthusrecruitedtotheendosomal membraneandpassesubiquitinatedcargostoESCRT-I, oneofwhosecomponents,Tsg101,alsorecognizesubiquitin.TherecruitmentoftheESCRTmachineryactsto bothclustertheubiquitinatedcargoproteinsonthe
endosomeandinducecurvatureoftheendosomalmembranetoformILVs.
ButILVsarestillabletoformintheabsenceof ESCRTs[8],soothermeansofgeneratingILVsmust exist,althoughthemechanismsfortheirgenerationare lessclear.GenerationoftheseESCRT-independentILVs requiresthetetraspaninCD63 aproteinabundanton ILVsbutwithunclearfunction[9] andmaybefacilitatedbycone-shapedbendingpropertiesoflipidssuch asceramide[10].
IfnotallILVsbecomeexosomes,whatdetermines thefateofanILV?
ThedestinyofILVsisdirectedbythefateoftheMVB theyresidein.Confusingly,inadditiontodifferenttypes ofILVs,therearealsodifferenttypesofMVBs[11]and whatregulatesthefateoftheseendosomesisanotherinterestingquestion.MVBshaveseveralpotentialfates (Fig.2)andcaneitherfusewithlysosomes(wherecontentsaredegradedandrecycled),fusewiththeplasma membrane(whereILVsarereleasedasexosomes),asI havealreadymentioned,orcontributetothegeneration ofspecialisedorganelles,suchasmelanosomes(inmelanocytes),Weibel-Paladebodies(endothelialcells),azurophilicgranules(inneutrophils)andsecretorygranules (inmastcells).ThelevelsofcholesterolonMVBsappear toplayapartinregulatingtheirfate,cholesterol-rich MVBsbeingdirectedtotheplasmamembraneforexosomerelease,whilecholesterol-poorMVBsaretargeted tothelysosome[12].
ButwhatregulatesthebalancebetweenexosomereleaseandalternativefatesofILVsremainsengimatic.
Whataboutdifferencesbetweencells:doallcells releaseexosomes?
Well,notallcellshaveanendomembranesystem,sono. Butmostmammaliancellscontainendomembranesand generateILVswithinMVBs,thoughremarkablylittleis knownaboutexosomereleaseinmostcelltypes.
Somecells forexample,theBcells,dendriticcells andmastcellsoftheimmunesystem appeartorelease exosomesconstitutively;infact,mostofthedatawe haveonexosomescomesfromimmunecells.Aswellas releasingexosomesconstitutively,thesecellsmayalso bestimulatedtosecreteexosomesbycellularinteractions.Forexample,murinedendriticcells,whichare specializedtoactivateTlymphocytes,secretehigher levelsofexosomesuponinteractionwithantigenspecificCD4+Tlymphocytes[13].Infact,lymphocyte interactionsgenerallycanbeaccompaniedbyexosome release;humanTcells(includingprimaryTcellsfrom blood,TcellclonesandJurkatcelllines)releaseexosomesuponactivationoftheirantigenreceptors[14]
Fig.2. ILVsaregeneratedbyinvaginationoftheendosomalmembraneandhavethreepossiblefates. Inset:intraluminalvesicles(ILV)areformed byinvaginationoftheendosomalmembranebyeitherESCRT-dependentorESCRT-independentmechanisms.Maturedendosomesaccumulate ILVswithintheirlumenandhavethreedistinctfates.Theymaydelivercontentthatcontributestothebiogenesisofspecializedlysosome-related organelles(forexample,melanosomes,Weibel-Paladebodies,azurophilicgranules),theymayfusewithlysosomesortheymayfusewiththe plasmamembranewherereleasedILVsarenowtermed ‘ exosomes ’
andBcellsreleasemoreexosomesuponengagement withantigen-specificCD4+Tcells[15].
Othercelltypescanbepushedtosecreteexosomesby meansofcalciumionophoresorotherstimuli[16,17], buttheextentofphysiologicalexosomesecretionin non-immunecellsislargelyunknown.
Whathappenswhenexosomesreachanacceptor cell?
Wedon’tknowexactly.Exosomesthattransfermembraneproteinsorluminalcontenttotheacceptorcell maybeengulfedwholeortheexosomemembranemay fusedirectlywiththehostplasmamembrane(Fig.3). Alternatively,exosomesmaynotneedtobetakenupby cellstoelicitaphysiologicalresponse:folliculardendritic cells,forexample,carryontheircellsurfaceexosomes thatbearMHC–peptidecomplexesandotherproteins thattheydonotexpressandaretherebyenabledtoactivateimmunecellswithwhichtheyinteract[18].
Forintercellulartransmission,variousmechanismsof phagocytosisandendocytosisofextracellularvesicles havebeendescribedandwhichmechanismoperates maydependuponvesiclesize,whichmayinturndependuponthecargocarriedbythevesicle.Inorderfor materialtobereleasedtoanacceptorcell,exosomes mustfusewiththehostcellandthistakesplaceviaeitherdirectfusionwiththeplasmamembraneora ‘backfusion’ stepfromwithinahostendocyticorganelleafter theexosomehasbeenengulfed.Theprocessofbackfusionisnotentirelyclear,althoughitappearstorequire theunconventionallipidLBPAandproteinAlix[19] (andisexploitedbyanthraxtoxinlethalfactortoescape fromendosomestothecytosol[20]).
Whetherexosomesfusewithtargetcellsoractviainteractionswithcell-surfaceproteins,orboth,isanother fundamentalcellbiologyquestionthatwillneedtobe addressedifwearetounderstandthefunctionsof exosomes.
Fig.3. Exosomeuptakebyrecipientcells.FusionofMVBswiththecellsurfacereleasesILVsasexosomes.Inorderforexosomestoelicita responsefromrecipientcellstheymighteitherfusewithplasmamembrane(a)orbetakenupwholeviaendocytosis(b),followingwhichthe exosomemustbedeliveredtothecytosol,forexample,viaaback-fusionevent(c).Alternatively,exosomesmayattachtothesurfaceofrecipient cellstoelicitasignallingresponse(d)
Sowhataretheconsequencesofallthis informationtransfer?Whatbiologicalfunctions havebeenestablishedforexosomes?
Therearemanyproposedfunctionsforexosomes,the best-establishedbeinginimmuneresponses.Exosomes isolatedfromBlymphocytesandbearingMHCclassII moleculeswereshowninearlyexperiments[4]toactivate Tlymphocytesinvitro,suggestingthattheywerecommunicatingwiththeTlymphocytesinjustthewaythatthe parentBcellsdid.Ihavealreadymentionedlaterworkby thesamegroup,whoshowedthatexosomesderivedfrom dendriticcells,whicharespecializedtoactivateTcellsin theinitiationofimmuneresponses,couldpromoteantitumourimmuneresponsesinmice[5],excitinginterestin thepossibilityofpracticalapplications.
Or,aswithfolliculardendriticcells,exosome-associated MHCIIcanbefoundonthesurfaceofcelltypesthatneitherexpressMHCIInorsecreteexosomes,indicatingthat exosomesaredeliveredfromonecelltypetoanother[18].
However,exosomesmayhaverolesotherthaninimmuneresponsesasseveralnon-immunecellssecrete exosomes.Theonlyphysiologicalrolesofarreportedfor non-immunecellsisinkeratinocyte-derivedexosomes, whichhavebeenshowntomodulatemelaninsynthesis byincreasingtheexpressionandactivityofproteins
withinthemelanosomesthatmodulateskinpigmentation[21].
Howexactlywouldexosomesfromonecell influencetheexpressionandactivityofproteins inanacceptorcell?
Exosomestransfernotonlyproteinandlipidsbut mRNAandmicroRNAintoacceptorcellsandthese RNAshavebeenshowninexperimentsinvitrotohave functionaleffectsinrecipientcells.Forexample,exosomesfrommicecanbetransferredtohumancellsand mRNAcanbetranslatedintomouseprotein[22].Similarly,microRNAs double-strandedRNAfragmentsthat canregulatespecificsetsofmRNA(andsoprotein levels) canactfunctionallyinacceptorcells.Themode ofactionofexosomeshasbeenafocusofspecialinterest incancerbiology.Exosomesfrombreastcancercell lines,forexample,havebeenshowntobeenrichedfor miRNAsrelativetonontumorigenicbreastcelllinesand exposureofnormalcellstoexosomesderivedfrom breastcancercelllinesincreasedbothcellsurvivaland proliferation,accompaniedbylossofexpressionofsome tumour-suppressorproteins[23].Exosomelevelsareelevatedintheserumofsomecancerpatientsversuscontrols.However,whetherthesevesiclesareexosomesor
otherformsofextracellularvesicle,oramix,is unclear Ihavealreadymentionedthispersistentproblem inexosomeresearch.
Soexosomescanalsocontributetodisease?
Yesindeed.Asexosomesprovideameansofintercellular communication,theymayalsoactasvehiclesfor ‘bad’ communicationorspread.AswellasmiRNAsinthecaseof cancer,exosomeshavebeenshowntocontainnumerous disease-associatedcargos forexample,neurodegenerativeassociatedpeptides,suchasAβ [24](inAlzheimer’ s disease),tau[25](innumerousneurodegenerativediseases), prions[26](intransmissiblespongiformencephalopathies), alpha-synuclein[27](insyn ucleinopathies,including Parkinson’sdisease)andsuperoxidedismutase1[28](in amyotrophiclateralsclerosis).Exosomeshavethusbeen suggestedtobepropagatorsofneurodegenerativeprotein spread,althoughsomecargosareeasiertoenvisagethan others.
Oftheneurodegenerative-associatedproteins,only someareintegralmembraneproteins,thatis,proteins insertedintolipidbilayers,ratherthancytosolic.Sorting ofproteinsintoILVs(andthusexosomes)iseasiertoenvisageformembraneproteins,wheretagssuchasubiquitinregulatewheretheyendup.Sofar,thepresence ofbothAβ [29]andPrPc[26]hasinfactbeenshownin ILVs,thoughthishasnotbeendemonstratedforother membraneproteins,suchasalpha-synucleinandtau.
Themechanismwherebycytosolicproteinsmaybe sortedtoILVs/exosomes,however,isnotclear.Inorder forcytosolicproteinstobecomeconcentratedinILVs, theywouldrequirepositiveincorporationandsorting, possiblybymembrane-associatedcomponentsonendosomes.Allwecansayisthatthereisevidencethatthis doesinfacthappen;cytosolicfactorssuchasmiRNAs areenrichedinexosomesrelativetocytosol,indicating thatsortingmustoccurwherebycertainmiRNAsare concentratedandothersarenot[30].
Themeansbywhichdisease-associatedfactorsspread betweencellsremainspoorlyunderstoodandexosomes wouldprovideameansforsuchtransmission.Thepresenceofexosomalproteins,suchasAlix,inassociation withAlzheimer ’ssenileplaquesstrengthensthecircumstantialcaseforexosomesasamediatorinsuchspread. Thehopeisthathavingameanstoregulateexosomereleaseandspreadmaybeusefulincombattingsomeof thesediseasesbutmuchmorebasicbiologyneedstobe establishedbeforethen.
NowI’mconfused—whatdetermineswhat exosomescontain?
Exosomeswillcontainwhateverissortedintothemduringtheirformation(asILVs).Formembraneproteins, thisusuallyoccursthroughubiquitination,whichactsas
asubstrateforrecruitmentoftheESCRTmachineryand subsequentgenerationofESCRT-dependentILVs.
Themechanismsthatconcentratecytosolicfactorsare currentlyunknown.AlthoughitseemsclearthatmiRNAs,forexample,areenrichedrelativetotheamountin theirparentcells,andarenotrandomlyincorporated intoexosomes,itisnotclearhowsomeareenriched morethanothers.Therearecurrentlyafewhypotheses formiRNAsorting,includingsortingviasumoylated heterogeneousnuclearribonucleoproteins[31]orbya miRNA-inducedsilencingcomplex(miRISC)[32].
Becauseofthedifficultiesinseparatingexosomesfrom otherextracellularvesicles,itislikelythatsomecargos reportedtobeenrichedin ‘ exosomes ’ mayinfactbe containedincontaminantvesiclesthatarenotexosomes.Whilemanyresearchersareverystringentabout applyingthelabels ‘ exosomes ’ and ‘extracellularvesicles’ correctly,othersunfortunatelyarenot.Inaddition,asI havesaidbefore,cytosolicproteinsarelikelytobefound inexosomepreparationsbecausetheexosomelumenis madeofcytosol.
Sohowexactlycanyoubesurethatagiven extracellularvesicleisanexosomeandnot somethingelse?
Thisisaninterestingquestionthathasacomplexanswer.Ideally,anintracellularcompartmentisidentified byaspecificbiologicalmarker,as,forexample,inthe caseoftheGolgi,nucleusormitochondria,allofwhich carryproteinsnotfound,orfoundatmuchlowerlevels, elsewhere.
OneproblemisthatILVs,andthusexosomes,represent anintermediatecompartmentofanintermediate.MVBs arenotstaticorganellesbutratherundergocontinuous maturation,inthecourseofwhichtheygainandloseproteins.TherewillneverbeanexclusivemarkerforexosomesbecauseanycargoontheILV/exosomemembrane mustfirstbeonthelimitingmembraneoftheendosome andanythingfoundinsidemustfirstcomefromthecytosol.AcargomaybeconcentratedonILVs/exosomesbutit willalsobeelsewhere.CD63couldbethoughtofasa pseudo-markerforexosomes.ILVsandexosomesare enrichedinseveralsuchtetraspaninsandmycolleagues andIhaveshowthatCD63isrequiredforESCRTindependentILVformation[9].Alixalsoappearstobe concentratedinILVs/exosomes[33],asdoesTsg101,a componentofESCRT-I,whichhasbeenusedasamarker ofexosomesinnumerousstudies[33,34],althoughthe presenceofTsg101inILVsorexosomesdoesnotfitwith conventionalmodelsofILVformation.AlthoughTsg101 isinvolvedinESCRT-dependentILVformation,asmentionedearlier,it,alongwithotherESCRTcomponents, shoulddisassociatefromtheendosomalmembraneprior toanILVpinchingofftheendosomalmembranetoallow
ittoparticipateinfurtherevents[35].Exactlywhen ESCRT-Icomponents ‘falloff ’ themembraneisunknown butitisconventionallythoughttobepriortoILVformation,soTsg101shouldremaincytosolicandavailablefor subsequentroundsofILVformation.Itispossiblethat someTsg101maybe ‘swallowed’ intotheformingILV lumen,butlevelsshouldbenegligible.
Soareyousayingthereisnoreliablemarkerfor endosomes?
Theremaynotbe notasinglereliableone.Ultimately, perhapsthebestmethodofdefiningexosomesbiochemicallymaybethroughacombinationofmarkers,includingtetraspanins,Alixandothers,withaconcomitant exclusionofresidentplasmamembraneproteins.AlthoughILVs/exosomeswillbytheirnaturecontainsome plasmamembraneproteinsandtheplasmamembrane willcontainsomeILV/exosomalproteins,itshouldbe possibletodefinerelativelevelsand/orenrichmentof proteinsofexosomesthatdistinguishthemfromother microvesicles.CargossuchasMHCIIfromBcellsand othercelltype-specificantigensmayalsohelptodistinguishexosomesfromotherformsofextracellularvesicle. Commonexosomalcargosincludetetraspanins(CD63, CD81,CD9),antigenpresentationmolecules(MHCI andMHCII)andothers(Alix,flotillin-1).Anonline databaseexists[36]whereproteins,lipidsandRNAare cataloguedfrompublishedandunpublishedexosomal studies.
Iftheyaresohardtocharacterizereliably,how areexosomesisolatedandstudied?
Exosomesarerarelyimagedbyconventionalmethodsas theyaretoosmalltoberesolvedbyfluorescencemicroscopyandtheirreleasemaybearareevent.Afewstudies haveimagedexosomereleaseoccurringincellcultures byvariouselectronmicroscopictechniquesbut,more commonly,exosomesarepooledfromcellularsupernatantoranimalfluids.Traditionally,theyhavebeen isolatedbydifferentialcentrifugationfromculture mediumwherebylargercontaminantsarefirstexcluded bypelletingoutthroughincreasingspeedsofcentrifugationbeforeexosomes,smallextracellularvesiclesand evenproteinaggregatesarepelletedatveryhighspeeds (~100,000× g)[37].Thesepreparationsthereforerepresentanenrichmentratherthanapurification.Enriched preparationsarecommonlyanalysedbybiochemistry, massspectrometryorelectronmicroscopy.Electronmicroscopyofisolatedfractionsas ‘wholemounts’ makeit possibletoimmuno-labelvesicles,withthelimitation thatisolatedpreparationsdonotprovidethesameinternalcontrolsaslabellingsectionsofcells.Remarkably littleattentionhasbeenpaidtothecharacterizationof exosomes,althougheffortsarebeingmadetorepairthis
omissionwithguidelinesandcriteriafordefininggroups ofextracellularvesicles[38].
Whatwouldyousayarethemostimportant issuesinexosomeresearch?
Withoutdoubtthesinglemostimportantissueisactuallyunderstandingthebiologicalsignificanceofthese structures.Withsolittleknownabouttheirbasic physiologicalfunctions,itmayseemhardtounderstand howexosomeshavebeenimplicatedinthepathogenesis ofsomanydisparatediseasestates.Fundamentalquestionsremainaboutexosomegeneration,fateandnormal functionbut,ultimately,inordertounderstandexosomes,onemustfirstunderstandILVs,afactthatistoo oftenoverlooked.Meanwhile,itisimportantthatpublicationsonexosomesgiveacarefulandexplicitaccount ofthecriteriausedfordistinguishingthemfromother extracellularvesiclestoavoidconfusingthefieldandencouragingscepticism.
Acknowledgements
TheauthorwishestothankScottieRobinson,PaulLuzioandPaulMannafor criticalreadingofthisarticle.
Competinginterests
Theauthordeclaresthathehasnocompetinginterests.
References
1.HardingC,HeuserJ,StahlP.Receptor-mediatedendocytosisoftransferrin andrecyclingofthetransferrinreceptorinratreticulocytes.JCellBiol.1983; 97(2):329–39.
2.PanBT,TengK,WuC,AdamM,JohnstoneRM.Electronmicroscopic evidenceforexternalizationofthetransferrinreceptorinvesicularformin sheepreticulocytes.JCellBiol.1985;101(3):942–8.
3.JohnstoneRM,AdamM,HammondJR,OrrL,TurbideC.Vesicleformation duringreticulocytematuration.Associationofplasmamembraneactivities withreleasedvesicles(exosomes).JBiolChem.1987;262(19):9412–20.
4.RaposoG,NijmanHW,StoorvogelW,LiejendekkerR,HardingCV,MeliefCJ, etal.Blymphocytessecreteantigen-presentingvesicles.JExpMed.1996; 183(3):1161–72.
5.ZitvogelL,RegnaultA,LozierA,WolfersJ,FlamentC,TenzaD,etal. Eradicationofestablishedmurinetumorsusinganovelcell-freevaccine: dendriticcell-derivedexosomes.NatMed.1998;4(5):594–600.
6.KimMS,HaneyMJ,ZhaoY,MahajanV,DeygenI,KlyachkoNL,etal. Developmentofexosome-encapsulatedpaclitaxeltoovercomeMDRin cancercells.Nanomedicine.2015;12(3):655–64.
7.McDonaldB,Martin-SerranoJ.Nostringsattached:theESCRTmachineryin viralbuddingandcytokinesis.JCellSci.2009;122(Pt13):2167–77.
8.StuffersS,SemWegnerC,StenmarkH,BrechA.Multivesicularendosome biogenesisintheabsenceofESCRTs.Traffic.2009;10(7):925–37.
9.EdgarJR,EdenER,FutterCE.Hrs-andCD63-dependentcompeting mechanismsmakedifferentsizedendosomalintraluminalvesicles.Traffic. 2014;15(2):197–211.
10.TrajkovicK,HsuC,ChiantiaS,RajendranL,WenzelD,WielandF,etal. Ceramidetriggersbuddingofexosomevesiclesintomultivesicular endosomes.Science.2008;319(5867):1244–7.
11.WhiteIJ,BaileyLM,AghakhaniMR,MossSE,FutterCE.EGFstimulates annexin1-dependentinwardvesiculationinamultivesicularendosome subpopulation.EMBOJ.2006;25(1):1–12.
12.MobiusW,Ohno-IwashitaY,vanDonselaarEG,OorschotVM,ShimadaY, FujimotoT,etal.Immunoelectronmicroscopiclocalizationofcholesterol usingbiotinylatedandnon-cytolyticperfringolysinO.JHistochem Cytochem.2002;50(1):43–55.
13.BuschowSI,Nolte-’tHoenEN,vanNielG,PolsMS,tenBroekeT,LauwenM,etal. MHCIIindendriticcellsistargetedtolysosomesorTcell-inducedexosomesvia distinctmultivesicularbodypathways.Traffic.2009;10(10):1528–42.
14.BlanchardN,LankarD,FaureF,RegnaultA,DumontC,RaposoG,etal.TCR activationofhumanTcellsinducestheproductionofexosomesbearing theTCR/CD3/zetacomplex.JImmunol.2002;168(7):3235–41.
15.MuntasellA,BergerAC,RochePA.Tcell-inducedsecretionofMHCclassIIpeptidecomplexesonBcellexosomes.EMBOJ.2007;26(19):4263–72.
16.SavinaA,FurlanM,VidalM,ColomboMI.Exosomereleaseisregulatedbya calcium-dependentmechanisminK562cells.JBiolChem.2003;278(22): 20083–90.
17.GuoBB,BellinghamSA,HillAF.Stimulatingthereleaseofexosomesincreases theintercellulartransferofprions.JBiolChem.2016;291(10):5128–37.
18.DenzerK,vanEijkM,KleijmeerMJ,JakobsonE,deGrootC,GeuzeHJ. FolliculardendriticcellscarryMHCclassII-expressingmicrovesiclesattheir surface.JImmunol.2000;165(3):1259–65.
19.BissigC,GruenbergJ.ALIXandthemultivesicularendosome:ALIXin Wonderland.TrendsCellBiol.2014;24(1):19–25.
20.AbramiL,LindsayM,PartonRG,LepplaSH,vanderGootFG.Membrane insertionofanthraxprotectiveantigenandcytoplasmicdeliveryoflethal factoroccuratdifferentstagesoftheendocyticpathway.JCellBiol.2004; 166(5):645–51.
21.LoCiceroA,DelevoyeC,Gilles-MarsensF,LoewD,DingliF,GuereC,etal. Exosomesreleasedbykeratinocytesmodulatemelanocytepigmentation. NatCommun.2015;6:7506.
22.ValadiH,EkstromK,BossiosA,SjostrandM,LeeJJ,LotvallJO.ExosomemediatedtransferofmRNAsandmicroRNAsisanovelmechanismof geneticexchangebetweencells.NatCellBiol.2007;9(6):654–9.
23.MeloSA,SugimotoH,O’ConnellJT,KatoN,VillanuevaA,VidalA,etal. Cancerexosomesperformcell-independentmicroRNAbiogenesisand promotetumorigenesis.CancerCell.2014;26(5):707–21.
24.RajendranL,HonshoM,ZahnTR,KellerP,GeigerKD,VerkadeP,etal. Alzheimer’sdiseasebeta-amyloidpeptidesarereleasedinassociationwith exosomes.ProcNatlAcadSciUSA.2006;103(30):11172–7.
25.SamanS,KimW,RayaM,VisnickY,MiroS,SamanS,etal.Exosomeassociatedtauissecretedintauopathymodelsandisselectively phosphorylatedincerebrospinalfluidinearlyAlzheimerdisease.JBiol Chem.2012;287(6):3842–9.
26.FevrierB,ViletteD,ArcherF,LoewD,FaigleW,VidalM,etal.Cellsrelease prionsinassociationwithexosomes.ProcNatlAcadSciUSA. 2004;101(26):9683–8.
27.EmmanouilidouE,MelachroinouK,RoumeliotisT,GarbisSD,NtzouniM, MargaritisLH,etal.Cell-producedalpha-synucleinissecretedinacalciumdependentmannerbyexosomesandimpactsneuronalsurvival.JNeurosci. 2010;30(20):6838–51.
28.GomesC,KellerS,AltevogtP,CostaJ.EvidenceforsecretionofCu,Zn superoxidedismutaseviaexosomesfromacellmodelofamyotrophic lateralsclerosis.NeurosciLett.2007;428(1):43–6.
29.Guduric-FuchsJ,O’ConnorA,CampB,O’NeillCL,MedinaRJ,SimpsonDA. Selectiveextracellularvesicle-mediatedexportofanoverlappingsetof microRNAsfrommultiplecelltypes.BMCGenomics.2012;13:357.
30.EdgarJR,WillenK,GourasGK,FutterCE.ESCRTsregulateamyloidprecursor proteinsortinginmultivesicularbodiesandintracellularamyloid-beta accumulation.JCellSci.2015;128(14):2520–8.
31.Villarroya-BeltriC,Gutierrez-VazquezC,Sanchez-CaboF,Perez-HernandezD, VazquezJ,Martin-CofrecesN,etal.SumoylatedhnRNPA2B1controlsthe sortingofmiRNAsintoexosomesthroughbindingtospecificmotifs. NatCommun.2013;4:2980.
32.FabianMR,SonenbergN.ThemechanicsofmiRNA-mediatedgene silencing:alookunderthehoodofmiRISC.NatStructMolBiol.2012;19(6): 586–93.
33.BobrieA,ColomboM,KrumeichS,RaposoG,TheryC.Diverse subpopulationsofvesiclessecretedbydifferentintracellularmechanisms arepresentinexosomepreparationsobtainedbydifferential ultracentrifugation.JExtracellVesicles.2012;1:18397.
34.TheryC,BoussacM,VeronP,Ricciardi-CastagnoliP,RaposoG,GarinJ,etal. Proteomicanalysisofdendriticcell-derivedexosomes:asecreted subcellularcompartmentdistinctfromapoptoticvesicles.JImmunol.2001; 166(12):7309–18.
35.HurleyJH.TheESCRTcomplexes.CritRevBiochemMolBiol.2010;45(6):463–87.
36.ExoCarta.http://www.exocarta.org.
37.TheryC,AmigorenaS,RaposoG,ClaytonA.Isolationandcharacterizationof exosomesfromcellculturesupernatantsandbiologicalfluids.Curr ProtocolsCellBiol.2006;Chapter3:Unit322.
38.LotvallJ,HillAF,HochbergF,BuzasEI,DiVizioD,GardinerC,etal.Minimal experimentalrequirementsfordefinitionofextracellularvesiclesandtheir functions:apositionstatementfromtheInternationalSocietyfor ExtracellularVesicles.JExtracellVesicles.2014;3:26913.