Cholesterol implications in plasmid DNA electrotransfer: Evidence for the involvement of endocytotic pathways
Christelle Rosazza
a,b,c, Emilie Phez
a,b, Jean-Michel Escoffre
a,b,1, Laurence Cézanne
a,b,2, Andreas Zumbusch
c,∗, Marie-Pierre Rols
a,b,∗∗aDepartmentofStructuralBiologyandBiophysics,CNRS,InstitutdePharmacologieetdeBiologieStructurale,205RoutedeNarbonne,F-31077Toulouse,France
bUniversityofToulouse,UPS,InstitutdePharmacologieetdeBiologieStructurale,F-31077Toulouse,France
cDepartmentofChemistry,UniversityofKonstanz,Universitätsstraße10,D-78457Konstanz,Germany
Keywords:
Genedelivery Electroporation Cholesterol Endocytosis Colocalizationanalysis
a b s t r a c t
ThedeliveryoftherapeuticmoleculessuchasplasmidDNAincellsandtissuesbymeansofelectricfields holdsgreatpromiseforanticancertreatment.Toallowfortheirtherapeuticaction,themoleculeshave firsttotraversethecellmembrane.ThemechanismsbywhichtheelectrotransferredpDNAinteracts withandcrossestheplasmamembranearenotyetfullyexplained.Theaimofthisstudyistounravelthe roleofcholesterolduringgeneelectrotransferincells.Weperformedcholesteroldepletionexperiments andmeasureditseffectsonvariousstepsoftheelectroporationprocess.Thefirsttwostepsconsisting ofelectropermeabilizationoftheplasmamembraneandofpDNAinteractionwithitwerenotaffected bycholesteroldepletion.Incontrast,geneexpressiondecreased.Colocalizationstudieswithendocytotic markersshowedthatpDNAisendocytosedwithconcomitantclathrin-andcaveolin/raft-mediatedendo- cytosis.CholesterolmightbeinvolvedinthepDNAtranslocationthroughtheplasmamembrane.Thisis thefirstdirectexperimentalevidenceoftheoccurrenceofendocytosisingeneelectrotransfer.
1. Introduction
Thecellplasmamembraneactsasaselectivebarriercontrolling moleculeexchangebetweenthecellanditsexternalenvironment.
Asmanytherapeuticmoleculesarenonpermeant,theirtargeted deliveryintolivingcellsandtissuesisanimportantgoalofmodern pharmacologyandtherapy(Escoffreetal.,2010b).Indeed,theaim ofmoleculedeliverymethodsistoefficientlyovercome thecell barrierstoallowfortheirtherapeuticaction.Astrictlocalizationof thepharmacologicalactivityoftherapeuticmoleculestoacellular targetsitewouldresultinasignificantreductionofitstoxicity,a reductionofitsdose,andwouldincreasetreatmenteffectiveness.
∗Correspondingauthorat:FachbereichChemie,UniversitätKonstanz,Univer- sitätsstraße10,Fach722,D-78457Konstanz,Germany.Tel.:+497531882357;
fax:+497531883870.
∗∗Correspondingauthorat:InstitutdePharmacologieetdeBiologieStructurale, CNRSUMR5089,205RoutedeNarbonne,F-31077Toulouse,France.
Tel.:+33561175811;fax:+33561175900.
E-mailaddresses:andreas.zumbusch@uni-konstanz.de(A.Zumbusch),marie- pierre.rols@ipbs.fr(M.-P.Rols).
1Presentaddress:UMRSInsermU930“ImagerieetCerveau”,CNRSERL3106,Uni- versitéFranc¸oisRabelaisdeTours,CHULeBretonneau,10TerbdTonnellé,37044 ToursCedex9,France.
2InmemoryofLaurenceDupou-Cezanne,acolleagueandafriend.
Inrecentyears,promisingnewpossibilitiesfortargeteddeliv- eryoftherapeuticmoleculeshavebeendeveloped.Someofthese relyontheelectropermeabilizationoftheplasmamembrane(an approachalsotermedelectroporation).Electropermeabilization,a physicalmethodthatconsistsoftheapplicationofelectricpulses oncellsandtissues,wasintroducedinthe1970sandsubsequently developedinthe1980sforgenedelivery(Neumannetal.,1982).
Ithastheadvantageofbeingveryversatile,highlyefficient,sim- ple,andlowincost(Golzioetal.,2004).Theseattractivefeatures havebeenpointedoutinseveralreviews(CemazarandSersa,2007;
Gehl,2008;HellerandHeller,2010;Miretal.,2003).Applications havebeensuccessfullydevelopedforantitumordrugs(Campana etal.,2009;Landstrometal.,2010;Miretal.,1998;Rolsetal., 2002)andgenedelivery(AiharaandMiyazaki,1998;Helleretal., 1996;Miret al.,1999; Rolset al.,1998; Titomirovetal.,1991).
Meanwhile,electrochemotherapyhasbeenacceptedinanumber ofcountriesasapalliativecaretreatment(Giardinoetal.,2006;
Sersaetal.,2008;Spugninietal.,2008)andclinicaltrialsofelec- trogenetherapyareunder investigation(Daud etal., 2008;Low etal.,2009).Inthelattercase,plasmidDNA(pDNA)isthether- apeuticagentusedtotransfergeneticinformation.Widerspread useof this approachis hampered bythe factthat verylittle is knownaboutthemolecularandcellularmechanismssupporting thereorganizationofthecellmembranewhichaccompaniesthe pDNAuptake(Teissieetal.,2005).Itisevidentthatgainingabetter http://dx.doi.org/10.1016/j.ijpharm.2011.05.024
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-211256
understandingoftheinternalizationandintracellulartraffickingof pDNAisaprerequisiteforsafeuseofgeneelectrotransferinclin- icalapplications.SeveraltheoreticalmodelspostulatethatpDNA crossestheplasmamembranethrough“electropores”anddirectly accessesthecytosolduringelectropulsation(Escoffreetal.,2009a).
However,todatesuchmodelshavenotbeensupportedbyexper- imentaldataperformedoncells.Experimentsperformedongiant unilamellarvesicles(GUVs)showthat electroporesmaybecre- ated(Portetetal.,2009)and thattheseelectroporesmayallow thedirectentranceofplasmidDNAintothevesicles(Portetetal., 2011).Experiments performedat thesinglecelllevelonmam- maliancellsshowthatthemechanismsofmoleculedeliveryare much morecomplex. Theyreportedphenomenologicaldescrip- tionsofsmallmoleculesandgeneelectrotransferprocesses.Small molecules(below4kDa)andmacromolecules(above4kDa)enter thecellthroughwelldefinedcellularregionsfacingtheelectrodes.
Smallmolecules(e.g.propidiumiodide,cytotoxicdrugssuch as bleomycinandcisplatin)enterthecellinmembraneregionsfac- ingboththecathodeandanode.Thisdiffusivefluxisdrivenbythe concentrationgradientdifferencebetweentheexternalmedium andthecytosoland/orbyelectrophoresis(Escoffreetal.,2010a;
GabrielandTeissie,1997;Puciharetal.,2008).Incontrast,pDNA uptakeonlyoccursin themembrane regionfacingthecathode.
ThecurrentunderstandingisthatpDNAuptakerequiresanumber ofconsecutivesteps(Golzioetal.,2002):duringtheelectropul- sation,the plasmamembrane is permeabilized,pDNA migrates electrophoreticallytowardsthepermeabilizedcells,andisinserted intothepermeabilizedmembrane. Afterelectropulsation,pDNA translocationacrossthemembraneoccurs,andthepDNAmigrates towardsthenucleusand crossesthenuclearenvelope(Escoffre etal.,2009a).Thus,pDNAdoesnotenterthecellduringelectropul- sation,butistransientlytrappedinthepermeabilizedmembrane.
Itsdistributioninthepermeabilizedmembrane isnothomoge- neous.Instead,itappearsin“competentsites”whosesizeranges from0.1to0.5mm(Faurieetal.,2004,2010;Golzioetal.,2002).
ThepDNAinteractsinayetunknownwaywithsomemicrodomains oftheplasmamembrane,thenatureofwhichisalsonotknown.
These“competentmicrodomains”havenotbeendetectedduring theelectrotransferofpDNAinpurelipidmembranes(Portetetal., 2011).Asecondimportantobstacleingeneelectrotransfercomes frompDNAtranslocationacrossthemembraneanditsintracellular traffickingintothecytosol.Inpreviousinvestigations,fluorescently labeledpDNAcouldonly bedetectedinsidethecytosolseveral minutesafterelectropulsation(Golzioetal.,2002;Jeromeetal., 2009).Thenit wasseen asfreepDNAand inaggregates. These resultssuggestedthatthepDNAcouldcrossthemembranethrough poresor/andviaendocytosis.Themodelofelectroendocytosishas beensuggestedbyseveralgroups(Antovetal.,2004;Chernomordik etal.,1990;Glogaueretal.,1993;Klenchinetal.,1991;Mahrour etal.,2005;Rolsetal.,1995;Rosazzaetal.,2011;Rosembergand Korenstein,1997).Thestimulationoffluidphaseendocytosishas beenreportedbymostofthem.Anincreaseoffluidphasemark- ersuptakesuchasLuciferYellowordextranhasbeenshownto occurafterelectropulsation.Moreover,theinternalizationofmem- branephospholipidswasincreasedandthefluidphase markers uptakeshowedtemperaturedependence(Antovetal.,2004).In addition,thecytoskeletonseemstohaveanactiveinvolvementin themacromoleculesuptake.Arecentstudyontheactincytoskele- tonimplicationsintheDNAelectrotransfershowedtheformation ofactinpatchescolocalizingwiththeDNAaggregatesatthemem- brane(Rosazzaetal.,2011).Moreover,analterationoftheactin networkinducedadecreaseinboththeamountofpDNAinter- actingwiththecellmembraneandthegeneexpressionlevel.Up todate,thepDNAtranslocationmechanismisnotyetfullychar- acterized,itispossiblethatbothsuggestedmodels,electropores andelectroendocytosis,occuratthesametimeaspDNAasbeen
detectedinbothaggregatedandfreeformsinthecytoplasm(Golzio etal.,2002).However,ratherlittledataconcerningtheintracellu- lartraffickingofelectrotransferredpDNAwhichcouldshedlight onthetranslocationmodehavebeenpublishedtodate.
Fromwhathasbeendetailedabove,wecansuggestthatthe membranecompositionislikelytoaffectitsinteractionwithpDNA molecules. This in turn could influence the pathway of pDNA entryintothecell (Rejmanet al.,2004).Inorder toverify this assumption,weinvestigated theroleof cholesterolin thegene electrotransferprocess.Cholesterolrepresents30%ofmembrane lipidsofmammaliancellsandhasamajorinfluenceonthebiophys- icalpropertiesoftheplasmamembrane,suchase.g.membrane fluidity and thickness (Maxfield and Tabas, 2005).It is mainly foundintheinnerleafletoftheplasmamembraneofcellsandis alsoenrichedinmembranedomainsinvolvedinalargevarietyof processessuchasproteintransport(Yoshimorietal.,1996),sig- naltransduction(SimonsandToomre,2000),andpathogenentry (Fivazetal.,1999;Lafontetal.,2004;Manesetal.,2003).More- over,cholesterolisinvolvedinclathrin-andcaveolin/raft-mediated endocytosis(DohertyandMcMahon,2009).Itplaysakeyrolein moleculeentryintocellsandcanthereforehaveamajorroleinthe interactionofpDNAwiththeplasmamembrane,thetranslocation ofpDNAthroughthemembrane,anditsintracellulartrafficking.
Thequestionsweaddresshereare:Ischolesterolinvolvedinthe electropermeabilizationprocessofthemembrane?Doesitpartici- pateintheformationofthepDNAinteractionwiththemembrane?
Whatisitseffectonelectroporationmediatedgenedelivery?How doesthepDNAcrosstheplasmamembrane?Inordertoanswer thesequestions,wehaveperformedcholesteroldepletionexperi- ments,whichallowustomeasureitseffectonthedifferentstepsof theelectroporationprocess.Inaddition,weperformedcolocaliza- tionstudieswithseveralendocytoticmarkerswhichgiveinsight intotheinternalizationpathwaysofpDNA.
2. Materialsandmethods
2.1. Cellculture
Thewild-type(Torontostrain)ChineseHamsterOvary(CHO) celllinewasselected forits ability togrowinsuspension in a spinner or platedonPetri dishes or on22mm×22mm micro- scopeglasscoverslips(Lab-Tek®II,NuncTM,Roskilde,Denmark).
CHO cells were grown in Eagle’s minimum essential medium (MEM1011,Eurobio,LesUlis,France)supplementedwith8%heat inactivatedfetal bovine serum(Gibco®,InvitroGenTM, Carlsbad, CA),0.584g/Ll-glutamine (Gibco®,InvitroGenTM,Carlsbad, CA), 3.5g/Ld-glucose(Sigma–Aldrich,St.Louis,MO),2.95g/Ltryptose- phosphate (Sigma–Aldrich, St. Louis, MO), 100U/mL penicillin (Gibco®,InvitroGenTM,Carlsbad,CA),1mg/Lstreptomycin(Gibco®, InvitroGenTM,Carlsbad,CA)andBMEvitamins(Sigma–Aldrich,St.
Louis,MO).Thecellswereroutinelysub-culturedevery2daysand incubatedat37◦Cinahumidifiedatmospherewitha5%CO2incu- bator(Golzioetal.,2002).
2.2. Electropulsationprocedure
ElectropulsationwasperformedusingaCNRScellelectropul- sator (Jouan, St. Herblain, France), which delivers square-wave electric pulses. The amplitude (U), the duration (T), the num- ber (N), and the frequency (f) of the electric pulsetrain were controlled independently. Anoscilloscope (Enertec,St. Etienne, France)monitoredthepulseshape.FortheCHOcells,theoptimum electropermeabilizationparametersare10pulsesof5msat1Hz frequencywithanelectricfieldstrengthbetween0.4and0.8kV/cm (Golzioetal.,2002).Stainless-steelelectrodeswereusedinorder
toavoidelectrochemicalreactionsofthemetal.Severaltypesof electrodeswereuseddependingontheproceduretobeapplied:
(i)parallelplateelectrodes(10mmlength,5mminter-electrode distance)wereusedtopulsecellsuspensionsand(ii)parallelrod electrodes(10mmlength,0.5mmdiameter,7mmgap)wereused topulsecellsunderfluorescencemicroscope(Mazeresetal.,2009).
Theelectropulsationswereperformedinalowconductivitybuffer (10mMK2HPO4/KH2PO4,pH7.4,1mMMgCl2,250mMsucrose), whichminimizedthetemperatureincreaseduetotheapplication oftheelectricfield.Cellsweremaintainedinthepulsationbuffer for5minaftertheapplicationoftheelectricpulsestoallowfor membraneresealing.
2.3. Cholesteroldepletion
Thecholesteroldepletionwasperformedusing5mMmethyl- b-cyclodextrin (MbCD) (Sigma–Aldrich, St. Louis, MO) in the culture mediumwithout serum.Cell suspensions werewashed withPBSandsuspendedinthedepletionmediumatadensityof 106cells/mL.Theywereincubatedinthespinnerfor15minat37◦C withlowagitation(70–100rpm).Controlcellswerehandledand incubatedunderthesameconditionsusingtheculturemedium withoutserum.Standard techniqueswereusedforquantitative analysis: thepercentageof cholesteroldepletionwasevaluated accordingtoCezanneetal.(1992);aftercelllysisandseveralcen- trifugations, themembrane proteinswere quantified usingthe Lowry method (Lowry et al., 1951); lipids were extracted and measuredashasfirstbeendescribedbyBlighand Dyer(1959);
phosphate measurementsweredoneaccording toRouseret al.
(1970),cholesterolmeasurementsaccordingtoZlatkisetal.(1953).
2.4. Electropermeabilization
A cell suspension (106cells/mL) wasincubated in a spinner either in the depletion medium for the MbCD treated cells or intheculturemediumwithoutserumforthecontrolcells.After the 15min incubation time, the cells were centrifuged (5min, 120g). For each electropermeabilization condition 5×105 cells weresuspendedin100mLpulsationbuffercontaining100mmol/L propidium iodide (PI). The pulse series were applied at field strengthsbetween0.1and1kV/cm,theothersparameterswere chosenashasbeendescribedabove.After5min,thecellswere transferredtoPBSbufferandanalyzedusingflowcytometry(FAC- Scan,BDBiosciences,LePontdeClaix,France)viatheFL2channel (560nm≤em≤600nm).Thepercentageoffluorescentcellsthere- forecorrespondstothepercentageofelectropermeabilizedcells (i.e.theefficiencyofelectropermeabilization)andthemeanflu- orescenceintensityreflectsthemeanquantityofPIincorporated intothecells(i.e.thelevelofelectropermeabilization)(Rolsand Teissie,1998).
2.5. InteractionpDNA/membrane
pEGFP-C1 plasmid (Clonetech, Palo Alto, CA) contains the EnhancedGreenFluorescentProtein(EGFP)geneunderthecon- trol of the cytomegalovirus promoter. Plasmids were prepared fromtransformedDH5˛EscherichiacoliusingtheMaxiprepDNA purificationsystemaccordingtomanufacturerinstructions(Qia- gen,Chatsworth,CA). ThispDNAwasstainedstoichiometrically with a 30.4×10−5mol/L TOTO-1 solution (Molecular Probes®, InvitroGenTM, Eugene, OR), for 1mg/mL pDNA solution during 60minonice(e.g.anaveragebasepairtodyeratioof5)(Escoffre etal.,2009b;Ryeetal.,1992).
AftertheMbCDincubation,thecellswerecentrifuged(5min, 120g)andsuspendedin100mLpulsationbuffercontaining2mg labeledpDNAatarateof2×105cellspercondition.Forthispur-
pose,eachsuspensionwasdepositedbetweentherod-electrodes whichwerepreviouslyfixedontheLab-Tek®-IIinstalledonthe fluorescence microscope. The electric field parameters were as describedabovewithstrengthsbetween0.4and1kV/cm.
Cells were observed with a Leica 100×, 1.3 numerical aperture oil immersion objective mounted on a Leica DMIRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany). The excitation source was a Leica mercury lamp 100 HBO. The wavelengths were selected using the Leica L4 filter block (450nm≤ex≤490nm; dichroic mirror pass band, 515nm≤em≤560nm).ImageswererecordedwiththeCellScan System fromScanalytics (Billeria,MA) equipped witha cooled charge-coupled devicecamera (PrincetonInstruments, Trenton, NJ). This digitizing set-up allowed for quantitative localized analysisof thefluorescenceemission along thecellmembrane.
Plot histograms detected local fluorescence intensity increase abovethebackgroundleveloutsideofthecells.Twocharacteristic parameterswereused:thepeakintensityandtheintegralunder thepeak.Bothweredirectlyrelatedtothenumberoffluorescent moleculeslocallypresent(Faurieetal.,2010).
CellswerealsotransferredinPBSbufferandanalyzedusingflow cytometry(FACScan,BDBiosciences,LePontdeClaix,France)via thechannelFL1(510nm≤em≤540nm).Thepercentageoffluo- rescentcellsgivesthepercentageofcellsbeingininteractionwith thelabeledpDNAandthemeanfluorescenceintensityassociated isproportionaltothemeanamountofpDNAininteractionwith thecells.
2.6. Geneelectrotransfer
Cellsuspensionswereculturedatadensityof106cells/mLin spinners,treatedornotwithMbCD,andcentrifugedtoremove themedia.Theyweresuspendedinthepulsationbuffercontain- ing2mgpEGFP-C1plasmidDNAand106cellspercondition.The pulse parameters were 10 pulses of 5ms at 1Hz with ampli- tudesvaryingbetween0.4and0.8kV/cm.Afterthe5minwaiting timetoallowcellstoreturntotheimpermeablestate,cellswere seeded in 35mm Petri dishes for 24h in the culture medium (seeSection2.1).Subsequently,theywerewashed,harvestedwith trypsin-EDTA,suspendedinPBS,andanalyzedusingflowcytom- etry(FACScan,BD Biosciences,LePont deClaix,France)viathe channelFL1(510nm≤em≤540nm).Thepercentageoffluores- centcellsgivesthepercentageofEGFPtransfectedcells(i.e.the efficiency of transfection) and the mean fluorescence intensity associatedisproportionaltothemeanamountofEGFPexpressed bythecellpopulation(i.e.theleveloftransfection).
2.7. Cellviability
Cell viabilitywasdetermined bytheability of cellsto grow and divideover a24hperiod(corresponding tomorethanone generation)(Kuengetal.,1989).Viabilitywasmeasuredbymoni- toringcellgrowththroughacolorationmethodwithcrystalviolet (Merck&Co.,WhitehouseStation,NJ).Aftertreatment,thecells wereculturedtoadensityof106cells/mLintheculturemedium on35mm Petridishesfor 24h. After3washingswithPBS,the cells were incubatedwith0.1% crystal violet in PBSfor 20min undergentleagitation(20rpm)atroomtemperature.Then,the cellswerewashed3timeswithPBSandlysedwith10%aceticacid (Sigma–Aldrich,St.Louis,MO)for10minundergentleagitationat roomtemperature.100mLofthelysatewasdilutedin2mLPBS andtheopticaldensity(OD)wasmeasuredat595nm.TheODis proportionaltotheamountofcellsandthemeasurementswere normalizedinrelationtotheuntreatedcells (controlcondition) (Golzioetal.,1998).
2.8. Intracellulartrafficking 2.8.1. Endocytoticmarkers
ThepDNAwascovalentlystainedwithCy3dyeusingtheLabel IT®NucleicAcidLabelingKit(Mirus®,Madison,WI)accordingto manufacturerinstructions. Theendocytotic markerswereAlexa Fluor® 647-transferrin (MolecularProbes®,InvitroGenTM, Carls- bad, CA) to test for the clathrin-mediated endocytosis path or Alexa Fluor® 647-cholera toxin subunit B (Molecular Probes®, InvitroGenTM,Carlsbad,CA)totestforthecaveolin/rafts-mediated endocytosispath.24hpriortoelectropulsation,2×105cellswere culturedonLab-Tek® IIslides.Then,thecellswereincubatedat 4◦Cfor30mintoinhibitanyendocytoticprocesses.Thecellswere subsequentlywashedwithpulsationbufferandpulsedin300mL pulsationbuffercontaining1mgCy3-labeledpDNAwiththe10mm lengthplate-electrodes.Five minutesafter electropulsation,the pulsationbufferwasremovedandicecoldculturemediumcontain- ingtheendocytoticmarker,either50mg/mLtransferrinor1mg/mL choleratoxinsubunitB,wasadded.Thecellswereagainincubated at4◦Cfor 30mintoallowfortheendocytotic markertointer- actwiththeplasmamembranewithoutendocytosistakingplace.
Afterwards,thecellswereincubatedfor15minat37◦Ctoinduce theendocytosis.
2.8.2. Fluorescencemicroscopy
Asolidstatelaser(CoboltJive561nm,75mW,CoboltAB,Stock- holm, Sweden) and a diode laser (HL6535MG 658nm, 90mW Hitachi,Thorlabs,Newton,NJ)wereusedtoexcitetheCy3orAlexa Fluor®647labeledmolecules.Thelaserpowerswereadjustedwith neutraldensityfilters.Toobtainhomogeneousillumination,the laserswerecoupledintoamulti-modefiber(0.22±0.02NA,Optro- nisGmbH,Kehl,Germany)whichwasshakentodestroycoherence andsuppressinterferenceeffects.Thefluorescencewascollected usinga100×/1.4NAoilobjective(HCXPLAPO,LeicaMicrosystems GmbH,Wetzlar,Germany),separatedfromexcitationlightwitha dualbanddichroicmirror(HCDualbandBeamsplitterz561/660, Semrock,Rochester,NY).Thefluorescenceemittedfromthegreen labels wasselectedusing a long-pass filter(Raman emitterRU 568,Semrock,Rochester,NY)andaband-passfilter(BrightLineHC 593/40,Semrock,Rochester,NY).Thefluorescenceemittedfrom theredlabelswasselectedusingalong-passfilter(Ramanemit- terRU664,Semrock,Rochester,NY).Imageswererecordedusing anEMCCDcamera(AndoriXON,AndorTechnologyPLC,Belfast, Ireland).Acquisitionsweresequentiallytakentoavoidcrosstalk.
2.8.3. Colocalizationanalysis
The image processing and analysis were performed using ImageJ(NIH,Bethesda,MD,USA)andaprocedureadaptedfrom (Lachmanovichetal.,2003).Indetail,pointnoiseonrawimages wasremovedbyapplyinga3×3pixelsmedianfilter.Toremove diffusebackground,anadditionalfilteringstepwasperformed.For thispurpose,firsttheshortmorphologypluginofImageJwasused withthesizesettoacirclewith15pixelsdiameter.Theresult- ingimagewassubtractedfromthepointnoisefilteredimage.This processallowsforthesuppressionofthebackgroundwithoutsig- nificantlychangingtherelativeintensitiesoftheparticles.Further objectsegmentationandquantificationwereperformedwiththe JACoPpluginofImageJ(BolteandCordelieres,2006).Thelatterwas usedtomanuallydefinethethresholdabovewhichallpixelsare consideredtobepartofanobject.Theobjectcountingmoduleofthe plugingivesthetotalnumberofparticlesandthatofcolocalizing onesforeachchannel.Twooptionsareavailablefordefiningcolo- calization.Thefirst,calledtheoverlapapproach,definesthecenter ofmassofeachobjectbelongingtothegroupA(e.g.greendetec- tionchannel)andtestsifitfallsintotheareacoveredbyanobject belongingtothegroupB(e.g.reddetectionchannel).Colocalization
thenisamatchbetweenthecentersofmassofgroupAobjectswith theareacoveredbygroupBobjects.Thedegreeofcolocalization isgivenbythepercentageofAobjectscolocalizingwithBobjects.
Thisoperationcanbeperformedseparatelyforeachchannel.The secondoptiontodefinecolocalizingobjectsiscallednearestneigh- bordistanceapproach.Aswiththeoverlapmethod,thecenterof massofeachobjectisdefined,butthistimeforbothgroups.The softwaremeasuresthedistancebetweenthecentersofmassofA groupobjectsandthecentersofmassofBgroupobjects.Asthe numberofobjectsoftendiffersfromonechanneltotheother,the programselectsthechannelwithfewerobjectsandsearchesthe nearestneighborinthesecondchannelwhichhasmoreobjects.
Ifthisdistanceisfoundtobebelowtheopticalresolutionofthe acquisitionsystem,thetwoobjectsareconsideredtobecolocal- ized.Thedegreeofcolocalizationisthengivenbythepercentage ofobjectsinthefirstchannelcolocalizingwithobjectsofthesecond channel.
2.9. Statisticalanalysis
Errorbarsrepresentthestandarderrorofthemean(SEM).The statisticalsignificancesofdifferencesbetweenthecontrolandthe MbCD treated cells for allexperimentswere evaluatedusing a pairedStudent’st-test.The degreeofsignificance is givenwith these following labels:NS, not significant; *p<0.05; **p<0.01;
***p<0.001.
3. Results
First, weanalyzed theeffect ofcholesterol depletiononthe differentstepsinvolvedinthepDNAelectrotransferprocess:mem- braneelectropermeabilization,pDNA/membraneinteraction,and geneexpression.Then,wedeterminedthemechanismofpDNA translocationthroughtheplasmamembraneanditsintracellular traffickingusingendocytoticmarkers.Forthat,cellswerepulsed underelectricfieldparameterswhichwereknownfromprevious studiestoleadtoefficientgeneexpression.
3.1. Effectofcholesteroldepletiononelectrotransfectionsteps Inorder todeterminetowhat extentthecholesterolhasan influenceongeneelectrotransfer,CHOcellswereincubatedwith methyl-b-cyclodextrin (MbCD). Indeed, cyclodextrinsare cyclic oligomersofglucosethathavetheabilitytosequesterlipophiles intheirhydrophobiccore(Pithaetal.,1988).Treatmentofcellcul- turewiththeMbCDdrugresultsindepletionofcholesterolfrom theplasmamembranefollowedbydissociationofproteinsfrom rafts(SimonsandToomre,2000)anddisturbanceofclathrin-coated vesiclesformation(Rodaletal.,1999).Therefore,MbCDcanaffect clathrin-andcaveolin/raft-mediatedendocytosis.Ourexperimen- talconditions inducedapproximately40% cholesteroldepletion witharather low reductionof thephospholipids/proteinsratio (12%).Thisdepletionrateremainedstableforupto2h.Thesecon- ditionsallowedpreservingthecellviabilityatahighlevel(upto 80%).
3.1.1. Effectofcholesteroldepletiononmembrane electropermeabilization
Theresultsregardingtheuptakeofpropidiumiodideintocon- trolandcholesteroldepletedCHOcellsexposedtoelectricpulse seriesareshowninFig.1.Intheabsenceofanelectricfield,25–30%
ofthecellswerefluorescentduetothepresenceofdeadcellsinthe population.Thispercentagewasthesameforthecontrolaswellas thecholesteroldepletedcells(Fig.1a).Theapplicationofelectric fieldpulsesresultedinthepermeabilizationofcells.Thepercentage ofelectropermeabilizedCHOcellswasnotaffectedbycholesterol
Fig.1.EffectofcholesteroldepletionontheelectropermeabilizationofCHOcells.
CellswereincubatedwiththedrugMbCDfor15minandexposedtoanelectric field.10pulsesof5msat1Hzwithstrengthsvaryingbetween0and1kV/cmwere applied.Themembranepermeabilizationwasdetectedbypropidiumiodideuptake andmeasuredusingflowcytometry.(a)Percentageofpermeabilizedcellsand(b) meanfluorescenceintensity.Thewhitedotsrepresentthecontrolcells,i.e.not treatedwiththedrugMbCD,theblackdotscorrespondtothetreatedcells.(c)Cell viabilityofthecontrolcells(whitebars)andthecholesteroldepletedcells(black bars).8independentexperimentswereperformed.
depletion.Theelectropermeabilizationthresholdwas0.3kV/cmfor bothcontrolandtreatedcells.Abovethatthreshold,anincreaseof theelectricfieldstrengthsfurtherincreasedthefractionofelec- tropermeabilizedcells.Foranelectricfieldstrengthof0.8kV/cm, nearly 90% of all cells are permeabilized. Higher electric fields donotleadtoafurtherincreaseinthenumberofpermeabilized cells.Theefficiencyofpermeabilization,relatedtotheamountof moleculeselectrotransferredintothecells,wasquantifiedbymea- suringthemeanfluorescenceintensityvalueofthecellpopulations (Fig.1b).Intheabsenceofelectricfields,thenativepermeability washigherforthecholesteroldepletedcellsthanforthecontrol ones.TheMbCDtreatmentdidnotaffectthemeanfluorescence intensityofthePI(datanotshown).Inthepresenceofelectricfields, the cell electropermeabilization was still higher for theMbCD
treatedcells,buttheeffectoftheelectricfield(theincreaseinflu- orescence,i.e.in theamountofelectrotransferredPImolecules) wasthesameasforthecontrolcells.Thedifferenceinpermeabil- itybetweentreatedanduntreatedcellsremainednearlyconstant forallelectricfieldstrengths.Inaddition,wedeterminedtheeffect ofelectricpulsesonthecellviability,whichwasmeasured24h later(Fig.1c).Asmentionedabove,cholesteroldepletionresultedin 20%decreaseincellviability.Theeffectofelectropermeabilization oncellviabilitywasthesameforcontrolandcholesteroldepleted cells.Inconclusion,cholesteroldepletiondidnotsignificantlyaffect theelectropermeabilizationprocess,i.e.theelectrotransferofsmall moleculesintocellsandthecellviability.Itonlychangesthenative permeabilityoftheCHOcells.
3.1.2. EffectofcholesteroldepletiononpDNA/membrane interaction
Videomicroscopyatthesinglecellleveloffersdirectaccessto theearlyeventsofpDNAdeliveryacrosstheelectropermeabilized membrane.ImagesofpDNA/membraneinteractionwereacquired usingfluorescencemicroscopywithTOTO-1labeledpDNAinthe minutesafterapplicationof thepulses. Aspreviouslydescribed (Golzioetal.,2002)andasshowninFig.2aandb,pDNAinteracted intheformofaggregateswiththeelectropermeabilizedpartofthe plasmamembranefacingthecathode.Thisprocessisstillpresent incholesterol depletedcells.pDNAdoesnotenterthecelldur- ingelectropulsation,butinsteaditistrappedatthepermeabilized membrane.OnlyseveralminutesafterelectropulsationispDNA detectedinside thecytoplasm. Consequently,the interactionof pDNAatthemembranelevelcanbequantified.AsshowninFig.2c, theamountofpDNAinteractingwiththepermeabilizedmembrane ofcholesteroldepletedcellswasnotsignificantlydifferentfrom thatofcontrolcells.Flowcytometryanalysisshowedsimilarresults (Fig.2dand e).Whatever theelectricfieldstrengthapplied,the numberofcellsinteractingwiththepDNAandthemeanlevelofflu- orescenceintensitywerenotaffectedbythecholesteroldepletion.
Inconclusion,theelectro-mediatedformationofpDNAaggregates withthecellmembraneisnotacholesterol-dependentprocess.
3.1.3. Effectofcholesteroldepletionongenetransferand expression
Tohighlightanyotherstepsofgeneelectrotransferthatmay beaffected by cholesterol,we measured EGFPgene expression ofviablecells,whichisthefinalstepofthemechanismofgene electrotransfer.Withoutelectropulsation,controlandcholesterol depleted cells did not expressEGFP (Fig.3a and b). Moreover, thepresenceoftheMbCDdrugdoesnotaffectthemeanfluores- cenceintensityoftheEGFP(datanotshown).Atthethreeelectric fieldstrengthvaluesleadingtomembranepermeabilization,cells expressedtheEGFPreportergene.Boththepercentageoftrans- fectedcellsandtheassociatedfluorescenceintensityofcholesterol depletedcellsweredramaticallylowerthanthoseofcontrolcells.
At0.4kV/cm,cholesteroldepletioninduceda3.5-folddecreaseof thetransfectionefficiencycomparedtothecontrolcondition.At 0.6kV/cm,thetransfectionefficiencyofcholesterol-depletedcells was5-foldlowerthanthatofcontrolcellsandthetransfectionlevel decreasedbyafactorof2.Theeffectofcholesteroldepletiononthe transfectionefficiencywaslessvisibleat0.8kV/cm,butthetrans- fectionleveldecreasedbyafactorof14.Cellviabilitywasthesame forcontrolandtreatedcellspulsedinthepresenceofpDNA(Fig.3c).
Theseresultsshowthatcholesterolcanplayamajorroleinthe pDNAtranslocationthroughthemembraneand/oritsintracellular trafficking.
Fig.2.EffectofcholesteroldepletiononthepDNA/membraneinteractionwithCHO cells.CellswereincubatedwiththedrugMbCDfor15minandexposedtoanelectric field.10pulsesof5msat1Hzwithfieldstrengthsvaryingbetween0and1kV/cm wereapplied.ThepDNAwaslabeledusingTOTO-1dyeandobservedusingwidefield microscopy.(a)Controlcellsand(b)treatedcells(c)quantificationofthisinteraction bymeasuringthefluorescenceintensityanddividingitbytheareaoftheinterac- tion(n=30cellspercondition).TheTOTO-1-pDNA/membraneinteractionwasalso quantifiedusingflowcytometrygivingaccessto(d)thepercentageofcellsinter- actingwithlabeledpDNAandto(e)theassociatedmeanfluorescenceintensity.
3independentexperimentswereperformed.Forallgraphs,thewhitebarscorre- spondtothecontrolcellsandtheblackonestothecholesteroldepletedcells.Scale bar=15mm.
Fig.3.Effectofcholesteroldepletionongeneexpressionafterelectroporationof CHOcells.CellswereincubatedwiththedrugMbCDfor15minandexposedtoan electricfieldinthepresenceofpEGFP-C1.10pulsesof5msat1Hzwithstrengths varyingbetween0and1kV/cmwereapplied.TheEGFPproteinexpressionwasmea- sured24haftertheapplicationoftheelectricfield.(a)Percentageofcellsexpressing theEGFPprotein,(b)meanfluorescenceintensityoftheEGFPprotein,(c)cellviabil- ity.Forallgraphs,thewhitebarscorrespondtothecontrolcellsandtheblackones tothecholesteroldepletedcells.4independentexperimentswereperformed.
3.2. Colocalizationstudywithendocytosismarkers
The effects of cholesterol depletion ongene electrotransfer, as well as the role of actin which we just reported (Rosazza et al., 2011), prompted us todetermine thepathway of pDNA entryintotheelectropermeabilizedcells.Indeed,bothactinand cholesterol are involved in endocytotic processes. In order to determinethemannerofpDNAentryafterelectropulsation,we performeddualcolorobservationsofCy3-labeledpDNAandtwo endocytoticmarkers:AlexaFluor®647labeledtransferrin(TF)to
Fig.4. ColocalizationofpDNAwithaclathrin-mediatedendocytosismarker,transferrinandwithacaveolin/raft-mediatedendocytosismarker,choleratoxinsubunitB.
Cy3labeledpDNAwaselectrotransferredontoCHOcellsviatheapplicationof10electricpulsesof5msat1Hzand0.4kV/cm.ObservationseitherwithAlexaFluor®647- transferrin,orwithAlexaFluor®647-choleratoxinsubunitBwereperformedusingwidefieldmicroscopy.(aandd)pDNA,(b)transferrin,(e)choleratoxinsubunitB,and (candf)mergeofthetwochannels.Thewhitelinesontheimagerepresentthecellshapes.Scalebar=5mm.
assess theclathrin-mediated endocytosisand AlexaFluor® 647 labeledcholeratoxinsubunitB(CTB)toevaluatethecaveolin/raft- mediatedendocytosis.
Inafirstqualitativeapproach,microscopydatashowthatthe pDNApartiallycolocalizeswithboththeTFandtheCTB(Fig.4).
Thismeans that pDNAis internalized, atleast in part,via two endocytotic processes. To determine the involvement of each pathwayinthepDNAinternalization,colocalizationquantification wasperformedusingobject-basedapproaches,whichanalyzethe spatialdistributionofthefluorescencesignals(Lachmanovichetal., 2003).Thecolocalizationanalysisdoesnotrelyonthecoincidence ofindividualpixelsbutonthecoincidenceofstructures.Therefore, eachpixelisnotconsideredasapartofanimagebutasapartof a uniqueobject. Object-based methodsalsodiscriminate better betweensignalscomingfromstructuresandthoseoriginatingfrom background.Thesemethodsareofspecialinterestfortheanalysis ofsubcellularstructureshavingspecificshapesandsizescloseto theopticalresolutionlimitofmicroscopes.Afterprocessing(Fig.5a andc),theimagesaresegmented(Fig.5banddred,patches).All thepixelsabovealimitvalueareconsideredtobepartofanobject.
Theedgesofthefluorescentstructuresarethendelimited.From each obtainedobject, thecenterofmass isdetermined(Fig.5b andd,greendots).Theanalysisusingtheoverlapapproachtests whetherthecentersofmassofobjectsinthefirstchannelfallin areascoveredbyobjectsofthesecondchannel(Fig.5bandd).This canbedoneseparatelyforbothchannels.Thenumberofcolocaliz- ingobjects(Fig.5bandd,yellowdots)amongthetotalamountof objectscountediscalculatedasthepercentageofcolocalization.
Theanalysisusingthenearestneighbordistanceapproachmea- suresdistancesbetweenthecentersofmassofobjectsinthefirst channelandthecentersofmassofobjectsinthesecondchannel (Fig.5f).Thesedistancesarecompared totheopticalresolution limitoftheacquisitionsystem.Twoeventscolocalizewhenthedis- tanceoftheircentersofmassisbelowtheopticalresolutionofthe
system(Fig.5f,blueandpurpledots).Thedegreeofcolocalization isalsogivenasapercentageofobjectsofchannelonecolocalizing withchanneltwo.Fig.5gshowsthetwodefinedpercentagesof colocalizationbetweenthepDNAandthetwoendocytoticmarkers.
Thepercentagesofcolocalizationdeterminedbythetwodifferent approachesyieldavalueofapproximately50%±7%ofcolocaliza- tionofthepDNAwiththeCTBandof25%±7%withtheTF.We thereforeconcludethat themain endocytoticprocess occurring duringpDNAelectrotransferiscaveolin/raft-mediatedendocytosis.
Clathrin-mediatedendocytosisisalsoobserved,butlessfrequently.
4. Discussion
Theobjectivesofourworkweretoinvestigatethepotential role of cholesterol in the process of pDNA electrotransfer and tocharacterize someroutesof itsinternalizationintocells. We firstshowedthatthenativecellpermeabilityis affectedbythe plasmamembranecholesterollevel.Inagreementwithprevious investigations,thisresultcanbeexplainedbyanincreaseinmem- branefluidity,whichisinfluencedbythecholesterollevel.Indeed, thedepletion or theaddition ofcholesterol in lipid vesicles or bilayerfilms,respectively,increasesordecreasestheirpermeabil- itywithrespecttowater,ions,glucoseandothersolutes(Lande etal.,1995;Mathaietal.,2008;Papahadjopoulosetal.,1972).Our datashowthat,whatevertheelectricfield strength,cholesterol depletionhasnosignificanteffectonpermeabilizationleveland efficiencyaccessedbyPIuptake.Thesedataare,toourknowledge, thefirstresultingfromcellexperiments.Previousdataontheeffect of cholesterol onmembrane electropermeabilization havebeen obtainedusingmolecular dynamics simulationsand usinglipid bilayersorvesiclesasmembranemodels.Theyshowedthatmem- braneelectroporationrequiredanenhancementorareductionof theelectricfieldstrengthwhenthecholesterolcontentincreasedor decreased,respectively(Fernandezetal.,2010;Kakorinetal.,2005;
Fig.5.AnalysisofthecolocalizationofpDNAwithaclathrin-mediatedendocytosis marker,transferrinandwithacaveolin/raft-mediatedendocytosismarker,cholera toxinsubunitB.Thequantificationwasperformedusingobject-basedmethods;
theoverlapapproachandthenearestneighbordistanceapproach(Lachmanovich etal.,2003).Thefirststep,commonforthetwomethodsofanalysis,istheimage processinggivingsuchimages:(a)pDNAchannel,(c)choleratoxinsubunitchan- nel,and(e)mergeofthetwochannels.Foracomparison,therawimagesareshown,
KoronkiewiczandKalinowski,2004;vanUitertetal.,2010).More- over, simulation datashowedthat pore formationkineticswas underthecontrolofcholesterollevel.Thesedifferencesbetween modelsandcellsclearlyshowthatthepermeabilizationofplasma membranesismorecomplexthanthatdescribed bymembrane models.Thiscanbeexplainedbythefactthatthecompositionin proteinsandinlipidsofaplasmamembrane(variousphospho- lipidsand proteins,membrane asymmetry,interactionwiththe cytoskeleton...)ismuchmorecomplexcomparedtomembrane models(oneorthreephospholipids)(vanMeeretal.,2008).
We further show that cholesterol depletion has no effect onpDNAinteractionwiththeplasmamembrane subsequentto theelectricfield application,a keystepofpDNAelectrotransfer (Escoffreetal.,2010a;Faurieetal.,2010;Golzioetal.,2002;Phez etal.,2005).Ifmembraneelectropermeabilizationisanecessary stepforpDNA/membraneinteractiontooccur,theelectrophoret- icallydriven insertionof thepDNAin theplasmamembrane is crucialforgenetransfer.Cholesteroldepletionseemstohaveno effectoneitherstep.Increasingtheelectricfieldstrengthindeed enhancesthepDNAinsertioninthemembraneinthesamepro- portionsforboththecontrolandthecholesteroldepleted cells.
TheseresultsshowthatthepDNA/membraneinteractionstepis acholesterol-independentprocess.Moreover,thecholesterolcon- tentcannotbeanimportantparameterdefining“competentsites”
forpDNA/membraneinteraction.
Theresultsdescribedaboveindicatethatcholesterolmightnot haveanyinvolvementinthefirststepsofthegeneelectrotransfer processbut,asgeneexpressionisconsiderablyaffectedincholes- teroldepletedcells, furtherstepscouldbeaffected.Indeed,our resultsshowthatbothtransfectionlevelandefficiencyweredra- maticallylowercomparedtothoseofthecontrol,forallelectric fieldstrengthsapplied.Cholesterolmayhaveaninvolvementin thepDNAtranslocation acrosstheplasmamembraneand/orits intracellular trafficking. Because cholesterol is mainly found in plasmamembranes,weproposethatitisthetranslocationstep thatis alteredbythereduction ofcholesterol content.Thishas beendemonstratedfortheendocytosisofseveraldifferentparti- cles(Kabouridisetal.,2000;Laietal.,2008;Norkin,1999;Norkin etal.,2001;Rejmanetal.,2004).
Thecolocalizationstudiesperformedinthisworkclearlyshow thatpDNAisendocytosed.pDNApartiallycolocalizedwithboth thecholeratoxinsubunitBandthetransferrin.Thismeansthat caveolin/raft-aswellasclathrin-mediatedendocytosisisusedby theelectrotransferredpDNAtoenterthecell.Thequantificationof therespectivepathwaysindicatesthataround50±7%ofthepDNA entersintothecellsviathecaveolin/raft-mediatedpathwayand around25±7%viatheclathrin-mediatedpathway.Whileprevious workssuggestedthatanendocytoticprocesscouldoccurduring geneelectrotransfer(Antovetal.,2005;Klenchinetal.,1991;Rols etal.,1995;Satkauskasetal.,2001),thisisthefirsttimethatthis
respectively,inFig.4d–f.Fortheoverlapapproach,theImageJpluginJACoPtested whetherthecentersofmassofeachgreenpatch(shownasagreendot)fallswithin theareacoveredbyaredpatch.(b)pDNAasredpatchesandcholeratoxinsubunit Basgreendots,(d)choleratoxinsubunitBasredpatchesandpDNAasgreenones.
Yellowcolorrepresentsthecolocalizingobjects.Forthenearestneighbordistance approach,theprogramteststhedistancebetweenthecentersofmassofgreen objectsandthecentersofmassofredobjects.(f)pDNAasgreendotsandcholera toxinsubunitBasreddots.Whenthedistanceisbelowtheopticresolutionlimit oftheacquisitionsystem,thedotsare,respectively,representedasblueandpurple dots.Inbothcases,thepercentagesofcolocalizingobjectswerethuscalculated.The analysisofmorethan100cellswitheachendocytoticmarkergavethepercentages showninthegraph(g).Theblackbarsrepresentthepercentagesofcolocalization withthecholeratoxinsubunitB,i.e.withthecaveolin/raft-mediatedendocytosis (CAV-MEonthegraph),thegreybarsrepresentthepercentagesofcolocalization withthetransferrin,i.e.withtheclathrin-mediatedendocytosis(CLA-MEonthe graph).Scalebar=5mm.(Forinterpretationofthereferencestocolorinthisfigure legend,thereaderisreferredtothewebversionofthisarticle.)
isclearlydemonstrated.Theseresultsmayexplain thedecrease ofgeneexpressionobservedwhencellsarecholesteroldepleted.
Sincecholesterolisinvolvedinendocytoticprocesses(Dohertyand McMahon,2009),itsdepletionisexpectedtoreducetheendocy- toticactivityandmay,inourcase,reducethepDNAuptakewitha subsequentlowerpDNAquantityreachingthenucleusandbeing availableforgeneexpressiontooccur.Thisfinallygivesoneexpla- nationfortheobservationofreducedlevelsofgeneexpressionin cholesteroldepletedcells.Oneshouldnotethatcholesteroldeple- tionmayinadditionreducethefurtherintracellulartrafficking, endosomalescape, and/ornucleartargetingoftheelectrotrans- ferredpDNAasit isthecasefortheadenovirustype2andthe choleratoxin(Imellietal.,2004;ShogomoriandFuterman,2001).
Around75%ofthepDNAduringelectrotransferisinternalized byconcomitantclathrin-andcaveolin/raft-mediatedendocytosis.
ThepDNAaggregatesinteractingwiththeplasmamembranehave sizesvaryingfrom100to500nm(Faurieetal.,2004,2010;Golzio et al., 2002).This wide range of sizesmay explain the several endocytoticpathwaystakenbypDNA.Astudyontheuptakeof microspheresshowedthatparticlesupto200nmwereinternalized mainlybytheclathrin-mediatedpathway.Withincreasingdiame- ters,ashifttoacaveolin/raft-mediatedendocytosiswasobserved andfor500nmmicrospheresthelatterwasthepredominantendo- cytoticpathway(Rejmanetal.,2004).Thus,particlesizeinitself candeterminewhichpathwayisfollowed.Theremaining25%of pDNAforwhichwecannotclearlyidentifytheinternalizationpath- waycouldenterbyoneoftheotherendocytoticprocessessuchas macropinocytosis.pDNAcould,however,stillentercellsviaelec- tropores.Thishypothesis,evenifnotsupportedbyexperimental dataatthepresentstage,stillhastobeconsideredasonepotential wayforpDNAtocrosstheplasmamembraneasispostulatedbased onsimulations(Smithetal.,2004)andonresultsbasedonpDNA electrotransferinGUVs(Portetetal.,2011).Inthislastpaper,it hasbeenshownthatthepredominantpathwayofelectromediated pDNAuptakeintoliposomesisundoubtedlytheelectrophoretic entranceinafreeformviadefectscreatedonthepoleofthevesicles facingthecathode.
ThefactthatpDNAinternalizationislargelytakingplacevia endocytosisallowsspeculationsabouttheintracellulartrafficking ofthepDNA.Intracellulartraffickingofendosomesiscellcytoskele- tonbased(MurrayandWolkoff,2003).Theactincytoskeleton,in additiontobeingrequiredfor anyendosomeformation,canbe usedfortheearlystepofthetransport.Furtherlongrangetrans- porttakes placeviathetubulinnetwork. Previousstudieshave showntheinvolvementofboththeactinandthetubulinnetworks inthemechanismofgeneelectrotransfer.Indeed,actinpolymer- izationoccursatthemembranesiteswherethepDNAaggregates areformedanditsalterationpriortogeneelectrotransferreduces thepDNAaccumulationatthemembraneandthepDNAexpression (Rosazzaetal.,2011).VaughanandDean(2006)andVaughanetal.
(2008)haveshownthatastabilizationofthemicrotubulenetwork enhanceselectrotransferredpDNAexpression.Theyalsodemon- stratedtheabilityofpDNAtointeractwiththemicrotubulesvia otherproteins.Ourfindingsareinagreementwiththesestudies, asanendocytoticprocessrequirestheparticipationoftheactinand thetubulinfilaments.It,inaddition,reinforcesthehypothesisofan activeintracellulartransportofthepDNA.
In conclusion, we show that although cholesterol has no significant effect on the electropermeabilization and on the pDNA/membrane interactionsteps,it isinvolved inlater stages of gene electrotransfer. We report here,for thefirst time, that 50%±7%ofpDNAisinternalizedbycaveolin/raft-mediatedendo- cytosisand25%±7%byclathrin-mediatedendocytosis.Thesedata giveimportantinsightintothemechanismofgeneelectrotransfer evenifthetranslocationstephasstilltobecharacterized.Ithasto beclarifiedhowtheremaining25%ofpDNAareinternalized.Other
endocytotic pathways may beinvolved as wellas the transfer through electropores as reported in giant lipid vesicles (Portet etal.,2011).
Acknowledgements
WethankElisabethBellardandAndreaNagyfortheirhelpand theproofreadingofthisarticle.Weacknowledgefinancialsupport fromtheAssociationFranc¸aisecontrelesMyopathies(toM.-P.R.), fromthe Ministèredes Affaires Etrangèreset Européennes and the Deutscher Akademischer Austausch Dienst (program PHC PROCOPE).
References
Aihara,H.,Miyazaki,J.,1998.Genetransferintomusclebyelectroporationinvivo.
Nat.Biotechnol.16,867–870.
Antov,Y.,Barbul,A.,Korenstein,R.,2004.Electroendocytosis:stimulationofadsorp- tiveandfluid-phaseuptakebypulsedlowelectricfields.Exp.CellRes.297, 348–362.
Antov,Y.,Barbul,A.,Mantsur,H.,Korenstein,R.,2005.Electroendocytosis:expo- sureofcellstopulsedlowelectricfieldsenhancesadsorptionanduptakeof macromolecules.Biophys.J.88,2206–2223.
Bligh,E.G.,Dyer,W.J.,1959.Arapidmethodoftotallipidextractionandpurification.
Can.J.Biochem.Physiol.37,911–917.
Bolte,S.,Cordelieres,F.P.,2006.Aguidedtourintosubcellularcolocalizationanalysis inlightmicroscopy.J.Microsc.224,213–232.
Campana,L.G.,Mocellin,S.,Basso,M.,Puccetti,O.,DeSalvo,G.L.,Chiarion-Sileni, V.,Vecchiato,A.,Corti,L.,Rossi,C.R.,Nitti,D.,2009.Bleomycin-basedelec- trochemotherapy:clinicaloutcomefromasingleinstitution’sexperiencewith 52patients.Ann.Surg.Oncol.16,191–199.
Cemazar,M.,Sersa,G.,2007.Electrotransferoftherapeuticmoleculesintotissues.
Curr.Opin.Mol.Ther.9,554–562.
Cezanne,L.,Navarro,L.,Tocanne,J.F.,1992.Isolationoftheplasmamembrane andorganellesfromChinesehamsterovarycells.Biochim.Biophys.Acta1112, 205–214.
Chernomordik,L.V.,Sokolov,A.V.,Budker,V.G.,1990.Electrostimulateduptakeof DNAbyliposomes.Biochim.Biophys.Acta1024,179–183.
Daud,A.I.,DeConti,R.C.,Andrews,S.,Urbas,P.,Riker,A.I.,Sondak,V.K.,Munster, P.N.,Sullivan,D.M.,Ugen,K.E.,Messina,J.L.,Heller,R.,2008.PhaseItrialof interleukin-12plasmidelectroporationinpatientswithmetastaticmelanoma.
J.Clin.Oncol.26,5896–5903.
Doherty,G.J.,McMahon,H.T.,2009.Mechanismsofendocytosis.Annu.Rev.Biochem.
78,857–902.
Escoffre,J.-M.,Mauroy,C.,Portet,T.,Wasungu,L.,Rosazza,C.,Gilbart,Y.,Mallet,L., Bellard,E.,Golzio,M.,Rols,M.-P.,Teissié,J.,2009a.Geneelectrotransfer:from biophysicalmechanismstoinvivoapplications.Biophys.Rev.1,177–184.
Escoffre,J.M.,Bellard,E.,Golzio,M.,Teissie,J.,Rols,M.P.,2009b.Transgeneexpres- sionoftransfectedsupercoiledplasmidDNAconcatemersinmammaliancells.
J.GeneMed.11,1071–1073.
Escoffre,J.M.,Portet,T.,Favard,C.,Teissie,J.,Dean,D.S.,Rols,M.P.,2010a.Electrome- diatedformationofDNAcomplexeswithcellmembranesanditsconsequences forgenedelivery.Biochim.Biophys.Acta.
Escoffre,J.M.,Teissie,J.,Rols,M.P.,2010b.Genetransfer:howcanthebiological barriersbeovercome?J.Membr.Biol.236,61–74.
Faurie,C.,Phez,E.,Golzio,M.,Vossen,C.,Lesbordes,J.C.,Delteil,C.,Teissie,J.,Rols, M.P.,2004.Effectofelectricfieldvectorialityonelectricallymediatedgenedeliv- eryinmammaliancells.Biochim.Biophys.Acta1665,92–100.
Faurie,C.,Rebersek,M.,Golzio,M.,Kanduser,M.,Escoffre,J.M.,Pavlin,M.,Teissie, J.,Miklavcic,D.,Rols,M.P.,2010.Electro-mediatedgenetransferandexpression arecontrolledbythelife-timeofDNA/membranecomplexformation.J.Gene Med.12,117–125.
Fernandez,M.L.,Marshall,G.,Sagues,F.,Reigada,R.,2010.Structuralandkinetic moleculardynamicsstudyofelectroporationincholesterol-containingbilayers.
J.Phys.Chem.B114,6855–6865.
Fivaz,M.,Abrami,L.,vanderGoot,F.G.,1999.Landingonlipidrafts.TrendsCellBiol.
9,212–213.
Gabriel,B.,Teissie,J.,1997.Directobservationinthemillisecondtimerangeofflu- orescentmoleculeasymmetricalinteractionwiththeelectropermeabilizedcell membrane.Biophys.J.73,2630–2637.
Gehl,J.,2008.Electroporationfordrugandgenedeliveryintheclinic:doctorsgo electric.MethodsMol.Biol.423,351–359.
Giardino,R.,Fini,M.,Bonazzi,V.,Cadossi,R.,Nicolini,A.,Carpi,A.,2006.Elec- trochemotherapyanovelapproachtothetreatmentofmetastaticnoduleson theskinandsubcutaneoustissues.Biomed.Pharmacother.60,458–462.
Glogauer,M.,Lee,W.,McCulloch,C.A.,1993.Inducedendocytosisinhumanfibrob- lastsbyelectricalfields.Exp.CellRes.208,232–240.
Golzio,M.,Mora,M.P.,Raynaud,C.,Delteil,C.,Teissie,J.,Rols,M.P.,1998.Control byosmoticpressureofvoltage-inducedpermeabilizationandgenetransferin mammaliancells.Biophys.J.74,3015–3022.
