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Nanoscale Organization of Tetraspanins during HIV-1budding by correlative dSTORM/AFM

Selma Dahmane, Christine Doucet, Antoine Le Gall, Célia Chamontin,Patrice Dosset, Florent Murcy, Laurent Fernandez, Desirée Salas, Eric

Rubinstein, Marylène Mougel, et al.

To cite this version:Selma Dahmane, Christine Doucet, Antoine Le Gall, Célia Chamontin, Patrice Dosset, et al..Nanoscale Organization of Tetraspanins during HIV-1 budding by correlative dSTORM/AFM.Nanoscale, Royal Society of Chemistry, 2019, 11 (13), pp.6036-6044. �10.1039/c8nr07269h�. �hal-02141165�

NanoscaleOrganizationofTetraspaninsduringHIV-1buddingbycorrelative

dSTORM/AFM

SelmaDahmane1≠,ChristineDoucet1≠,AntoineLeGall1,CéliaChamontin2,PatriceDosset1,Florent

Murcy1,LaurentFernandez1,DesiréeSalasPastene1,EricRubinstein3,4,MarylèneMougel2,Marcelo

Nollmann1,Pierre-EmmanuelMilhiet1*

1CentredeBiochimieStructurale(CBS),INSERM,CNRS,UniversitédeMontpellier

2IRIM,CNRS,UniversityofMontpellier,Montpellier,France

3Inserm,U935,Villejuif,France

4UniversitéParisSud,InstitutAndréLwoff,Villejuif,France

*towhomcorrespondenceshouldbeaddressed:pem@cbs.cnrs.fr

#Thesetwoauthorsequallycontributedtothework.

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SUMMARY(150words)

Membranepartitionandremodelingplayakeyroleinnumerouscellmechanisms,especiallyinviral

replicationcycleswherevirusessubvert theplasmamembranetoenterandescape fromthehost

cell.SpecificallyassemblyandreleaseofHIV-1particlesrequirespecificcellularcomponents,which

are recruited to the egress site by the viral protein Gag.We previously demonstrated that HIV-1

assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81,which are key

playersinmanyinfectiousprocesses,formingenrichedareaswherethevirusbuds.Inthisstudywe

correlated super resolution microscopy mapping of tetraspanins with membrane topography

delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is

specificallytrappedwithinthenascentviralparticles,especiallyatbudstips,andthatGagmediate

CD9andCD81depletionfromtheplasmamembrane.Inaddition,weshowedthatCD9isorganized

assmallmembraneassembliesoffewtensofnanometersthatcancoalesceuponGagexpression.

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INTRODUCTION

Acommonfeatureoflivingorganismsisthepresenceofalipidbarrierdelimitingcells.Yetthislipid

membraneallows communicationbetween the cell interior and its environment. This ismediated

eitherbyproteincomplexesembeddedinthelipidbilayer,abletotransducesignals,orbyexchange

ofmaterialthroughmembranevesicles.Thislatterphenomenoninvolvesasequenceofmembrane

remodelingevents,which includemembranedeformationand lateral reorganizationofmembrane

components. Indeed, the plasma membrane can be envisioned as a mosaic of micro and nano-

domains of distinct lipid and protein compositions. These domains are dynamic and their lateral

organization leads to specific local properties of the plasma membrane. Remodeling of this

organizationisinvolvedinnumerousprocessessuchascellularadhesion,endo-andexocytosis,cell

fusionormigration.

Viral cycles involve membrane remodeling during virus entry and egress, two critical steps for

infection.Inaddition,theseeventsarearchetypesofcoordinatedreorganizationofhostmembrane

componentsandmembranedeformation.Understandingtheirorchestrationisthusofinterestwith

respect to infectiousmechanisms andmembranebiology in general. InHuman Immunodeficiency

type 1 virus (HIV-1), viral egress is initiated by the structural protein Gag that is necessary and

sufficienttoreleasevirus-likeparticles(VLPs)1.Gagisexpressedasapolyproteinthatwillbecleaved

after particle release. Gag is targeted to the inner leaflet of the plasma membrane where it

multimerizes, induces membrane curvature (budding sites) and finally membrane fission by

recruitinghost factors suchas theESCRTmachinery 2. Lipidsof thehostplasmamembraneplaya

key role in thisprocess 3.Among these, sphingolipidsandcholesterol 4,5 areknown to formorbe

enrichedindifferenttypesofmicrodomainsthatcouldbehaveaspre-formedrecruitmentplatforms

4. It was proposed that HIV-1 Gag proteins can sense cholesterol and acyl chain environment in

membranes. HIV-1 also hijacks host proteins to achieve egress. This is the case of the ESCRT

machinery,asalreadymentioned.Butotherproteinsmaybe involved,amongstwhichproteinsof

thetetraspaninfamily.

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Tetraspanins belong to a family of proteins characterized by four transmembrane regions and a

specificfoldinthelargerofthe2extracellulardomains.Allhumancelltypesexpressseveralofthese

proteinswhich play an essential role inmultiple cellular processes ranging from cellmorphology,

migration, cell-cell fusionandsignaling 6. Tetraspaninsaremolecularorganizerswithin theplasma

membrane forming a dynamic network of protein-protein interactions at the cell surface by

interactingwith one another andwith other transmembrane proteins (integrins, Immunoglobulin

superfamilyproteinsandothers)7–9.This interactionnetworkisreferredtoasthetetraspaninweb

orTetraspanin-enrichedmicrodomains (TEM)10.A fractionof tetraspaninsandassociatedproteins

concentrate into microscopically visible structures named tetraspanin-enriched areas (TEA) or

platforms11,12. Interestingly,singlemoleculemicroscopieshaverevealedthat tetraspaninsarealso

organized in dynamic nano-clusters 12,12–14. How these two levels of organization participate in

tetraspaninfunctionsisnotclear.

Severalstudieshaveshowncolocalizationofseveraltetraspanins(CD9,CD63,CD82andCD81)with

HIV-1GagandEnv,inseveralcelltypesincludingTcells15–17.TEMswerethusproposedtoconstitute

gateways for HIV-1 assembly and budding. More recently, we have demonstrated using single

molecule trackingexperiments thatbothCD9andCD81are specifically recruitedand sequestered

within Gag assembly sites. This supports that viral components do not cluster at pre-existing

microdomainsbutratherpromotetheformationofdistinctdomainsenrichedintetraspaninsforthe

executionof specific functions, yetnot fullyelucidated 18. Tetraspaninknockdownor inhibitionby

specific antibodies also revealed that tetraspanin down-regulation decreases virus entry and

replication in macrophages 19,20. In addition several studies pinpointed a potential role of

tetraspanins in modulating HIV-1 infectivity through their incorporation into the released viral

particles. Overexpression of tetraspanins in virus-producing cells led to the production of virions

with less infectivity21,22.Thepresenceoftheseproteinsatexitsitesalsoreducedtheformationof

syncitia invirus-producingcellsandcell-to-cellfusioninducedbythevirus21,23,24.Conversely,CD81

and CD82 levels are down-regulated by the HIV-1 accessory proteins Vpu and Nef, which induce

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proteinsequestrationinintra-cellularcompartmentsanddegradation,leadingtodecreasedlevelsat

theplasmamembrane25.TheseobservationsraiseimportantquestionsconcerningtheroleofCD9

andCD81inthedifferentstepsofviralreplication,fromGagrecruitmentattheplasmamembrane

tobuddingandreleaseofviralparticles.Inparticular,althoughCD9andCD81,areinvolvedinmany

membrane remodeling events 26, it is not clear whether they could play a role in membrane

deformation and/or fission during HIV-1 egress. In addition, characterizing the effect of Gag on

tetraspaninorganizationmayhelpunderstandtherelativeimportanceofthemicro-andnanoscale

organizationofthetetraspaninweb.

Herewedeveloped andused correlativemicroscopy combining dSTORM (direct stochastic optical

reconstruction microscopy) with AFM (atomic force microscopy) 27, two advanced microscopy

techniquesallowinglateralresolutionofafewtensofnanometers,wellbeyondlightdiffractionlaw.

MembranetopographyincludingbuddingsiteswasdelineatedbytheAFM(forarecentreview,see

28)whereas CD9mappingwas analyzed using direct StochasticOptical ReconstructionMicroscopy

(dSTORM),a typeofSMLMbasedonphotoswitchingof fluorophores29,30 .Ourresultsshowthat i)

GagexpressioninducestheconcentrationofCD9andCD81nanoclusterswithinGagassemblysites.

While the distribution of these clusters is dramatically altered, their intrinsic nanoscale structure

doesnotchangemuchuponGagexpression; ii)CD9concentrateswithinnascentviralparticles. In

mostcases, it localizesat thevery tipofviralbudsand isexcludedfromthebasesof thebudding

sites.This supportsa role inmembranecurvature inductionor sensing rather than fission; iii)Gag

mediatesspecificdepletionofbothCD9andCD81fromcellsurface,suggestingthatCD9andCD81

depletion isduetotheiraccumulation inGag-inducedVLPbuddingsites.Thishappenseven inthe

absenceoftheregulatoryproteinsVpuandNefanddependsonVLPsrelease.Beyondthefieldsof

tetraspanin and infection, this study demonstrates that this type of correlative microscopy is an

incredible asset to depict at the nanoscale tight coordination between protein distribution /

recruitmentandmembraneremodeling.

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RESULTS

NanoscaleorganizationofCD9duringGagassembly

WehadpreviouslystudiedthedynamicsofCD9attheplasmamembranebySingleParticleTracking.

MostCD9harborsBrownianmotionwithproteinsthatsometimescanbetransientlyconfinedwithin

tetraspanin-enriched areas 12. In striking contrast, CD9 gets permanently trapped within HIV-1

assembly sites in cells expressing Gag 18. This suggests that Gag interferes with CD9 interacting

network. Yet how this affects CD9 nanoscale organization is not clear. To investigate this we

performeddSTORMexperiments to compareCD9distribution in control andGag-expressingHeLa

cells.CellsweretransfectedwithequimolarratiosofpGagandpGAG-GFP,inducingthebiosynthesis

ofVLPsmimickingHIV-1infection31andstainedwithanti-CD9antibodieslabelledwithAlexa64724h

or48hafter transfection. Importantly, imagingwasperformed in fixedcells since tetraspaninsare

very dynamic 7. The localization precisionwas below 30 nm (Fig. S1A)32. dSTORM images of non-

transfected HeLa cells showed a sparse distribution of CD9 molecules on the basal membrane

surface(Fig.1A,leftcolumn).ThemeandensityofCD9localizations(i.e.detectedevents)incontrol

cellswas1192µm2±127 (sem) (Fig. 1BandTable S1).UponGagexpression (Fig. 1A, secondand

third columnsandFig. S1B),Gag-GFP foci assembledat theplasmamembrane (white), consistent

withprevious reports,andCD9partitioned intoGag-enrichedareas (Fig.1A).Themeandensityof

localizationsincells24hand48haftertransfectionwithpGAG-GFPdecreasedto793±141and658

±151perµm2,indicatingareductioninCD9levelsuponGagexpression.Interestingly,usingamask

basedonGag-GFPsignal,wenotedadramatic increase inthe localizationdensitywithinGag-GFP-

enrichedareasat thecostofsurroundingareas (8430±1655versus428±63 localizations/µm2 in

areasdevoidofGag,48hafter transfection) (Fig.1BandTableS1).Asexpected fromFig.1A,CD9

localizationdensityinGag-GFPfociwascorrelatedtoGag-GFPintensity(Fig.S1C,Fig.S1D,andTable

S4forKendall'staucorrelationcoefficients).

To refine our analysis of CD9 lateral reorganization, single molecule localizations were analyzed

using a segmentation procedure based on Voronoï diagrams. This method allows a precise and

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automatic segmentation and quantification of protein organization (Fig. 1A). A framework named

"SR-Tesseler"33wasusedtoestimatethesize(innm2)ofCD9clustersattheplasmamembrane(for

moredetails,seetheSupplementalMaterialssection).Incontrolcells,themeanareaofCD9clusters

or assemblieswas 3710± 1513nm2 (Fig. 1C and Table S2) that corresponds to a disk of 68.7nm

diameter.AsignificantincreaseofthismeanareawasobserveduponGagexpression,upto5471±

2198nm2forcellsanalyzed48haftertransfectionthatcorrespondstoadiskof83.5nmdiameter.

ThisincreasewasevenmorepronouncedwhenconsideringonlyGag-enrichedareasusingthemask

methoddescribedabove:6527±2763nm2forcells48hpost-transfectionversus3710±1513nm2

forcontrolcells,correspondingtoadiskof91.2diameter(seeTableS2andthemirrorhistogramsin

Fig. 1C). Gag expression thus induces an enlargement of CD9 nanodomains, supporting that Gag

modulatesCD9 interactingnetwork.However the increase inCD9cluster sizes remainsmoderate,

eveninareasofveryhighCD9densitysuchasGagassemblysites.Interestingly,70-90nmdiameter

fitswiththesizerangeofHIV-1buddingsites34.ThissuggeststhatCD9maybeconfinedwithinthe

buddingsites.Toverifythis,wewantedtopreciselycorrelateCD9localizationwiththetopography

ofGag-inducedbuddingsites.

CorrelationbetweenGagassemblywithinVLPbudsandCD9dSTORM

Wefirst implementedcorrelativeTIRFandAFMimaging.Toensurethatapicalcellularmembranes

imagedbyAFMwere in the TIRFevanescent field,we focusedon thin regionspresent at the cell

periphery (Fig. 2). Topographic images of the cell surface revealed membrane protrusions that

overlappedwithGag-GFPfoci,suggestingtheywereVLPs.Somefluorescentareasdidnotcoincide

with membrane protrusion, likely because Gag assembly occurred on the basal membrane not

accessibletotheAFMtip(seetheasterisk inFig.2).Virus-likebudswereonaverage104±49nm

high and 162 ± 75 nm wide, which is in accordance with measurements derived from super

resolution microscopy (see the review 34 or electron microscopy studies 35. As expected the size

distribution of virus-like buds was heterogeneous (Fig. S2A), reflecting the progression of the

buddingprocess.Asamatteroffact,thesurfaceofbudsmeasuredbyAFMcorrelateswithGag-GFP

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content (Fig.2E). Inaddition,AFMallowed furthercharacterizationofVLPs formationasshown in

Figure 2F where the AFM tip could delineate 2 budding sites that cannot be differentiated with

conventionalTIRFmicroscopy.ThisillustrateswellthegaininresolutionobtainedbyAFM.

We thus combined dSTORM and AFM on cells expressing Gag-GFP to get more details on CD9

organization in Gag-enriched domains (Fig. 3). CD9 clusters characterized by dSTORM overlapped

wellwiththeshapeofGag-GFPbuddingsitesdelineatedbytheAFMtip(Fig.3Ato3D).Inadvanced

buds(sphericalbudattachedtothemembranebyaneck),theneckareawasdevoidofCD9(e.g.Fig.

3C).InnascentbudsCD9waslocalizedattheverytipofmembraneprotrusionsandmostlyexcluded

from the basis of budding sites (Fig. 3F and see the gallery of budding sites in Fig. S3). This

demonstrates that CD9 is indeed trapped within the nascent bud. Moreover, CD9 seems to

preferentially associatewithmembrane regions of high positive curvature. As described in Fig. 1,

some CD9 clusters were also observed in membrane areas devoid of Gag-GFP proteins. Since

calculationofCD9densitiesfrom2Dprojectedareas(asinfig.1)mayhaveintroducedadrawback,

especially in buds,we calculated the number of CD9 localizations divided by themembrane area

extracted from AFM images (see supplemental Materials). Taking into account the membrane

topography, we confirmed that CD9 density is higher in Gag-GFP budding sites (3836 ± 934

localizations/µm2)comparedtomembraneregionswhereGagproteinsareabsent(505±111)(Fig.

S2B).

GagreducesthecellsurfaceexpressionofbothtetraspaninsCD9andCD81

Interestingly,quantificationofourdSTORMdatasuggests thatCD9 levelat theplasmamembrane

globallydecreasedwhenGag isexpressed.Toconfirm this,weanalyzedcell surfaceexpressionof

bothCD9andCD81onHeLacellsexpressingornotGag-GFPusingFACS.Cellswerethenfixedand

stained with labeled anti-CD9 or anti-CD81. 30-40% of cells were positively transfected and we

defined3typesofpopulations:untransfectedcells(GFP-)andcellswithintermediate(GFP+)orhigh

(GFP++)levelsofexpression(Fig.4A).WhileGFP-transfectedcellshadcomparableCD9levelsinthe

3populations,CD9surfacelevelsweredecreasedby70-80%incellsstronglyexpressingGag-GFP,as

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comparedtoGFP-negativecellsfromthesamesample.Intra-sampleratiosofCD9andCD81levelsin

GFP++ or GFP+ versus GFP- cells were averaged from 4 independent experiments (Fig. 4B). The

valuesconfirmedthatcellswithhighGagexpressionaredepletedofCD9andCD81.Incontrast,we

foundthatthecellsurfacelevelsofCD46,anon-rafttransmembraneproteinwithlittleassociation

with tetraspanins, remains unaffected upon overexpression of Gag proteins (data not shown),

indicating thatGag specifically affectsCD9andCD81 surface levels. These levels are known tobe

down-regulated in an HIV-1 infection context due to their degradation and intracellular

sequestration by the viral proteins Vpu and Nef 25. Since these proteins are not present in our

system,wewonderedifthedepletionisduetothehighexcisionrateofmembranebudsenrichedin

CD9andCD81.Totestthis,weusedaGagmutantprotein(Gag∆NC),whichassemblesattheplasma

membranebutisimpairedinVLPsrelease36.WhenexpressedinHeLacells,Gag∆NCdidnotreduce

tetraspanincellsurfacelevels(Fig.4C).ThissuggeststhatCD9andCD81downregulationatthecell

surfaceisduetotetraspaninescapefromtheplasmamembranewhenVLPsarereleased.Thelateral

reorganizationoftetraspaninsbyGagthushasnanoscopicaswellasmacroscopicconsequences.As

ofnow,theimpactofthisdepletionontheviralcycleand/orthehostcellfateisnotclear.However,

we confirmed that depletion of CD9 and/or CD81 from HeLa cells by siRNA did not affect VLPs

release(Fig.S4A-B).Butinterestingly,wenotedacompensationeffect:CD9ismoreconcentratedin

VLPswhenCD81isdepleted,andthereverseisalsotrue.ThissuggeststhatGaginducesanoverall

tetraspaninconcentrationinVLPsduringviralegress.

DISCUSSION

Tetraspaninshavebeendescribedasorganizersoftheplasmamembraneofeukaryoticcells,playing

a key role in membrane remodeling especially in the infection context. In this study we used

dSTORM/AFMcorrelativemicroscopyto investigatehowHIV-1Gagaffects the lateralorganization

oftheCD9tetraspaninthatbecomestrappedwithintheviralparticle.

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We characterized CD9 localization in control cells using dSTORM and reported a clustered

organization.Othertetraspaninshavebeenpreviouslycharacterizedbyhigh-resolutiontechniques,

namely STED 14 and dSTORM 13,37. Interestingly, their localizations were also clustered, yet with

slightlylargersizes(100-150nmwide)thanfoundhereforCD9(67nm).However,eventhoughCD9

transientlyassociateswiththetetraspaninscharacterizedinthesestudies(e.g.CD82,CD81,CD53),

theirdistributionisnotexpectedtobeidentical.Infact,theareaofCD81clustersmeasuredinthe

presentworkwasslightly largerthanthatofCD9(diskof81nmdiameter,Fig.S5).Thisdifference

fits well with the slower diffusion of CD81 compared to CD9, as measured by single molecule

tracking38.Inaddition,itisprobablethatthetetraspaninclustersdifferincompositionandsizefrom

onecelltoanother.Takentogether,clustersizesmeasuredinthepublicationscitedabovefallinthe

samerangeandsupportamodelwherebytetraspaninsdiffuseintheplasmamembrane,embedded

insmallassembliesthatcouldcontainothertetraspanins,someproteinpartners,andlipids7,14.

CD9 and CD81 being recruited at budding sites during HIV-1 egress suggested that functional

platforms could originate from the gathering of these small assemblies. Indeed we confirm here

that,uponexpressionofGag-GFP inamodel systemrecapitulatingHIV-1-inducedVLPproduction,

CD9andCD81lateralorganizationisdramaticallychanged(respectivelyFig.1andFig.S5A)andthe

two tetraspanins are highly concentrated in regions of Gag assembly, forming large tetraspanin-

enrichedareas. Surprisingly theaverage cluster sizedidnot radically changeuponGagexpression

(Fig.1CandFig.S5B),suggestingthatthelargeCD9andCD81assembliesobservedinGag-enriched

areas are composed of tetraspanin clusters that gather but do not fully coalesce (they can be

resolvedbydSTORM).Thislocalconcentrationoftetraspaninsiscompensatedbyadecreaseintheir

density in surrounding regions (as compared to control cells, Fig. S1E), indicating that CD9

enrichment inGag+ areas is due to lateral reorganization of CD9 rather than protein recruitment

fromintracellularcompartments.Thismodelisingoodagreementwithourpreviousworkshowing

that CD9, which diffuses in a Brownian motion in the plasma membrane, is trapped within Gag

assemblysites 18.Gag ismost likely thedriving forcethatconcentrates tetraspaninclusters,which

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probably co-segregatewith othermembrane partners that could facilitate budding. As previously

suggested, lipid compositionof thesemembrane assemblies could also play a key role in building

largemembraneplatformssinceGagproteinsarerecruitedbyPIP2lipidsinacholesterol-dependent

manner4,39.

CorrelativedSTORM/AFMoncellsexpressingGag-GFPshowedthatCD9concentratesattheverytip

ofnascentVLPswithalmostcompleteexclusionfrombudsnecksatlatestages.CD81hadpreviously

been observed mainly localized at the tips of elongated Influenza viruses using EM 40. In this

particularcase,CD81wasproposed toalsoplaya role in fissiondue to itspresenceatbothends,

includingthesideattachedtothehostcell.This isquitedifferentfromourobservationswithCD9,

which is thusmost likely not involved in bud scission. But CD9 seems to have the propensity to

partitionwithinpositivelycurvedmembranes.This is ingoodagreementwiththepresenceofCD9

andCD81intubularstructuressuchasmembraneprotrusionsandfilipodia(datanotshownand41).

ThecorrelationbetweenCD9densityandmembranecurvaturecouldeitherreflectaroleofCD9in

membrane remodeling and deformation, especially positive curvature, or its sensitivity to

membranecurvature.Atthattimeit isdifficulttodiscriminatebetweenthesetwomodelsbutthe

firsthypothesis ismore likely sincewehavealsoobservedCD9andCD81 in flatmembranes. It is

thenmoretemptingtospeculatethattheseproteinsareimportantintheformationofhighlycurved

membranesencounteredduringvirusbudding, in theproductionofexosomes frommultivesicular

bodies42,aswellas incell fusionprocessobservedduringthegametefusionwhereassociationof

CD9with highmembrane curvature regions has been reported previously in oocytes 43 or during

myoblastfusion44.Interestingly,proteinconfinementwithinmembranedomainshasbeenproposed

toinducebendingofthelipidbilayer,evenintheabsenceofanyspecificproteinfunctionaldomain

45,46. Importantly, the smaller the protein, the stronger the effect 45. These observations are

particularlyinterestingregardingtetraspanins:indeed,theyaresmallproteins(~20kDa)andtheyare

transientlyorpermanentlyconfinedwithinmolecularplatformsattheplasmamembrane(reviewed

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in38.Inthisregard,modulationoftheirconfinementthroughinteractingpartners(inthecaseofthis

study,byGag)couldmediatetheirabilitytocurvemembranes.

We then performed single or dual depletion of CD9 and CD81 in HeLa cells and measured VLP

production (Fig. S4A-B). Similarly to what has already been described 21, CD9 and CD81 are not

essentialforVLPsreleaseinHeLacells.However,weobservedthatCD9silencingledtoanincrease

ofCD81expressionwithinVLPs,andviceversa,suggestingthatonetetraspanincouldcompensate

for the loss of another. Interestingly, this effectwas not observed in cell extracts, emphasizing a

specific role of tetraspanins in viral particles. Theother sideof the coinof this redundancy is the

difficultytoclearlyestablishtheirfunctionalrole.

MarkusThali'sgrouphasdemonstratedthat,despitetheirenrichmentatviralexitsites,theoverall

levelsoftetraspaninsaredecreasedinHIV-1-infectedcells.MorespecificallyCD81down-regulation

in HIV-1 infected cells was explained by its degradation in proteasomal and lysosomal pathways.

TheseprocesseswereshowntodependuponHIVproteinsVpuandNef25.Here,wereportthatGag-

GFP expression could also trigger CD9 and CD81 depletion in the absence of Vpu and Nef. In

addition,tetraspaninlevelsremainnormaluponexpressionofaGagmutantimpairedinVLPrelease

36,suggestingthatCD9andCD81depletionisdirectly linkedtoVLPreleasefromhostcells.Evenif

thefractionofcellularplasmamembraneescapingthroughthisprocessremainslow(asassessedby

unaffectedlevelsofCD46),CD9andCD81globalproteinlevelsareimpactedmostprobablybecause

oftheirhighconcentrationwithinGagassemblysites.Interestingly,tetraspaninlevelsinfluenceHIV-

1lifecycleatdifferentstagesandinoppositemanners.Indeed,CD81potentiatesHIV-1transcription

22,45 through its association to SAMHD1 46, while overexpression of tetraspanins at the surface of

virions, including CD9 and CD81, decrease their infectivity 21,22. Tetraspanins thus appear as key

elementsinthemodulationofHIV-1virulence.

TakentogetherourresultsshednewlightontheinvolvementofCD9andCD81duringHIV-1egress

using a new type of correlative microscopy that is suitable to investigate virus-host interactions,

providing topographic details and molecular mapping at the nanoscale in native conditions. The

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acute description of tetraspanin distribution at a nanoscopic and microscopic scale in the 3D

topography landscape probed by AFM allow us to make new hypotheses regarding tetraspanins

functions in membrane bending. More generally such correlative microscopy appears as an

outstanding technique to analyze membrane remodeling and protein partitioning in critical

biologicalprocesses.

Conflictsofinterest

Therearenoconflictstodeclare.

Authorcontributions

Conceptualization, M.N. & P.E.M.; Methodology, S.D., A.L.G., C.D. & P.D.; Validation, S.D., C.D.,

M.M.,M.N.&P.E.M.;Formalanalysis;S.D.,C.D.,A.L.G.&P.E.M.;Investigation,S.D.,C.D.,C.C.,F.M.,

L.F.&D.S.P.,Resources,M.M.,E.R.andP.E.M.;Writing-OriginalDraft,S.D.,C.D.&P.E.M.;Writing-

Review&Editing,C.D.E.R.,M.N.&P.E.M.;Visualization,C.D.&P.E.M.;Supervision,M.M.,M.N.&

P.E.M.;Fundingacquisition,M.N.&P.E.M.

Acknowledgments

We acknowledge the support from France-BioImaging (FBI, ANR-10-INSB-04), the French

Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INBS-05), the European Research

CouncilStarting(ERC-Stg-260787),theAgenceNationalepourlaRecherche(ANR-15-CE11-0023)and

theGIS IBISA (Infrastructures enBiologie Santé etAgronomie). LF andDSPwere recipients of the

FrenchMinistry of Education and Research. LF was a FRM fellow and SD salary was paid with a

Sanofi-Pasteurcontract.WearegratefultoZhannaSantybayevaforcreatingthecartooninFigure5,

MarkusThaliforprovidingtheGag-GFPplasmids,andHeikoHaschke(JPKcompany)forhistechnical

helpandhelpfuldiscussion.

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FIGURELEGENDS

Figure1 -GagproteinsrecruitandreorganizethetetraspaninCD9attheplasmamembrane. (A)

RawCD9dSTORMlocalizations(reddots,2firstrows)andlocaldensitymapobtainedfromtheSR-

Tesseler framework (third row; the colour scale represents local densities in logarithmic scale) in

HeLacellsexpressingHIV-1Gag-GFP(whitesignal inmicrographs).Fromlefttoright:controlcells;

cellsexpressingGag-GFPfor24hor48h;themiddlerowshowzoomedareasoutlinedintheupper

images.Scalebarsare5µm(upperrow)and1µm(bottomrows);(B)dSTORManalysis.CD9density

(numberoflocalization/µm2)incontrolcells(red)orincellsexpressingGag-GFPproteins24h(blue)

or 48h (green) after transfection. In themirror histograms below the X axis, empty and hatched

histogramsrepresentthedensityoutsideandwithinGag-GFPpositiveareas,respectively.Errorbars

areSEMandnisthenumberofanalyzedcells.*,**and***indicatepvaluesbelow0.05,0.001and

0.0001respectively,asdeterminedbytheMann–WhitneyU-test(forexactpvalues,seeTableS1);

(C)HistogramsofsizedistributionofCD9clustersinnm2forthe3conditions.Thelegendissimilarto

B.

Figure 2 - Nanoscale imaging of HeLa cells expressing Gag-GFP using correlated fluorescence-

atomic forcemicroscopy. Gag-GFP fluorescence image (AandC)andAFMtopographic images (B

andD)werecompared(48hpost-transfectionhere).Thecircleshighlightsomecorrelationbetween

fluorescenceandthemembraneprotrusiondelineatedbytheAFMtip.TheasteriskpointsoutaGag

assemblywherenomembraneprotrusionwasobservedbyAFM.(E)NormalizedGag-GFPsignalasa

functionofthebudsurfacemeasuredbyAFM.(F)Profileplotofthetopography(orange line)and

fluorescencesignal(greenline)alongthesectionindicatedbytheblacklineontheAFMimage.Scale

barsare5(AandB)or1µm(CandD).TheAFMzcolourscalesare6.6µm(upperAFMimage)and

800nm(lowerimage).

Page 21 of 44 Nanoscale

19

Figure 3 - CD9 recruitment at HIV-1 budding sites. HeLa cells expressing HIV-1 Gag-GFP were

immuno-stainedwith anti-CD9 coupled toAlexa-647and imagedbyAFM (first row), conventional

fluorescence (second row)anddSTORM(third row)TIRF illumination:AandD)AFM3D imagesof

twodifferentcells(thedottedlinedelineatesthezoomedareasshownbelowandtheinsetsarethe

correspondingGag-GFPsignal fluorescence images);BandE)overlaysof theGag-GFPpicturewith

thereconstructeddSTORMimageofthetetraspaninCD9.Scalebarsare500nm(AandD)or200nm

(B,C,EandF).ThecolourzscaleshowninAandDis300nm.

Figure4-GagreducestetraspaninlevelsatthecellsurfaceofHeLacells.

A,BandC)SurfaceexpressionofCD9andCD81measuredbyflowcytometry48haftertransfection

withGag-GFPorGFP(Control).A)Representativeflowcytometry2Ddotplotsofgatedlivingcells;

three cell populations were defined based on their GFP intensity (GFP-, GFP+ and GFP++).

Percentages indicatetherepresentativefractionofeachpopulation;B)MeanCD9andCD81levels

were quantified for each population and normalized to GFP- levels. Data were averaged from 3

independentexperiments.Errorbarsarestandarddeviations.C)SurfacelevelsofCD9andCD81in

HeLacellstransfectedwithGFPalone(grey),withGag-GFP(greenline)orwiththeGag∆NCmutant

(redline).BlackarrowheadsindicateHeLacellswithCD9orCD81depletion.

Figure5–ModeloftetraspaninlateralorganizationintheHIV-1context.Thisschemerepresents

howthetetraspaninsCD9andCD81(inblueandyellow)arelaterallysegregatedwithinGag-induced

budding sites (in green). This leads to decreased CD9 and CD81 levels in the surrounding plasma

membrane(inlightgrey),resultinginanetproteinlosswhenVirus-LikeParticlesareexcised.

Page 22 of 44Nanoscale

HeLa WT 24h post-transfection 48h post-transfectionHeLa cells expressing Gag-GFPA

CD9

dens

ity(n

loca

lizat

ion/

µm2 )

0

5000

10000

B

*****

****

1000

C

0

12

CD9

Clus

ter s

ize

(nm

2 )

8000

4000

0

4000

8000

***

***

***

WTn=58

24 hn=22

48 hn=24

Page 23 of 44 Nanoscale

0.0 0.2 0.4 0.6

Normalized Gag-GFP signal

Bud

surf

ace

(µm

2)

*

0.8 10

1

0

50

100

100

200

00.5 1

Hei

ght (

nm)

Distance (nm)

Fluo

resc

ence

(a.u

.)

BA

AC

B

D

E

F*

Fluorescence AFM

Page 24 of 44Nanoscale

A

B

C

D

E

F

Page 25 of 44 Nanoscale

CD9

Gag CtlRe

lativ

e su

rfac

e ex

pres

sion

CD9

CD81

CD81

CD9

GagGFP GFP

0

1

Coun

ts (a

rbitr

ary

units

)

GFP+ GFP++

102101 103

102101 103

2

4

6

102101 10310-1102101 10310-1

103

103

102

102

10

10

1

1

10-1

10-1

++++++

0

GFP-58.2%

GFP-58.7%

GFP-59.2%

GFP-61.4%

GFP+19.6%

GFP+20.0%

GFP+6.24%

GFP+5.86%

GFP++14.8%

GFP++14.2%

GFP++26.3%

GFP++27.2%

Control GAG GAG ∆NC

CD81

Rela

tive

surf

ace

expr

essi

on

Gag Ctl

1

0 0Coun

ts (a

rbitr

ary

units

)2

4

6

Fluorescence Intensity

Fluorescence Intensity

A B C

Page 26 of 44Nanoscale

Gag-GFP CD81CD9 Cytoplasm

Plasma membrane

Page 27 of 44 Nanoscale

1

SupplementalMaterials

NanoscaleOrganizationofTetraspaninsduringHIV-1buddingbycorrelative

dSTORM/AFM

SelmaDahmane1*

,ChristineDoucet1*

,AntoineLeGall1

,CéliaChamontin2

,PatriceDosset1

,Florent

Murcy1

,LaurentFernandez1

,DesiréeSalasPastene1

,EricRubinstein3,4

,MarylèneMougel2

,Marcelo

Nollmann1

,Pierre-EmmanuelMilhiet1#

1

CentredeBiochimieStructurale(CBS),INSERM,CNRS,UniversitédeMontpellier

2

IRIM,CNRS,UniversityofMontpellier,Montpellier,France

3

Inserm,U935,Villejuif,France

4

UniversitéParisSud,InstitutAndréLwoff,Villejuif,France

#towhomcorrespondenceshouldbeaddressed

*Thesetwoauthorsequallycontributedtothework.

MATERIALSANDMETHODS

Plasmidsandantibodies

Thecodon-optimizeduntaggedGag,Gag-GFP,Gag∆NC,pNL4-3∆env,andpMA-YFP∆envconstructs

have been previously described1,2

. Full lengthmAbs raised against CD81 (TS81), CD9 (SYB-1) and

CD46(11C5),werelabeledwithAlexa647aspreviouslydescribed3

.AlexaFluor594-conjugatedgoat

anti-mouseantibodieswerefromMolecularProbes.ForVLPproductionanalysis(Fig.S5),HeLacells

Page 28 of 44Nanoscale

2

were transfected 14 h before imaging using X-tremeGENE 9 DNA transfection reagent (Roche),

accordingtothemanufacturer’sprotocol,withtwoHIV-1pNL4-3∆envplasmidsencodinguntagged

andYFPtaggedGagatmolarratio1:1.ThismixtureisrequiredtogetVLPmimickingHIV-1.

Cellcultureandsamplepreparation

HeLacellsweregrowninDMEM(Gibco)supplementedwith10%FCS(Gibco).Forimaging,cellswere

seeded.on25mmroundglasscoverslipsplacedin6-wellplates(2.105

cells/well)(Marienfeld).Prior

touse,coverslipswererinsedwithacetone,ethanol,andwater,thensonicatedin1MKOHfor20-30

minutes in a water bath. Coverslips were then extensively rinsed in MilliQ water, air-dried and

plasma-cleanedfor20minutes.Theywerethencoatedwithcollagen.Beforetransfection,cellswere

placed in freshmedium.Cellsweretransfectedusing2µgDNAperwellwithanequimolarratioof

Gag-GFPandGag.Cellswereplacedinfreshmedium4-6hoursaftertransfectionandanalysed24-

48hpost-transfection.For immunostaining,cellswere incubated for15minat37°CwithAlexa647-

conjugatedprimaryantibody(1.5μg/mL),washedandfixedwith4%paraformaldehydeinPBSfor20

minatroomtemperature(fixationincreasesthemembranespringconstantandthusfacilitateAFM

imaging).Afterfixation,cellswerewashedwithPBSandincubatedfor10minwith1/1000dilutionof

100nmfluorescentbeadsemittingatfourwavelengths(TetraSpeckMicrospheres,Invitrogen)used

as fiducial marks. For dSTORM imaging, an oxygen-scavenging PBS-based buffer included 10%

glucose, 0.04 mg/mL glucose oxidase, and 0.5 mg/mL catalase, supplemented with

mercaptoethylamine(MEA)(allfromSigma).

ImageacquisitiononAFM-SMLMCorrelativeMicroscope

The setupwasbuilt as a combinationof aNanowizard3microscope (JPK, Berlin) togetherwith a

homemade objective-type TIRF inverted optical microscope (Zeiss, Le Pecq, France) equipped for

singlemoleculelocalizationmicroscopywithanoil-immersionobjective(Plan-Apochromat100x,1.4

DIC, Zeiss). A 1.5x telescope was used to obtain a final imaging magnification of 150-fold

correspondingtoapixelsizeof107nm.Fourlaserswereusedforexcitation/photo-activation:405

nm (OBIS, LX 405-50, Coherent Inc.), 488 nm (OBIS, LX 488-50, Coherent Inc.), 561 nm (OBIS, LX

Page 29 of 44 Nanoscale

3

561-50,Coherent Inc.), and640nm (OBIS, LX640-100,Coherent Inc.). Laser lineswereexpanded,

and coupled into a single beam using dichroic mirrors (427, 552 and 613 nm laser MUXTM,

Semrock). An acousto-optic tunable filter (AOTFnc-400.650-TN, AA opto-electronics) was used to

modulate laser intensity. Light was circularly polarized using an achromatic quarter wave plate

(QWP).Twoachromatic lenseswereusedtoexpandtheexcitationlaserandanadditionaldichroic

mirror (zt405/488/561/638rpc,Chroma) todirect it towards theback focalplaneof theobjective.

Fluorescence light was spectrally filtered with emission filters (ET525/50m, ET600/50m and

ET700/75m, Chroma Technology) and imaged on an EMCCD camera (iXon Ultra897, Andor

Technologies). Themicroscopewas equippedwith a piezo TipAssistedOptics (TAO)module (JPK,

Berlin)allowing100x100x10µmsampledisplacementinx,yandzdirection,respectively.

Toensurethestabilityofthefocusduringacquisition,home-madeautofocussystemwasbuilt.4%of

theredlaserwasdeviatedfromtheopticalpathusingaglassplateanddirectedatthesample/glass

coverslip interface. This beam was then reflected towards the objective lens and redirected

following the same path as the incident beam and guided to a home-made QPD allowing its

transversedisplacementstobedetectedandcorrectedbytheTAOstage.Camera, lasersandfilter

wheelwerecontrolledwithasoftwarewritteninLABVIEW(NationalInstruments).

For dSTORM acquisitions, two laserswere used to illuminate the cells. 1kW/cm2

of 641 nm laser

illuminationwasusedforimagingand0-0.1kW/cm2

of405nmforconversionfromthedarkstate.

The641nm lasercontinuously illuminated thesampleduringdataacquisition,while theactivation

laserwaspulsed for50ms. The intensityof activationwasprogressively increased throughout the

acquisitiontoensureaconstantamountofsimultaneouslyactivatedfluorophoreswithinthelabeled

structures.Forimageacquisition,onaverage25,000frameswererecordedatarateof50ms/frame.

CellswerefurtherimagedwithAFMafterreplacementofthedSTORMoxygen-scavengingbufferby

PBS.AFMimagingwasperformedwithaNanowizard3(JPKBerlin,Germany)usingtheQuantitative

Imaging mode with MLCT cantilevers (Nano-Bruker, Palaiseau, France). To achieve the best

combination between AFM and fluorescence images, we used the built-in software calibration

Page 30 of 44Nanoscale

4

DirectOverlayTM

which is using the accuracy of theAFM closed loop scanning systemenabling the

overlayofbothmicroscopiesathighresolutionprecision,typically10to30nm.

dSTORMdataprocessingandanalysis

Post-acquisition imageanalysiswasperformedusing theMultipleTargetTracking (MTT)algorithm

described elsewhere4

generating tables containing the x-y particle coordinates of eachmolecule

detectedduring theacquisition. Lateraldrift correctionwasperformedasdescribedpreviouslyby

following the trajectoryof the fiducialmarks andemploying custom softwarePALMcbswritten in

MATLAB(MathWorks)5

.Theexperimentaldriftcorrectionprecisionwastypically3-10nm.

ClusterizationanalysiswasdonebyatessellationapproachusingamodifiedversionoftheVoronoi

tesselation algorithmdeveloped by Levet et al.6

. Single-molecule localizations are first converted

intoaVoronoidiagram.Briefly10umx10umregionsof interest(ROI)wereselectedmanuallyand

Voronoidiagramswereretrievedusingthe‘voronoi’function.Localdensitieswerecalculatedasthe

inverse value of the corresponding voronoi cells area. For each ROI, a density histogram of

experimental localizationswasgeneratedandcomparedtothedensityhistogramofanequivalent

number of randomly distributed localizations. The histograms intersection defined a threshold D.

Localizationswereconsideredtobeclusteredwhenexhibitingalocaldensityd>1.6D.

A binary map of clustered localizations was generated and localization clusters were then

segmented.Amaskwascreated,basedonGFPfluorescence,todefineareascorrespondingtoGag

assembly sites.Using thismask, clusterswere sorteddependingon their co-localizationwithGag.

Clusters areaswere then calculated for clusterswithinoroutsideGagassembly sites.All analyses

werecarriedoutinMatlab.Graphicalrepresentationsandstatisticalanalyseswereperformedusing

Prism.

FACS

48hpost-transfection,cellsweretrypsinizedandrinsedtwiceincoldPBS.Cellswerethenincubated

withappropriateantibodiesdilutedinPBS+3%serumat1.5µg/mLfor30minutesonice.Cellswere

rinsed in PBS + 3% serum and incubated with Alexa647 anti-Mouse (Molecular Probes) for 30

Page 31 of 44 Nanoscale

5

minutes on ice. Cells were then rinsed in cold PBS, fixed in 4%PFA for 10 minutes at room

temperature,andrinsedtwiceincoldPBS.FACSanalysiswascarriedoutonaMACSQuantanalyzer

(Miltenyi). All data were acquired using the same detector settings and gating parameters. Data

were analyzed with FlowJo and the ratio of tetraspanin levels in transfected (GFP+ or GFP++) /

untransfected(GFP-)cellswerecalculated.Datawereaveragedfrom4independentexperiments.

QuantitationofVLPproductioninHeLacellsdepletedornotofCD9andCD81

HeLacellswereseeded in6wellplatesandco-transfectedwith1µgofpNL4-3∆Env,1µgpMaYFP-

∆Env,100pmolsiRNA(either,scrambledoragainstCD9and/orCD81).48haftertransfection,culture

supernatantswere collected and submitted to ultracentrifugation at 30,000g for 90minutes on a

sucrosecushion.Pelletswereresuspendedin40µlofDMEMwithoutserumandstoredat-80°Cuntil

SDS-PAGEanalysis.Cellswerescrapedoniceandpelleted.Eachpelletwasresuspendedin100ulof

TNE-Triton (10mMTrispH7.5,150mMNaCl,5mMEDTA,1%Triton),complementedwithprotease

inhibitor cocktail (EDTA-freeComplete,Roche)and incubatedon ice for10minutes, vortexing2-3

times.Lysateswerespunfor10minutesat11,000g;supernatantswerecollectedandstoredat-80°C

untilSDS-PAGEanalysis.

CD9,CD81andp24contentsinbothcellextractandsupernatantwereanalyzedbywesternblotting

using anti-tetraspanin antibodies described above and anti-p24 antibodies (Serotec), revealed by

peroxidase-conjugatedgoatanti-mouseantibodies fromJackson ImmunoResearch.Quantitationof

western-blottingsignalswasperformedwithFIJI.

Silencing RNA oligonucleotides were from Ambion: Oligo sc (UAGAUACCAUGCACAAAUCC dTdT),

siCD9 (GCAGAAATCCTGCAATGAAdTdT)andsiCD81(CACGUCGCCUUCAACUGUAdTdT).

Page 32 of 44Nanoscale

6

FIGURELEGENDS

FigureS1-dSTORMclusteranalysisofCD9inHeLacells

(A) Localization accuracy: frequency distributions of dSTORM localization precision in HeLa cells

undernaive(WT),24hor48hGag-GFPexpression.

(B)Gag-GFPfluorescencesignalacquiredbyTIRFmicroscopyfromHeLacellsexpressingHIV-1Gag-

GFPfor24hor48h(showninFig.1A).Scalebars,10µm.

(C)Left:TIRFimageofGag-GFPfociattheplasmamembraneofaHeLacells48hpost-transfection

(left);relativeGFPintensityispseudo-coloredaccordingtotheassociatedcolorscalebar(arbitrary

units).Right:thecorrespondingmoleculardensitymapofCD9-Alexa647;densityispseudo-colored

accordingtotheassociatedcolorscale.

D) Normalized CD9 density (ratio of molecular density of CD9 within Gag-GFP domains to total

molecular density) correlated to the normalized intensity of Gag-GFP assembly sites (48h post-

transfection,numberofcells=8).Thecorrelationcoefficientis0.44withar2

of0.33.

E)BoxandWhiskersrepresentation(5-95%percentile)ofareasdepletedofCD9clusterscalculated

fromdSTORManalysisincontrolcells(red)orincellsexpressingGag-GFPproteins24h(blue)

or48h(green)aftertransfection.ErrorbarsareSEMandnisthenumberofanalysedcells.

**indicatespvaluebelow0.001ascomparedtoWT,asdeterminedbytheMann–Whitney

U-test.

FigureS2-SizeofbuddingsitesmeasuredbyAFM

(A) Size distribution of HIV-1 Gag-GFP particles. Height and diameter of GFP-positive buds were

measuredbyAFM(n=60).Thelinerepresentsthelinearregressionbetweenthese2parameters.

(B) Distribution of CD9 “true” density in Gag-GFP domains (Gag+) compared to regions of the

membrane where Gag-GFP protein is absent (control). The true density is the number of CD9

dSTORMlocalizationsdividedbythebudmembranearea,measuredfromAFMtopographicimages.

Page 33 of 44 Nanoscale

7

Thecontroldistributioniscalculatedfromareasrandomlyselectedinmembraneregionsdevoidof

Gag-GFP(n=53);thesedensitieswerecalculatedfroma3pixelsx3pixelsROI,whichistherangeof

thebudarea.

FigureS3-Galleryofcorrelativeimagesofbuddingsites

Panelofsixrepresentativeimagesofbuddingsites indifferentcellsexpressingGag-GFP(670nmx

670 nm zooms): first row, AFM topography images; second row, AFM signal is overlaidwith CD9

dSTORMlocalizations(reddots);thirdrow,idemwithGFP-Gagsignalinaddition(green).Thecolor

scalebarforAFMis350nm.

FigureS4-CD9andCD81aredispensableforVLPsrelease

The acute enrichment of CD9 and CD81 into Gag-induced budding sites questioned about the

functional role of CD9 and CD81 inmembrane remodeling and/or bud fission. Since tetraspanins

share a number of interactors, CD9 and CD81 may be at least partially redundant, we thus co-

depletedCD9andCD81bysiRNAapproachandmeasuredVLPproductionaswellasCD9andCD81

expressionwithinbothcellsandVLPs.

A)Cellswereco-transfectedornot(control-Ctl)withGag(pNL4-3∆)andsiRNAtargetingCD9,CD81,

both tetraspanins or scrambled (Sc) SiRNA. Expression of p24, CD9 and CD81 were analyzed by

western blotting. The supernatant is representative of VLP particles released in the extracellular

medium.Bracketshighlight theexpressionofCD81andCD9whendownregulatingCD9andCD81,

respectively.

B) Relative expression of CD9 (white box) and CD81 (grey box) in supernatants as compared to

controlcells(scrambledSiRNA).Nosignificantdifferencewereobservedincellextracts.Importantly

no significant difference in p24 expression was observed in both cell extracts and supernatants.

QuantitationwasperformedwithFIJI and statistical analysis inPrismusingANOVA test combined

withaTurkey'smultiplecomparisontest;*indicatesapvaluebelow0.05.

Page 34 of 44Nanoscale

8

FigureS5-GagproteinsrecruitandreorganizethetetraspaninCD81attheplasmamembraneof

HeLacells

(A)dSTORM imagesofCD81 inanaïveHeLa cell (top) andaGag-GFPexpressingHeLa cell at48h

post-transfection(down)withregions inwhiteboxes(10μm×10μm)enlargedonthesecondrow.

Scalebarsare5µm(firstrow)and1µm(secondrow).

(B) Histograms representing the CD81 cluster size in nm2

inWT cells (red) or GFP-Gag-expressing

cells48haftertransfection(green). InthemirrorhistogramsbelowtheXaxis,emptyandhatched

histograms represent the density outside and within Gag-GFP positive areas, respectively. ***

indicates a p value below0.0001 for comparisonof CD81 cluster sizes insideGagdomains versus

clustersizesoutsidethesedomains,asdeterminedbytheMann-WhitneyU-test.

Page 35 of 44 Nanoscale

9

TableS1:CD9density

wt Gag-24h Gag-48h

Mean±sem 1192±127 793±141 658±151

p-values 0.0141*

0.0004**

0.0141

0.0004

Out In Out In

Mean 748 6073 429 8430

± ± ± ± ±

SEM 161 1387 63 1655

p-values

0.0032*

0.0032

<0.0001***

<0.0001***

<0.0001***

0.0004**

0.0003

0.0004

<0.0001

<0.0001***

<0.0001

<0.0001

0.2812 0,2812

CD9 density is expressed as the number of localization events per µm2

(± sem). p values were

calculated using a non parametric two-tailed Mann-Whitney U-test. *, **, and *** respectively

indicatepvaluesbelow0.05,0,001and0.0001.Eachpvaluecorrespondingtoapairofdatasetsis

indicatedtwiceonthesamelineinthecorrespondingdatasetcolumn.

Page 36 of 44Nanoscale

10

TableS2:CD9clustersizes

wt Gag-24h Gag-48h

Mean±sem 3710±1513 4428±1606 5471±2198

Max 248100 244551 163721

Median

p-values

1603

<0.0001***

<0.0001***

2519

<0.0001

<0.0001***

2976

<0.0001

<0.0001

wt Out In Out In

Mean±sem 3710±1513 4378±1586 4423±1648 4737±1670

6527±2763

Max 248100 244551 144715 92393 163721

Median

p-values

1603

<0.0001***

<0.0001***

2519

0.8453

2519

<0.0001

0.8453

2748

<0.0001***

3320

<0.0001

<0.0001

CD9clustersizeisexpressedinnm2

(±sem).pvalueswerecalculatedusinganonparametrictwo-

tailedMann-WhitneyU-test. *, **, and *** respectively indicate p values below0.05, 0,001 and

0.0001.Eachpvaluecorrespondingtoapairofdatasetsisindicatedtwiceonthesamelineinthe

correspondingdatasetcolumn.

Page 37 of 44 Nanoscale

11

TableS4:CorrelationbetweenGFP-GagintensityandCD9density(Kendallmethod)

Cell

number

Tau p-value

1

2

3

4

5

6

7

8

0.73

0.19

0.72

0.61

0.85

0.46

0.34

0.57

3.10-15

0.047

3.10-15

5.10-10

2.10-16

2.10-6

0.00066

8.10-10

Bibliography

1C.Chamontin,P.Rassam,M.Ferrer,P.-J.Racine,A.Neyret,S.Lainé,P.-E.MilhietandM.Mougel,

NucleicAcidsRes.,2015,43,336–347.

2S.Nydegger,M.Foti,A.Derdowski,P.SpearmanandM.Thali,Traffic,2003,4,902–910.

Page 38 of 44Nanoscale

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3C.Espenel,E.Margeat,P.Dosset,C.Arduise,C.LeGrimellec,C.A.Royer,C.Boucheix,E.Rubinstein

andP.-E.Milhiet,J.CellBiol.,2008,182,765–776.

4A.Sergé,N.Bertaux,H.RigneaultandD.Marguet,NatMeth,2008,5,687–694.

5J.-B.Fiche,D. I.Cattoni,N.Diekmann, J.M.Langerak,C.Clerte,C.A.Royer,E.Margeat,T.Doan

andM.Nöllmann,PLoSBiol.,2013,11,e1001557.

6F.Levet,E.Hosy,A.Kechkar,C.Butler,A.Beghin,D.ChoquetandJ.-B.Sibarita,Nat.Methods,2015,

12,1065–1071.

Page 39 of 44 Nanoscale

24h post-transfection 48h post-transfectionHeLa cells expressing Gag-GFP

A

BC

Normalized Gag-GFP signal

Nor

mal

ized

CD

9 de

nsity

0 1

1

Freq

uenc

y

Localization precision (nm)0 20 40 600

1000

2000

3000

4000

5000

0 20 40 600

1000

2000

3000

4000

5000

Localization precision (nm)Fr

eque

ncy

0 20 40 600

1000

2000

3000

4000

5000

Localization precision (nm)

Freq

uenc

y

HeLa WT

24h post-transfection

HeLa cells expressing Gag-GFP

48h post-transfection

Distance (nm)

Dis

tanc

e (n

m)

150 200 250 150 200 250

Distance (nm)

250

300

350

0 200 104

D

WT

24h

48h

Areas depleted of CD9 clusters (107 nm2)

9 10 11 12

E

**

n =15

n =16

n =16

Page 40 of 44Nanoscale

Height (nm)

Dia

met

er (n

m)

0 2000

200

400

Gag +Control

CD9 density (number of localization/µm2)

B

A

0 2000 4000 6000 8000 100000

10

20

30

Freq

uenc

y

Page 41 of 44 Nanoscale

AFM

AFMCD9 (dSTORM)

AFMCD9 (dSTORM)

Gag-GFP

Page 42 of 44Nanoscale

CD9

CD81

p24

pNL4-3∆

SiRNA

+ + + + - + + + + -CD9

CD9-CD81

CD81CtlSc CD9

CD9-CD81

CD81CtlSc

SupernatantCell extract

CD9

CD9-CD81

CD81ScSiRNA

Rel

ativ

e Ex

pres

sion

as c

ompa

rd to

Sc

0

0.4

0.8

1.2

*

*

A

B CD9CD81

Page 43 of 44 Nanoscale

A

HeL

a ex

pres

sing

Gag

-GFP

HeL

a W

T

B

CD81

Clu

ster

siz

e (n

m2 )

8000

4000

0

4000

8000

48hWT

n=12 n=9

***

Page 44 of 44Nanoscale