Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

Nikola Hülsbusch

, Gonzalo P. Solis

, Vladimir L. Katanaev

, Claudia A.O. Stuermer

aDepartmentofBiology,UniversityofKonstanz,78467Konstanz,Germany

bDepartmentofPharmacologyandToxicology,UniversityofLausanne,CH-1005Lausanne,Switzerland

a r t i c l e i n f o

Keywords:

Reggie/flotillin Focaladhesion Integrin Rab11 Cargotrafficking Targeteddelivery

a b s t r a c t

Reggies/flotillinsareimplicatedintraffickingofmembraneproteinstotheirtargetsitesandinthereg- ulationoftheRab11a-dependenttargetedrecyclingofE-cadherintoadherensjunctions(AJs).Herewe demonstrateafunctionofreggiesinfocaladhesion(FA)formationand␣5-and␤1-integrinrecyclingto FAs.Downregulationofreggie-1inHeLaandA431cellsbysiRNAandshRNAincreasedthenumberofFAs, impairedtheirdistributionandmodifiedFAturnover.Thiswascoupledtoenhancedfocaladhesionkinase (FAK)andRac1signalingandgaininplasmamembranemotility.Wildtypeandconstitutively-active(CA) Rab11arescuedthephenotype(normalnumberofFAs)whereasdominant-negative(DN)Rab11amim- ickedtheloss-of-reggiephenotypeincontrolcells.Thatreggie-1affectsintegrintraffickingemerged fromthefasterlossofinternalizedantibody-labeled␤1-integrininreggie-deficientcells.Moreover,live imagingusingTIRFmicroscopyrevealedvesiclescontainingreggie-1and␣5-or␤1-integrin,trafficking closetothesubstrate–nearmembraneandmakingkiss-and-runcontactswithFAs.Thus,reggie-1in interactionwithRab11acontrolsRac1andFAKactivationandcoordinatesthetargetedrecyclingof␣5- and␤1-integrinstoFAstoregulateFAformationandmembranedynamics.

Introduction

Integrinsareheterodimericreceptors,composedof␣-and␤- subunitswhichareinvolvedintheformationofFAs.Integrin␣- and␤-subunitsassembleinacell-typespecificmannerandserveas receptorsfordistinctextracellularmatrixproteins,solubleligands andRGD-containingproteins(Margadantetal.,2011).

Duringcellmigration,integrinsandFAsundergorapidturnover (Margadantetal.,2011),aprocessthatincludesrecyclingandthe targetedre-deliveryofintegrinsandintegrin-interactingcompo- nentstonewlyformingfocalcontactsand nascentFAs(Caswell etal.,2009).IntegrinrecyclingrequiresEHD1- (epsinhomology domaincontainingprotein1)positivecargovesicletraffickingcon- trolled by Rab and Rho-GTPases and the cytoskeleton(Caswell etal.,2009;GrantandDonaldson,2009;Jovicetal.,2007).Rab11, akeyplayerincargorecycling,isthereforeindispensableforFA

Abbreviations:AJ,adherensjunction;Ab,antibody;CA,constitutivelyactive;DN, dominantnegative;FA,focaladhesion;FAK,focaladhesionkinase;FN,fibronectin;

PLL,poly-l-lysine;wt,wildtype.

∗Correspondingauthors.Tel.:+497531882236.

E-mailaddresses:Nikola.Huelsbusch@uni-Konstanz.de(N.Hülsbusch), Claudia.Stuermer@uni-Konstanz.de(C.A.O.Stuermer).

formationand cellmigration (Arjonenetal., 2012;Bridgewater etal.,2012;Evaetal.,2010).IntegrinandE-cadherintraffickingare alsoinfluencedbySrcandFAK(Caneletal.,2013).Thesekinases seemtoregulatewhethercellsincreasetheirmigratoryactivityby integrinactivationortheirengagementincell–celladhesionbyE- cadherinactivation(Caneletal.,2013).Howintegrinsaretargeted totheplasmamembraneandFAsisstillincompletelyunderstood.

Thestreak-likeFAsresembletosomeextentadherensjunctions (AJs) which are adhesive structures that are dependent on E- cadherin,thehomophiliccelladhesionproteininvolvedincell–cell interaction.Celladhesionbetweenepithelialcellsdependsonthe dynamic turnover of E-cadherin and AJs which involvesRab11 andthebalancedactivationofRhoGTPases(GrantandDonaldson, 2009;IvanovandNaydenov,2013).Weandothershaverecently recognizedthatthereggie/flotillinproteinsparticipateincelladhe- sion(Guillaumeetal.,2013;Kurrleetal.,2013;Solisetal.,2012).

OurworkhasshownthatreggiesregulateAJformationandthe Rab11a-dependentturnoverofE-cadherininA431cells(Solisetal., 2012,2013)andN-cadherindeploymentinaxonalgrowthcones ofhippocampalneurons(Bodrikovetal.,2011).Reggies/flotillins formoligomericclustersinlipidraftsatthecytoplasmicfaceof theplasma membrane and at specifictrafficking vesicles (Solis et al., 2007, 2013). We identified reggies as associates of the Rab11a-positive tubulo-vesicularrecycling compartment where

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-306483 Erschienen in: European Journal of Cell Biology ; 94 (2015), 11. - S. 531-545

https://dx.doi.org/10.1016/j.ejcb.2015.07.003

theyco-clusterwithmembranedeformingBARproteins,SNX4and EHD1 and directlyinteractwithRab11a and SNX4 (Soliset al., 2013).Reggie-deficientcellspresentedabnormalAJsandmigrated fasterinawound-closureassay(Solisetal.,2012).Thiscorrelated withabnormalE-cadherintraffickingand changesintheactiva- tionstateofRhoandRabfamilyGTPasesafterreggie-1knockdown (Solisetal.,2012,2013).

Theacceleratedcellmigrationafterreggiedownregulationand theinteractionwithCAP(c-cbl-associatedprotein),acomponent of FAs, which is knownto bind reggie-2 (Kimura et al., 2001;

Kiokaetal.,2002;Langhorstetal.,2008a),suggestedthatreggies mightalsoplayaroleinFAformation(SchmidtandDikic,2005).

Indeed,a proteomicanalysispreviously recognizedthatreggie- 1andreggie-2arepartoftheFAcomplex(Kuoet al.,2011).In addition,thedownregulationof reggieand overexpression of a dominantnegative(DN)reggie-1constructdisturbedthelocaliza- tionofoverexpressedprionproteininstructuresresemblingFAs (Schrock et al.,2009), affectedSrc and FAKand impairedaxon growth(Langhorstetal.,2008a;Munderlohetal.,2009).Asreggies were,furthermore,implicatedinthedeliveryoftheTcellreceptor totheTcellcap(Stuermeretal.,2004)andinRab11a-dependent E-cadherinrecycling(Solisetal.,2013),weconcludedthatreggies mightregulateaswellintegrinrecyclingandFAdynamicsthrough Rab11a(Stuermer,2010).

Here, we investigated whether reggie-1 is involved in the regulationofintegrindependentFAsinanalogytoitsroleinAJfor- mation,andwhetherintegrintraffickingdependsontheinteraction ofreggiewithRab11a.Indeed,downregulationofreggie-1bysiRNA andshRNAaffectedFAnumber,distributionandturnoverinHeLa andA431cellsandincreasedmembranemotilityonfibronectin (FN)incorrelationwithariseinRac1andFAKactivity.Weshow(by ColibriandTIRFmicroscopyinliveHeLacells)thatreggie-1traffics togetherwith␣5-and␤1-integrin.Thereggie-dependentincrease inFAnumberwasrescuedbyaRab11awildtype(wt)andaRab11a constitutivelyactive(CA)mutantconstruct,andwasmimickedby Rab11DN.

Thus, we show that the influenceof reggie onthe Rab11a- dependentrecyclingprocesshasextensiveeffectsonFAnumber anddistribution,integrintrafficking,membranemotilityandthe regulationofcell-matrixadhesion.

Materialsandmethods Reagentsandantibodies

AllcellculturereagentswerepurchasedfromGibcoBRL.Mono- clonalantibodies(mAbs)againstPaxillin,FAK,␤1-integrin,Rac1, reggie-2(flotillin-1)andreggie-1(ESA)werefromBDBiosciences, mAbagainst␤1-integrin(12G10)fromBiorad,mAbagainstGFP fromRoche, polyclonal antibody(pAb) againstR1 (clone 1680) from Sigma,pAb against ␣5-integrin fromSanta Cruz Biotech- nology, pAbs against ␣-tubulin and GAPDH from Abcam, pAb againstRab11FIP1(RCP)wasakindgiftfromRytisPrekeris(Uni- versity of Colorado, Denver), anti phospho-pAbs against pFAK (Y576/577),Pyk2andpPaxillinwerepurchasedfromCellSignaling.

ThePAK-1PBDagaroseconjugatedRac1-GTPassayreagentwas purchasedfromUpstateandallsecondaryAbsforWesternblots andimmunostainingfromJacksonImmunoResearch.

Plasmids

The R1-mRFPand R1-EGFP-rescue vectorshave beenprevi- ouslydescribed(Langhorstetal.,2007;Solisetal.,2007,2012).

ThemCherry-Rab11aconstructandthe␣5-integrin-EGFPvector werekindlyprovided by RichardEva(Universityof Cambridge,

UK)andDonnaWeb(UniversityofVirginia,Charlottesville,USA).

The ␤1-integrin-GFP was a kind gift from Sue Craig (Univer- sityof Manchester, UK). EGFP-Rab11a Q70L and S25N mutants werekindlyprovidedbyStephenFerguson(UniversityofWest- ernOntario,Canada)andthemRFP-Rab11amutantswerecreated byreplacingtheEGFP-ORFbymRFPfromthepmRFP-C1vector (Solis et al., 2012). The GFP-Paxillin construct was a gift from KennethYamada(Bethesda,Maryland,USA).TheEGFP-Rab4awt wasobtainedfromMarciScidmore(CornellUniversity,NY,USA), theECFP-Rab4aDNwasakindgiftfromRalfJacob(Universityof Marburg,Germany)andmRFP-Rab8awtfromJohanPeränen(Uni- versityofHelsinki,Finland).

Cellculture

HeLaandA431cellswereculturedinMEMandDMEM,respec- tively and supplemented with 10%FCS, penicillin/streptomycin and l-glutamine. VectortransfectionwasperformedwithLipo- fectamine(LifeTechnologies)for48handsiRNAtransfectionwas carriedoutwiththeNanofectinesiRNAtransfectionreagent(PAA) for72h,allaccordingtothemanufacturer’sinstructions.Labeled (AlexaFluor546) siRNA duplexes against R1 (R1.0) or luciferase fromfirefly(GL2)werepurchasedfromQuiagenaccordingtotarget sequencesdescribedpreviously(Solisetal.,2007).

Stabletransfectedcelllines

HeLaandA431cellswithstableknockdownwereobtainedby usingshRNAplasmids(Solisetal.,2012).Cellswereselectedin normalgrowthmediumsupplementedwith10␮g/mlpuromycin.

StablytransfectedHeLaandA431cells(shLucandshR1.0)were describedpreviously(Solisetal.,2012,2013).AsecondHeLacell lineforthedownregulationofreggie-1(shR1.1)wasobtainedusing theprimers5sensestrand,5-gatccccGTTCATGGCAGACACCAAGtt- caagagaCTTGGTGTCTGCCATGAACttttta-3,and3antisensestrand, 5-agcttaaaaaGTTCATGGCAGACACCAAGtctcttgaaCTTGGTGTCTGC- CATGAACggg-3.AnnealedprimerswereclonedusingtheBamHI andHindIII sitesof thepRetroSuper vector.Cellswereselected andfurtherculturedin10␮g/mlpuromycin.

Immunofluorescenceandmicroscopy

HeLaandA431cellswerefixedwithPFA,washedwithPBSand permeabilizedwithPBScontaining0.01%Triton-X-100.Afteraddi- tionalwashing steps,cellswere blockedwith1% bovineserum albumin(BSA)inPBS.Immunostainingwasperformedovernight withthecorrespondingAbs.Afterthree washeswithPBS,cells wereincubatedwiththesecondaryAbsfor1.5h,followedbywash- ingstepswithPBSandMilliQwater.Thecoverslipsweremounted withMowiol(Sigma-Aldrich)anddriedat4^◦C.Microscopywas performedbyaPlan-Apochromat63×/1.4oilobjectiveoranApoc- hromat 40×/1.2Wobjective ata confocalmicroscope (LSM700, Zeiss,Jena).Forthelivestaining,cellswerewashedwithcoldPBS, incubatedwiththeAb,dilutedincoldmedium,at4^◦Cfor1h,fixed andimmunostainedwithoutpermeabilizationasdescribedabove.

Spreadingassay

HeLa shLucand shR1 cells weretrypsinized(0.05% Trypsin- EDTA,Gibco)for3min,centrifuged,resuspendedinnormalgrowth mediumandplatedontoFN-coatedcoverslips.

After2hofspreading,cells werefixedwithPFA,permeabili- zedandimmunostainedwithpaxillinandphalloidinasdescribed above.Thesizeofmorethan340shLucandshR1cells,respectively,

wasmeasuredinthreeindependentexperimentswithAxiovision Rel.4.8.Statisticalanalysiswasperformedusingthepairedt-test.

Liveimagingofoverexpressedproteins

Cells were seeded onto ␮-dishes with glass-bottom (ibidi) coated with FN and transfected as described above. After48h cells were recorded with a Colibri Cell Observer SD system, a widefield fluorescence microscope equipped with LED as light source(470nmand555nmat100%power)withan␣-PlanFluar 100×/1.45objectiveat37^◦Cand5%CO₂everysecondfor5min tomonitorco-traffickingoftwodifferentproteinsorevery30sec for1htorecordthemovementofFAswithaAxioCamHRm(all fromZeiss).DatawereanalyzedinAxiovision4.8(Zeiss)orImageJ (NationalInstitutesofHealth,Bethesda,MD).Forthequantifica- tionofFAturnoverallFAsofeachrecordedcellwereanalyzedand dividedintotheindicatedcategories(5independentexperiments withatleast3cellsperconstructperexperiment).Alternatively, cellswererecordedeveryothersecforupto5minat37^◦Cinculture medium(bufferedwith25mMHEPES)attheZeissAxioObserver Z1,equippedwithaTIRFsystem,usinga␣-PlanFluar100×/1.45 objectivewithdiodelasers488nmand543nm(Zeiss,Bioimaging center(BIC)UniversityofKonstanz).Videoswereanalyzedwith ZEN2012(Zeiss)orImageJ.

Liveimagingandanalysisofcellmotility

Cellswereseededon␮-dishes(ibidi)coatedwithFNandgrown overnightat37^◦Cand5%CO₂.Picturesweretakenevery30sfor1h inbrightfieldwitha40×objectiveat37^◦Cand5%CO₂.Moviesand kymographsoflinesacrossthecellswereanalyzedwithImageJ.

Thespeedofmovementofthecelledgeswasmeasuredusingthe plugin“KymoLineROI”(ElisaMay,BICUniversityofKonstanz).100 cellspercelllineweremeasuredinthreeindependentexperiments.

Statisticalanalysiswasperformedusingthepairedt-test.

Biochemicalanalysis

ToanalyzetheconcentrationofspecificproteinsinshLucand shR1HeLaandA431lines,cellsweregrowntoconfluencyonplas- ticcellculturedishes(Greiner).Toquantifythephosphorylated proteins,HeLacellsweregrownonFNcoatedglasscoverslipsand lysedwithice-cold lysis buffer(20mM TrisHClpH7.5 100mM NaCl,5mMMgCl22mMEDTA,1%Triton-X-100and10%glycerol) containingHALTprotease-phosphataseinhibitorcocktail(Thermo Scientific).Thelysedcells(inlysisbuffer)werehomogenizedwith a27Gneedle.Extractswerecentrifugedandthesupernatantwas boiledat95^◦Cfor5min.ProteinswereseparatedbySDS-PAGEand transferredtoanitrocellulosemembranebyWesternblot.Mem- braneswereblockedandanalyzedwiththecorrespondingprimary andsecondary Abs.Fortheanalysisofbiotinylated ␤1-integrin, cells were incubated on ice with 1mg/ml sulfo-NHS-SS-biotin (Pierce)inPBSfor30min.Freesulfo-NHS-SS-biotinwasquenched bytwowasheswithice-coldsolution,containing100mMglycine, 0.1mMCaCl₂and1mMMgCl₂.Biotinylatedproteinsfromcleared cellextracts,lysedasabove,werecollectedwith25␮lofstrep- tavidin beads at 4^◦C overnight and analyzed by Western blot withanAbagainst␤1-integrin.Whereindicated,cellswereincu- batedfor5hat37^◦Cinnormalmediasupplementedwith10␮M cycloheximide to stop protein synthesis prior to biotinylation.

Quantification of proteins from Western blots was done with ImageJ.

Rac1-GTPassay

Lysateswereclearedbycentrifugation.Proteinconcentration wasmeasuredandadjusted.Asmallamountofeachsamplewas savedfor proteinanalysis ofthe input.Lysateswereincubated with10␮goftheRac-Assay-ReagentPak-1PBD agaroseconju- gate(Upstate)at4^◦Cfor1h. Agarosebeadswerewashedthree timeswithlysisbuffer,resuspendedin2×samplebufferandboiled for 5min prior toseparation onSDS gels. Proteinswere trans- ferredtoa nitrocellulose membrane,blocked, incubatedwitha primaryAbagainstRac1(BDBioscience)andanalyzedasdescribed above.

QuantificationofFAs

HeLaandA431shLucandshR1cellswereseededonFN-coated glasscoverslips and fixedafter24hincubation at37^◦C and5%

CO2.ForsiRNAtreatment,thecellsweretransfectedandseeded after48hontoFNorPLL-coatedglasscoverslips.Afteradditional 24hincubation at37^◦C, cells werefixed.For DNAtransfection, shLucand shR1 cells wereseeded 6hafter transfectionto FN- coatedcoverslipsandincubatedfor48hpriortofixation.Cellswere thenimmunostainedwiththepaxillinAbasdescribedaboveand recordedattheLSM.ThenumberofFAspercellwascountedand thesizeofeachcellwasmeasuredwithImageJ.Eachexperiment wasperformedatleastthreetimesand100to150cellspercon- structandexperimentwereanalyzed.ThenumberofFAspercell inHeLacellswasnormalizedtothesizeofthecells(␮m²).InA431 cells,thenumberofFAspercellwasquantified,sinceitwasalready shownthatshLucandshR1arenotdifferentinsize(Solisetal., 2012).Statisticalanalyseswereperformedasindicatedbyapaired t-testorone-wayANOVA.

Pulsechaseexperimentswithˇ1-integrinAb

CellsgrownonFN-coatedcoverslipswereserumstarved for 2h and incubatedwith the12G10 ␤1-integrin Ab (5␮g/ml) in serumfreemediumfor1hat37^◦C.Cellswerewashedthreetimes withPBSandchasedfortheindicatedtimeswithnormalgrowth medium.AfterthreewasheswithPBS,cellsurface-boundAbwas removed withcoldacidic buffer(0.5MNaCl, 0.2Macetic acid) for10sfollowedbythreeadditionalwasheswithcoldPBS.Then, cellswerefixedwithPFA,permeabilizedandimmunostainedas describedabove.Forquantification,themeanfluorescenceinten- sitypercellofcombinedZ-stackswasdeterminedwithImageJfrom threeindependentexperiments(100–150cellsforeachtimepoint, constructandexperiment).Statisticalanalysiswasperformedwith one-wayANOVA.

Drugtreatment

CellsseededonFNweregrownasdescribedabove.Thecells werethenincubatedfor1hinmediumcontaining1mMamiloride orDMSOascontrol.ToanalyzeandquantifythenumberofFAs, cellswerefixedandtreatedasdescribedabove.

Results

Reggie-1knockdownaffectsFAs

InHeLacells,FAsaretypicallylocalizedatthecellperiphery, asexemplifiedbyimmunostainingswithanti-paxillinAb(Fig.1A).

In thesecells,reggie-1andreggie-2resideatintracellular vesi- clesandattheplasmamembrane(Langhorstetal.,2008b;Solis etal.,2013).Paxillindoesnotco-localizetoanysignificantextent withreggie-1andreggie-2exceptfor smallareaswhere reggie

Fig.1.Reggie-1siRNAaffectsthenumberanddistributionofFAsinHeLacells.(A)Immunostainingofpaxillinandreggie-1(R1)inHeLacellsonfibronectin(FN)shows paxillininFAsinthecellperipheryandR1insmallvesiclesandpunctawhichoccasionallyco-localizewithFAs(arrowheads).Theboxedareaisenlarged(right).Asimilar patterncanbeobservedwithEGFP-paxillinandreggie-2(R2)(B).Reggie-1siRNAtreatedcells(labeledsiRNAinred)onFN(C)orpoly-L-lysin(PLL)(D)havemoreFAsin ascattereddistribution(CandDlowerpanels)comparedtoGL2siRNAcontrolcells(CandDupperpanels).Theboxedareasareenlarged(right).(E)QuantificationofFAs per␮m²showedsignificantlymoreFAsinreggie-1knockdowncellsonFN(n=3,**p<0.01,pairedt-test,errorbars,SEM)andonPLL(n=4,**p<0.01,pairedt-test,error bars,SEM)comparedtoGL2controls.Combinedz-stacksfromimmunostainingwithanAbagainstR1proveefficientdownregulationofreggie-1byR1siRNA(red).Cells containingthesiRNAaremarkedbyarrowheads(F).Allscalebars,10␮m.NucleiarelabeledbyDAPI(blue).

vesiclesseemtopartiallyoverlapwithpaxillin(orEGFP-paxillin) positive FAs (Fig. 1A, Bzoom ins). Formerresultsshowed that theknockdown of reggie-1leads tothe degradation ofreggie- 2inHeLacells (Solisetal.,2007,2013).Toinvestigatewhether theknockdownof reggie-1andlossof reggie-2affectsFAs, we treatedcellswith(AlexaFluor546-labeled)siRNAagainstreggie-1

(R1)orluciferase(GL2)ascontrol.Immunostainingsconfirmedthe reductionofreggie-1insiRNAtreatedcells(Fig.1F),showingthe efficiencyofthesiRNA.ThenumberanddistributionofFAswere determinedbythepaxillinAb(Fig.1C–E).

R1siRNA ledtoachange ofFAsonfibronectin (FN)andon poly-l-lysine (PLL) (Fig. 1C and D lower panels): The paxillin

clustersbecamedisorganizedandmorewidelydistributedcom- paredtoGL2siRNAtreatedandwildtypeHeLacells,whereFAs residedmainlyatthecellperiphery(Fig.1A,CandD).Toquan- tify this effect thenumber of FAs per␮m² wasdetermined in reggie-1siRNAtreatedandGL2siRNAcontrolcells.Reggie-1knock- down led to a 34% increase in number of FAs on PLL and to a 46% increase on FN compared to control siRNA treated cells (Fig.1E).

Tosubstantiatethiseffectandtoexploretheunderlyingmech- anismsweusedcelllineswithstableknockdownofreggie-1(shR1 cells) (Soliset al.,2012, 2013).Twodifferent shRNAsequences againstreggie-1(shR1.0andshR1.1)wereusedtoexcludeunspe- cificside effects.TheshRNAsequenceagainstluciferase(shLuc) servedascontrol.Immunostainings(Fig.2A),and Westernblots (Fig.2C) withthe reggie-1Ab showed that shR1.0 and shR1.1 were efficient in downregulating reggie-1 and both led tothe

Fig.2.Stableknockdownofreggie-1affectsFAsinHeLaandA431cells.(A)HeLacellsonFNstablytransfectedwithshR1.0andshR1.1losemostoftheirreggie-1specific immunostaining(red).FAslabeledbypaxillin(white)weremorenumerousandscatteredinshR1.0aswellasinshR1.1cells,comparedtotheshLuccontrol.Boxedareasare enlarged(below).(B)QuantificationofFAsper␮m²revealedthatshR1.0andshR1.1havesignificantlymoreFAsthanshLuccells(n=3,***p<0.001,one-wayANOVA,error bars,SEM).(C)WesternblotanalysisofshLuc,shR1.0andshR1.1cellsshowthatreggie-1wasdownregulated,whichledtoadegradationofreggie-2,whereas␤1-integrin, paxillinandFAKremainedunchanged.(D)Paxillin(white)localizedinA431cellsonFNpredominantlyatcelledgesandoccasionallyinFAstructures.shR1.0cellshad significantlymorepaxillin-labeledFAspercellwhichwerewidelyscatteredandnotconfinedtotheperiphery.Theboxedareasareenlarged(right).QuantificationofFA numberpercellrevealedsignificantlymoreFApercellinshR1.0cellscomparedtoshLuccells(n=3,**p<0.01,pairedt-test,errorbars,SEM)(E).Reggie-1downregulation wasefficientinshR1.0cells(F).Allscalebars10␮m.NucleiarelabeledbyDAPI(blue).

degradationofreggie-2.Thepaxillinstaininginthesecellsdemon- stratedthatbothreggie-1shRNAsaffectedFAstoasimilarextent asthesiRNAagainstreggie-1:FAsbecamemorenumerous,disor- ganizedandscatteredintheabsenceofreggie-1(Fig.2A).Similar totheR1siRNAtreatedcells,thenumberofFAsper␮m²increased by30and37%,respectively,inshR1.0andshR1.1cellsovercon- trols(Fig.2B).Thiswasnotcausedbychangesintheexpressionof paxillin,␤1-integrinorfocaladhesionkinase(FAK),asshownby Westernblots(Fig.2C).

Toassure thatthe phenotype afterloss ofreggie-1 wasnot confinedtoHeLa cells,we usedA431cells witheithera stable knockdownofreggie-1(shR1.0)orluciferase(shLuc),seededthese cellsonFNandimmunostainedthemwithpaxillinandreggie-1 (R1)Abs.StainingandWesternblotsshowedefficientdownreg- ulation of reggie-1 in shR1.0 cells (Fig. 2D and F). A431 cells are epithelial cells that, in contrast to HeLa cells, form strong E-cadherin-dependent cell–cell contacts and do not form FA- structurestothesameextentasHeLacells. Paxillinlocalizedin thesecells either toruffles or,toa lower extent,toFAs atthe periphery,wherethecellswerenotincontactwithoneanother (Fig.2D).Reggie-deficientA431cellshadmorepaxillin-labeledFAs andreducedpaxillininruffles(Fig.2D).ThenumberofFAspercell increasedby56%inshR1.0cellscomparedtoshLuccells(Fig.2E).

The formation of FAs is integrin-dependent, implying that reggie-1downregulationmightaffectintegrintrafficking,aspre- viously seen for E-cadherin (Solis et al., 2013). Therefore, we transfectedHeLa shLucand shR1.0cells(henceforth shR1cells) withan␣5-integrin-EGFPconstructandusedTIRFmicroscopyto recognize the proteinin FAsin thesubstrate-near region.This approachshowed,thatshR1cellsdisplayedmoreintegrin-EGFP- labeled FAs at the substrate-near membrane than shLuc cells (Fig.3A),similartotheresultobtainedwithpaxillinstaining.This effectwasalsovisible,whenshLucandshR1cellswereimmuno- stainedlivewithanAbagainst␤1-integrin(Fig.3B).Tocontrolthat these␤1-integrin-clustersrepresentFAs,shLucandshR1cellswere transfectedwithEGFP-paxillinandstainedliveagainst␤1-integrin.

Fig.3Dshowsthatscattered␤1-integrinclustersinshR1cellsare indeedpositivefortheFA-markerpaxillin(zoomin).Thus,inthe absenceofreggie-1,FAsaredisorganizedasseenwithpaxillinand

␣5and␤1-integrin.

Asanadditionalcontrolforthespecificityoftheeffect,shR1 cellsweretransfectedwithareggie-1rescueconstruct(R1-rescue) which is resistant tothe shRNA (Munderloh et al., 2009). The non-rescued cells (Fig. 3C and box 2) displayed the shR1-FA phenotype showingan increased number and widelyscattered paxillin-labeledFAswhereastherescuedcellsshowedfewerFAs positionedatthecellperipherymuchascontrolcells(Fig.3Cand box1;forquantificationseeFig.8HandI).WesternblotsofshR1 comparedtoshLuccellsshowednodifferenceinthetotallevelof

␣5-integrinorinthephosphorylationstateofpaxillinbutasig- nificantincreaseof phosphorylated(Y576/577)FAKand aband aboveFAK(Fig.3E).Theexpressionlevelof␣5-integrin-EGFPis also not different in shLuc and shR1 cells (Fig. 3E lower pan- els).Thesedatashowthattheknockdownofreggie-1ledtoan increasein FAnumber, changeinshape andscattereddistribu- tion,togetherwithincreasedphosphorylationofFAK,suggesting that reggiesare involved in the regulationof FAstructure and position.

Reggie-1downregulationaffectsplasmamembranemovement andspreading

TheregulationandturnoverofFAsisimportantforcellmotil- ity.Therefore,weaskedwhetherreggie-1downregulationandthe ensuingchangeinFAnumberanddistributionwouldaffectmem- branemotility.shLucandshR1cellsonFN-coatedibidi␮-dishes

weremonitoredfor1h(Movie1).Therateofmembranemove- mentwasanalyzedbykymographsthatweretakenforeachcellin thevisualfield(Fig.4A).Themembranedynamicsonbothsidesof thecellweremeasuredandquantified(Fig.4A,B).Thekymographs andMovie1showthatshR1cellsmovemoreactivelycomparedto shLuccells(Fig.4Aarrowheads).Aquantificationofthismovement showedthatthemembraneintheperipheryofshR1cellsmoved twiceasmuchasinshLuccells(Fig.4B).Thus,theriseofFAnumber andtheirabnormaldistributioninshR1cellsislinkedtoenhanced membranedynamics.

Toassesswhethertheeffectofreggie-1knockdownmightnot onlyaffectcellsubstrateadhesionduringsteadystatebutalsothe earlystagesofcell-substratebinding,weperformedaspreading assayandlabeledthecellswithphalloidinandpaxillinantibody.

After2hofspreadingonFNbothcelllineswerespreadbutshR1 cellswereonaverageabout100␮m² smallerthancontrol cells (Fig.4C,D),inagreementwithpublisheddatashowinganimpaired spreadingofreggie-1siRNAtreatedHeLacells(Neumann-Giesen et al.,2007).Stainingfor paxillin revealed thatshLuc cells had manylargepaxillinclustersthatwerelocalizedpreferentiallyat theperipheryofthecellswhereasshR1cellshadalessorganized patternofpaxillinstainingappearingasverysmalldots(Fig.4C zoomins).

RoleofRac1activation

Cell movementand spreadingare influenced byRacactivity (Nobes and Hall,1999)and Racwassuggestedtocontributeto FAregulationthroughadhesioncomponentstabilization(Steffen etal.,2013).ToanalyzewhethertheactivationofRac1wasaffected in shR1 cells, we determined the amount of GTP-bound Rac1 byaffinity-purificationwiththeRac1-bindingproteinPak1-PBD, boundtoagarose.Indeed,whilebothshLucandshR1cellshadsim- ilaramountsoftotalRac1(Input),shR1cellshadtwiceasmuch Rac1-GTPthanshLuccells(Fig.4E).Therefore,weusedtheRac1 blockeramiloride(Koivusaloetal.,2010)toassesswhetherRac1 inhibitioncanchangetheeffectofreggie-1knockdownonFAs.The numberofFAsinshR1cells(butnotinshLuccells)wasreduced tonearnormal,when cells weretreatedfor 1hwithamiloride (Fig.4F,G).TreatmentofshLucandshR1cellswithDMSOascontrol showedthetypicalincreaseinnumberofFAsinshR1,compared toshLuccells.Thus,reggiescontributetothecontroloftheactiva- tionofRac1whichisconnectedtothecontrolofFAnumberand distribution.

RacactivitywasimplicatedinvesicletraffickingandRacitself wasimpliedtobesubjectedtorecyclingtotheplasmamembrane duringcellmigration(Palamidessietal.,2008).Thisandthenotion thatFAturnoverisstronglydependentonvesicletraffickingand integrindelivery(Bridgewateretal.,2012),ledustoask,whether andhowreggiesmightaffectFAturnoverandintegrintrafficking andrecycling.

Reggie-1trafficstogetherwith˛5-andˇ1-integrin

Tocorroboratethatreggie-1regulatesFAturnoverthroughits influenceonintegrintraffickingandrecycling,weco-transfected HeLa cells withreggie-1 coupledto mRFP(R1-RFP) and either

␣5-integrin-EGFP or ␤1-integrin-EGFP, because ␣5␤1-integrin is a receptor for FN. Recordings of moving vesicles with the Colibri system revealed that R1-RFP at vesicles move together with ␤1-integrin, as well as with ␣5-integrin (Fig. 5A, B and Movie2).

Wewereinterestedinco-traffickingofreggie-1andintegrins attheplaneofFAsandthereforeusedTIRFmicroscopyonliving cellstoobserveintegrintraffickingatthesubstrate-nearregion.

Themoviesshowedthat ␤1-integrin-EGFP(Fig.6A,Movie3)as

Fig.3.Reggie-1knockdownaffectsthedistributionof␣5-and␤1-integrinsandFAKphosphorylation.(A)TIRFimagesofoverexpressed␣5-integrin-EGFPinHeLashLuc andshR1cellsonFNshowmoreFAsinshR1cells.␤1-integrinliveimmunostainingshowsitslocalizationatthemembrane.InshLuccellsFAspreferentiallyresideatthe periphery,whileshR1cellsshowascattereddistributionofintegrinstaining(B).Boxedareasareenlarged(right).shR1cellshadmoreandscatteredFAscomparedtoshLuc cells.shR1cellsweretransfectedwithaR1-rescue(shRNAinsensitive)construct(Reggie-1-EGFP-rescue,showninred)andimmunostainedwithapaxillinAb(white).The boxedareasareenlarged(1and2below)showingFAsintransfected(1)anduntransfected(2)cells(C).TheincreaseofFAnumberwasalsovisibleinshLucandshR1cells overexpressingGFP-Paxillinandstained(live)witha␤1-integrinAb.Theboxedareasareenlarged(right)and␤1-integrinclustersarepositivefortheFAmarkerpaxillin (arrowheads)(D).Allscalebars,10␮m.NucleiarelabeledwithDAPI(blue).(E)shLucandshR1cellsgrownonFNshowednodifferencesintotal␣5-integrinlevelsand paxillinphosphorylation(pPaxillin)butthephosphorylationofFAKatY576/577inshR1cellswasincreased.(n=5,±SEM,*p<0.05,pairedt-test).TransfectedshLucand shR1cellsshowednodifferenceintheexpressionlevelof␣5-integrin-EGFP.

wellas␣5-integrin-EGFP(Fig.6B,Movie4)arelocalizedinFAsat theperiphery(Fig.6AandBzoomins,upperpanels),aswellasin movingvesicles.VesicleshadbothR1-RFPand␣5-integrin-EGFP orR1-RFPand␤1-integrin-EGFP,respectively(Fig.6A,BandMovie 3and4).WhenFAswereidentifiedwithEGFP-paxillinincellsco- transfectedwithR1-RFP,paxillinclusteredatFAsbutwasnotseen inmovingvesicleswithreggie-1.However,R1-RFPvesiclesoften contactedina“kiss-and-runmode”theEGFP-paxillinlabeledFAs (asshowninFig.6CandMovie5).

ThusR1-RFPvesiclescontactpaxillin-positiveFAs,andreggie- 1co-trafficswith␣5-and␤1-integrin-EGFPinvesicles,suggesting aninvolvementofreggie-1inintegrin-dependentFAformationand remodeling.

Reggie-1downregulationaffectstheturnoverofFAs

FAs are subject of constant remodeling and turnover (Bridgewater et al., 2012).To determine whetherthe turnover

Fig.4. Reggie-1affectsmembranedynamics,spreadingandRac1activation.Liveimagingin30sintervals(underbrightfield)for1hshowsthatshR1cellsmovemorethan shLuccells(A)(Movie1).Therightpanelsshowkymographsoftheregions1and2,markedbytheredlines.Thearrowheadsindicatetheextentofmembranedynamicsof thetwocellsover60min.ThemembranemotilityofshR1cellsishighercomparedtoshLuccells(B)(n=3,**p<0.01,pairedt-test,errorbars,SEM).(C)Spreadingassaywith shLucandshR1cellsfor2honFN.shR1cellsaresmallerandshowlessorganizedpaxillinclusters,comparedtocontrolcells.Boxedareasareenlarged.(D)Quantificationof thecellsizeafter2hofspreading(n=3,**p<0.01,pairedt-test,errorbars,SEM).(E)Rac1isoveractivatedinshR1cells(Rac-GTPassay),whereastotalRac1isequalinshLuc andshR1cells(input).Rac1-GTPwas(twofold)increasedinshR1cells(lanes1and2)(n=3,±SEM,*p<0.05,pairedt-test).(FandG)InshLucandshR1cells,treatedwith theRac1inhibitoramiloridefor1h,thenumberandlocalizationofFAswasrestoredtovaluesobservedinshLuccells(F).Boxedareasareenlarged(right).Quantification ofFAsper␮m²,revealedsignificantlyfewerFAsinshR1cellswithamiloridethaninDMSOtreatedshR1cells(G)(n=3,***p<0.001,**p<0.01,*p<0.05one-wayANOVA, errorbars,SEM).Allscalebars10␮m.

and remodelingofFAs depends onreggie-1,wemonitoredthe EGFP-paxillinlabeledFAsin shLucandshR1cellsonFNfor1h.

ForallFAsofeachrecordedcell,weanalyzed,whethertheywere stable(Fig.7A;redarrows),newlyformedand/orelongating(blue arrows),ordisappearingand/orretracting(greenarrows)(Table1,

Fig.7AandMovie6).ThereweresignificantlymoreEGFP-paxillin labeledFAs per ␮m² in shR1 than in shLuc cells (Table 1). In addition,shR1cellsshowedsignificantlymorestable,aswellas highernumbersofdisappearingand/orretractingFAs.Thenumber ofnewlyformedand/orelongatingFAswasalsoincreased,even

Fig.5.Co-traffickingofreggie-1with␣5-integrinand␤1-integrin.LiveimagingofHeLacellsonFN,transfectedwithR1-RFPand␤1-integrin-EGFP(A)orR1-RFPand

␣5-integrin-EGFP(B).Vesicletraffickingwasrecordedin1sintervalsover5minwithaColibrisystem.Theboxedareasareenlarged(right)andshowtimelines(inseconds) ofmovingvesiclesintheredandgreenchannelsseparately.ArrowspointtovesiclecontainingbothR1-RFPand␤1-integrin-EGFP(A)orR1-RFPand␣5-integrin-EGFP(B).

Scalebars,10␮m.ThefigurecorrespondstoMovie2.

Fig.6.Reggie-1andintegrinsco-trafficinthesubstrate-nearregionandcontactFAs.LiveimagingofHeLacellsonFNtransfectedwithR1-RFPand␤1-integrin-EGFP(A), R1-RFPand␣5-integrin-EGFP(B)orR1-RFPandGFP-Paxillin(C).Vesicletraffickingwasrecordedin2sintervalsupto5min.Theboxedareasareenlarged(A,BandCtothe right)andtimelines(inseconds)oftheredandgreenchannelsareshownseparately(AandB).ExamplesofvesiclescontainingR1-RFPtogetherwith␤1-integrin-EGFP(A) orR1-RFPtogetherwith␣5-integrin-EGFP(B)aremarkedbyarrowheads.GFP-Paxillin(whichisnotatvesicles)demarcatesFAs(C).ThetimelineshowsR1-RFPvesiclesthat contactoroverlap(arrowheads)withFAs(green,thresholdedimage).Scalebars,10␮m.(A)correspondstoMovie3,(B)toMovie4and(C)toMovie5.(Forinterpretationof thereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

though this was not significant, probably because of thesmall samplesize(Table1).Cellswithknockdownofreggie-1showa higherextentofFAremodeling,comparedtoshLuccells(Fig.7A, Movie6).Thus,reggie-1seemstoinfluencebothFAnumberand remodeling.

Reggiesregulateintegrinrecycling

Todeterminewhetherreggie-1influences␤1-integrintraffic- king,weconductedapulsechaseexperimentinshLucandshR1 cells.Cellswereincubatedwiththe␤1-integrinAb12G10for1h

Fig.7.ReggieknockdownaffectsFAturnoverandintegrintrafficking.LiveimagingofHeLashLuc(upperpanel)andshR1cells(lowerpanel)transfectedwithGFP-Paxillinon FN(A).Cellswererecordedfor60minandpicturesweretakenevery30s.ImagestotheleftshowFAsinshLucandshR1cellsattime0(beginningoftherecording).Theboxed areasareenlarged(right)andtimelines(inseconds)areshown.StableFAsaremarkedbyredarrows,emergingand/orelongatingFAsbybluearrowsandretractingand/or disappearingFAsbygreenarrows.TheremodelingofFAsinshR1cellsishigher,comparedtoshLuccells.ThefigurecorrespondstoMovie6.(B)Pulsechaseexperimentwitha

␤1-integrinAb(12G10).shLucandshR1cellsendocytosedthesameamountofAbafterthepulse(0min).Ablabelinginz-stacksdisappearedfasterinshR1comparedtoshLuc cellsasquantifiedin(C)(n=4,*p<0.05,***p<0.001,one-wayANOVA;errorbars,SEM).ThesignalofshLuccellsafter1hpulse(0min)wassetto100%.(D)Total␤1-integrin (lowerpanel)andsurfacelevels(upperpanel)donotdifferinshLucandshR1cells,asshowninabiotinylationexperimentincontrolcellsandafterblockageofprotein synthesiswithcycloheximide(n=3±SEM).(E)Abagainst␤1-integrinaddedtoliveHeLacellsfor1hat37^◦Cco-localizedwithRab11FIP1andwithEGFP-Rab11awt(F)at theperinuclearrecyclingcompartment.(G)HeLacellstransfectedwith␣5-integrin-EGFPandmCherry-Rab11ashowedco-localization(arrowheads)inthesubstrate-near region(TIRFimages).Theboxedareaisenlarged(right).(H)EGFP-Rab11aCAco-localizedwithendogenousreggie-1(R1)invariousvesicles(arrows).Allscalebars,10␮m.

ThenucleiarelabeledwithDAPI.

Table1

QuantificationofFAtunover.

NumberofFAs Appearingand/orelongatingFAs Disappearingand/orretractingFAs StableFAs

shLuc shR1 shLuc shR1 shLuc shR1 shLuc shR1

0.031±0.0048 0.050±0.0013^** 0.0145±0.0043 0.0173±0.0046 0.0126±0.0027 0.0210±0.0032^** 0.010±0.0013 0.0186±0.0054^* FAsper␮m²;^*p<0.05;^**p<0.01.

HeLashLucandshR1cellsweretransfectedwithEGFP-PaxillinandallFAsweremonitoredover1handdividedintothethreecategories.shR1cellsrevealedsignificantly morestableanddisappearingand/orretractingFAsandtendedtohavemoreappearingand/orelongatingFAs␮m².ThetotalnumberofFAswasalsoenhancedinshR1cells, comparedtothecontrol,asseenbefore(n=5,^**p<0.01,^*p<0.05,pairedt-test,mean,±SEM).

at37^◦Cinserumfreemedium,followedby30or60minchasein normalgrowthmediumandanacidicwashtoremoveallexter- nallyboundAb(Fig.7B).After1hpulse␤1-integrinAbwasseen insmallvesiclesandtoalargerextentintheperinuclearrecycling compartmentofshLucandshR1cells.Fluorescenceintensitymea- surementsofwholecellstacksshowedthattherewasnodifference betweencontrolandreggie-1deficientcellsafter1hpulse(Fig.7C).

However,after 30 and60minchase,shLuc cells retainedmore

␤1-integrin Ab labeling than shR1 cells (Fig. 7B, C), indicating fasterrecyclingof␤1-integrininreggie-depletedcells.Toexclude thatdifferentcell-surfacelevelsof␤1-integrinmightberesponsi- ble forthedifferences inrecycling,cell-surface␤1-integrinwas biotinylated under normal growthconditions as wellas under cycloheximidetreatmenttoblockproteinsynthesis.Thesurface levelsof␤1-integrininshLucandshR1cellswerenotsignificantly differentunderbothconditions(Fig.7D).Inadditiontherewasno enhanced degradationof ␤1-integrinin shR1cells, astotal ␤1- integrinwas similarin shLucand shR1 cells withand without cycloheximidetreatment(Fig.7Dlowerpanel).Altogether,these resultsindicate that knockdownof reggie-1 affects ␤1-integrin recyclingwithoutaffecting␤1-integrinmembraneexpressionand degradation.

Reggie-1controlsthenumberofFAsviaRab11a

Reggie-1 interacts withRab11a and is involved in recycling ofE-cadherinand thetransferrin receptor(Solisetal.,2013)so thatreggiesmightaswellplayaroleinRab11-dependentintegrin recycling.Indeed␣5-and␤1-integrinsco-localizewithRab11aand Rab11FiP1(alsoknownasRabcouplingprotein,RCP)(Fig.7E–G).

Followingexposureoflivingcellstotheactivating␤1-integrinAb for1hat37^◦C,labelingwasconcentratedintherecyclingcom- partmenttogetherwithRab11FIP1andEGFP-Rab11a,respectively (Fig.7E,F).When␣5-integrin-EGFPwasco-transfectedtogether withmCherry-Rab11a,theyco-localizedinvesicles closetothe substrate-near region of the cell, as seen by TIRF microscopy (Fig.7G).Accordingtopreviousresults,inwhichreggiewasshown tointeractwithRab11a andtoregulate itsactivity(Solisetal., 2013),overexpressedEGFP-Rab11aCAinparentalHeLacellswas locatedprimarilyatvesiclesintheperinuclearregionandinthe cellperiphery(Fig.7H).ManyofthevesicleswithEGFP-Rab11aCA werealsolabeledbyareggie-1Ab(Fig.7H).

Todeterminewhethertheoverexpression ofRab11awt,the Rab11a CA and DN forms might affect the number of FAs in shLucandshR1cells,wetransfectedthecellswiththeconstructs andstainedforpaxillin.mRFP-C1 servedasa controlvector.As expected,transfectionwiththecontrolvectorresultedinthenor- malnumberofFAsinshLuccellsandintheabnormalpatternand highernumber,describedaboveinreggie-deficientcells(Fig.8A, I).However,transfectionwiththeRab11awtconstructaswellas theRab11aCAmutantsignificantlyreducedthenumberofFAsin reggie-1-deficientcells buthadnovisibleeffectonthenumber ofFAsincontrolcells(Fig.8B,C,I).Arescueofthephenotypein shR1cellscouldalsobeachievedwithashRNAinsensitivereggie-1

construct,showingthespecificityoftheeffectofreggie-1knock- down(Fig.8H,I).Bycontrast,transfectionofshLuccellswiththe Rab11aDNmutantincreasedthenumberofFAssignificantlyin shLuccells,butithadnoeffectontheFAsinshR1cells(Fig.8D, I).IntegrinsareknowntoberecycledthroughRab11-,Rab8-and Rab4-dependentpathways(Bridgewateretal.,2012;Margadant etal.,2011;Sharmaetal.,2009).TofindanexplanationwhyFA turnoverandrecyclingofintegrinsinshR1cellsareincreased(as showninFig.7)eventhoughRab11aactivityisimpaired,weexam- inedifRab4aorRab8a-dependentrecyclingmightcompensatethe reducedactivationofRab11a.shLucandshR1cellsweretransfected witheitherRab4awt,Rab4aDNorRab8awtconstructsandthe numberofFAswasquantified(Fig.8E–I).Indeed,overexpression ofRab4awtledtoanincreaseinnumberofFAsinshLuccellsmim- ickingthephenotypeobservedinshR1cells,whereasit didnot furtherincreaseFAnumberinshR1cells(Fig.8E,I).Ontheother handRab8awtoverexpressiondidnotshowanyeffect(Fig.8G,I).

Interestingly,theRab4aDNconstructrescuedthenumberofFAsin shR1cellsandslightlyelevatedthenumberofFAsincontrolshLuc cells(Fig.8F,I).

Thus,Rab11awtandCAconstructsrescuedthereggie-1knock- downphenotype,whereastheRab11aDNmutantcausedshLuc cellstoacquirethephenotypeofreggie-1knockdowncells.Thisis inlinewithourpreviousdata,wherethesamemutantsrescued and,respectivelymimickedtheeffectofreggie-1knockdownon E-cadherinrecycling(Solisetal.,2013).Thisisconsistentwiththe interactionofreggie-1andRab11aandemphasizestheirshared roleinrecyclingof␣5␤1-integrin.

Discussion

Thereggieproteinsresideattheplasmamembraneandatvesic- ularcarriersinvolvedinmembraneproteintrafficking(Stuermer, 2010)andfunctioninRab11a-dependentcargorecyclinganddeliv- erytospecificsites.Thisfunctionpromotes,accordingtoourearlier work, proper E-cadherin recycling, cell adhesion and adherens junctionturnover(Solisetal.,2012,2013)whilereggie-1down- regulationimpairscelladhesion.Ourpresentresultssubstantiate theroleofreggiesinmembraneproteinrecyclinganddemonstrate thatreggiesfunctioninRab11a-controlledandFA-directedtraffic- kingof␣5-and␤1-integrin.Concretely,usingpaxillintovisualize FAs,weshowinreggie-deficientcellsthatFAsincreasedinnumber, losttheirpreferentialaccumulationintheperipheryandacquired ascattereddistribution.

Moreover, reggie downregulation enhanced the dynamic turnover of FAs and increased membrane dynamics coincident withanelevation ofFAKand Rac1 activity(Rac1-GTP). In con- trasttoviewsimplyingreggiesinlipidraft-mediatedendocytosis (Ait-Slimaneetal.,2009;Payneetal.,2007),reggie-deficientcells showed nodifference in Ab-labeled ␤1-integrin internalization in a pulsechaseassay,but lostthelabelingfaster than reggie- expressingshLuccontrolcells. Notably,thespecificdefectsthat resulted from loss of reggie-1,such as increase in FA number anda disperseddistribution,wererescuedbyincreasedRab11a

Fig.8.TheFAphenotypeafterreggie-1downregulationiscross-rescuedbyRab11a.DifferentRab-constructswereoverexpressed(showninred)inHeLashLucandshR1 cells(A–G).FAsaremarkedbypaxillin(white).TransfectionwithanmRFP-C1(control)constructdidnotaffecttheFAsineithershLucorshR1cells(A).shR1cellshadstill moreandscatteredFAs.TransfectionwithmCherry-Rab11a(Rab11wt)hadnoeffectonthenumberofFAsinshLuccells(left)butreducedthenumberofFAsinshR1cells (right)andchangedtheirdistribution(B).AnmRFP-Rab11a-CA(Rab11CA)constructdidnotaffectFAsinshLuccells,butrestoredinshR1cellsthenumberofFAstocontrol levels(C).mRFP-Rab11a-DNmutant(Rab11DN)increasedthenumberofFAsinshLuccellsbutnotinshR1cells(D).AnEGFP-Rab4aconstruct(Rab4wt,showninred)also increasedthenumberofFAsincontrolcells(E).AnECFP-Rab4a-DNconstruct(Rab4DN,showninred)slightlyelevatedthenumberofFAsinshLuccellsandreducedthe numberofFAsinshR1cellsbacktocontrollevel(F).OverexpressionofmRFP-Rab8awt(Rab8wt)didnothaveaneffectonthenumberofFAsinshLucorshR1cells(G).The reggie-1-EGFPrescueconstruct(R1-rescue,showninred)restoredthenumberofFAsinshR1cellstocontrolvalues(H).QuantificationofthenumberofFAsper␮m²(I)of shLucandshR1cellstransfectedasdescribedaboverevealedthattheRab11wt,Rab11CA,Rab4DNandR1-RescueconstructswereabletorescuethephenotypeinshR1 cells.Rab11DNandRab4wtmimickedtheeffectofreggie-1knockdownonFAsinshLuccells(I)(n=3,***p<0.001,**p<0.01,*p<0.05,one-wayANOVA,errorbars,SEM).