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RESEARCHARTICLE Sorbin and SH3 domain-containing protein 2 (SORBS2) is a component of the acto-myosin ring at the apical junctional complex in epithelial cells KarinFredriksson-Lidman*,ChristinaM.VanItallie,AmberJ.Tietgens,James M.Anderson LaboratoryofTightJunctionStructureandFunction,NationalHeartLungandBloodInstitute,National a1111111111 InstitutesofHealth,Bethesda,Maryland,UnitedStatesofAmerica a1111111111 a1111111111 *[email protected] a1111111111 a1111111111 Abstract SORBS2isascaffoldingproteinassociatedwithAbl/Argnon-receptortyrosinekinasepath- waysandisknowntointeractwithactinandseveralothercytoskeletalproteinsinvarious OPENACCESS celltypes.PreviousBioIDproximitylabelingoftightandadherensjunctionproteinssug- Citation:Fredriksson-LidmanK,VanItallieCM, gestedthatSORBS2isacomponentoftheapicaljunctioncomplexofepithelialcells.We TietgensAJ,AndersonJM(2017)SorbinandSH3 askedwhetherSORBS2playsapreviouslyunappreciatedroleincontrollingperijunctional domain-containingprotein2(SORBS2)isa componentoftheacto-myosinringattheapical actinandtightjunctionbarrierfunction.Usingsuperresolutionimagingweconfirmedthat junctionalcomplexinepithelialcells.PLoSONE12 SORBS2islocalizedattheapicaljunctioncomplexbutfartherfromthemembranethanZO- (9):e0185448.https://doi.org/10.1371/journal. 1andlocatedpartiallyoverlappingboththetight-andadherensjunctionswithaperiodic pone.0185448 concentrationthatalternateswithmyosinIIBinpolarizedepithelialcells.Overexpressionof Editor:MichaelKoval,EmoryUniversitySchoolof GFP-SORBS2recruitedalpha-actinin,vinculinandN-WASP,andpossiblyCIP4tocellular Medicine,UNITEDSTATES junctions.However,CRISPR-Cas9knock-outofSORBS2didnotalterthelocalization-or Received:June29,2017 immunofluorescentstainingintensityoftheseorseveralotherjunctional-andcytoskeletal Accepted:September12,2017 proteins.SORBS2knock-outalsodidnotaffectthebarrierfunctionasmeasuredbyTER Published:September29,2017 anddextranflux;nordiditchangeactin-dependentjunctionre-assemblyasmeasuredby Ca2+-switchandLatrunculin-Bwash-outassays.ThekineticsofHGF-inducedcellscatter- Copyright:Thisisanopenaccessarticle,freeofall copyright,andmaybefreelyreproduced, ingandwoundhealing,anddextranfluxincreaseinducedbyPDGFalsowereunaffectedby distributed,transmitted,modified,builtupon,or SORBS2knock-out.SORBS2concentrateswithapicaljunctionalactinthataccumulatesin otherwiseusedbyanyoneforanylawfulpurpose. responsetoknock-downofZO-1andZO-2.InspiteofourfindingthatSORBS2isclearlya TheworkismadeavailableundertheCreative componentoftheapicaljunctioncomplex,itdoesnotappeartoberequiredforeithernormal CommonsCC0publicdomaindedication. tight-oradherensjunctionassembly,structureorfunctionorforgrowthfactor-mediated DataAvailabilityStatement:Allrelevantdataare changesintightjunctiondynamics. withinthepaperanditsSupportingInformation files. Funding:Thisresearchwassupportedbythe DivisionofIntramuralResearch,NationalInstitutes ofHealth(US),ZIAHL006207,Dr.JamesM. Introduction Anderson. Tightjunctions(TJ)formacontinuousapicolateralparacellularbarrierbetweenepithelialcells Competinginterests:Theauthorshavedeclared thatnocompetinginterestsexist. thatenablesselectiveandregulatedmovementofsolutesbetweentheapicalandbasolateral PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 1/24 SORBS2isacomponentoftheapicaljunctionalcomplex compartments.TodatenumerousproteinshavebeenidentifiedattheTJ[1],includingthe transmembraneclaudinfamilyofproteins[2–4],occludin[5]andthescaffoldingproteins ZO-1,-2and-3[6],whichconnectthetransmembraneproteinstotheactincytoskeleton. DespitethemanyidentifiedTJproteinstherearemostlikelymanymoreproteinsthatplay importantrolesforTJregulationandfunction.InanefforttofindnovelTJinteractingpro- teins,wepreviouslyutilizedaproximity-dependentbiotinylationmethod(BioID)[7],fol- lowedbyproteomicanalysisofbiotinylatedproteinsneighboringZO-1andoccludin[8,9]. Amongthemostabundantproteinsrecoveredwhenanengineeredbiotinligasewasfusedto theN-terminusofZO-1wastheactin-bindingandsignalingproteinSorbinandSH3domain- containingprotein2(SORBS2,alsocalledArgBP2);itwasnotdetectedinasimilaranalysisof proteinsproximaltotheC-terminusofZO-1[9](S1Table).SORBS2wasalsoidentifiedas proximaltoboththeN-andC-terminusofoccludin[8](S1Table),althoughwithrelatively lowerabundance.TheselectiveidentificationofSORBS2asclosetotheN-butnottheC-ter- minusofZO-1raisedthepossibilitythatSORBS2mighthaveafunctionalspatiallyspecific roleinregulatingpropertiesoftheTJ. SORBS2wasoriginallycharacterizedasaninteractorofArgkinase[10]andwassubse- quentlyidentifiedasamemberofathree-proteinfamily,includingCAP((c-Cbl-associated protein)/ponsin/SORBS1)andvinexin(SORBS3)[11].AllthreeshareanN-terminalsorbin homologydomain(SoHo),whichhasbeenreportedtointeractwithflotillin[12–14],and threeC-terminalSH3domains[11,15].SORBS1,SORBS2andSORBS3localizeatfocaladhe- sionsandSORBS1andSORBS2alsolocalizeatadherensjunctionbasedcell-cellcontacts [15–20].Inaddition,SORBS2isalsoassociatedwithactinstressfibers[10,19,21].Morerele- vantly,SORBS2waspreviouslyco-localizedwithZO-1attightjunctionsinmurineepithelial NMuMGcells;however,arolefortightjunctionSORBS2wasnotdefined[19].SORBS2has beenreportedtonegativelyregulateAblkinase[22],recruitPyk2/Cblcomplextothelipidraft compartments[14]andbindtoAkt[23].Inaddition,evidencesuggeststhatSORBS2isalso involvedincelladhesion,actin/cytoskeletalorganization[10,24,25]andmigrationoftumor cells[17,25].TogethertheseobservationsraisetheinterestingpossibilitythatSORBS2might havearoleinasignalingpathwayregulatingperijunctionalactin,possiblythroughatyrosine kinasepathwayinepithelialcells. Inthepresentstudy,wesoughttobetterdescribethelocalizationofSORBS2relativeto establishedepithelialtightandadherensjunctions(AJ)proteinsusingsuperresolutionmicros- copyandtoexploreapotentialroleattheTJbyfunctionalanalysis,bothatbaseline,andafter growthfactortreatment.Wecomparedtwocontrolepithelialcelllinesderivedfromtwodif- ferenttissues,namelyhumanintestineandcaninekidney,withthesamecelllinesinwhich SORBS2hadbeendeletedbyCRISPR-Cas9.Weusedawiderangeofstandardassaystoevalu- atebarrierpropertiesandperijunctionalactinregulation.WecouldshowthatSORBS2isa componentoftheperijunctionalactomyosinringanditisbothrecruitedtoactin,andcan concentrateactinwhenitisover-expressed,consistentwithitsbeinganactinbindingprotein. ItslocalizationclearlyoverlapsboththeTJandAJ,butitdoesnotappeartobeessentialeither fornormaltight-oradherensjunctionassembly,structureorfunctionorforgrowthfactor- mediatedchangesintightjunctiondynamics. Materialsandmethods Cellculture,CRISPR-Cas9knock-outandtransfections Madin-DarbyCanineKidney(MDCKII)epithelialcells(WTtet-off(TO)MDCKIIcells,BD Biosciences,WTMDCKIIcells(CCL34),AmericanTypeCultureCollection,Manassas,VA andZO-1andZO-2doubleknock-downTOMDCKIIcells[26])wereculturedunder PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 2/24 SORBS2isacomponentoftheapicaljunctionalcomplex standardcultureconditionsinDMEM(Mediatech,Manassas,VA;4.5g/lglucose),1xpenicil- lin/streptomycin(Mediatech)and10%tet-testedfetalcalfserum(FCS;AtlantaBiological, Norcross,GA).ZO-1andZO-2doubleknock-downcellswerealsoculturedwiththeaddition ofdoxycycline(50μg/ml,Sigma-Aldrich,St.Louis,MO)forexperimentstoturnofftheZO-1 rescueconstruct.HEK293Tet-Off1Advancedcellline(Clontech,MountainView,CA)was culturesasdescribedabove.HumanintestinalepithelialcelllineSKco15,akindgiftfromDr. AsmaNusrat(UniversityofMichigan),wasculturedinthesamemediaasMDCKIIcellsbut withtheadditionof10mM4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid(HEPES, Mediatech),pH7.4,and1×nonessentialaminoacids(NEAA;Mediatech)and10%FCSunder normalcultureconditions.HumanrecombinantHGFandPDGF-BBwasobtainedfrom PeproTechInc(RockyHill,NJ).ForallSKco15experimentscellsweregrownoncollagen coatedsurfaces(rattailcollagen,Sigma-Aldrich).BothMDCKIIandSKco15cellswerepas- sagedevery4–7days.WhenculturedonTranswellfilterinserts(Corning,Corning,NY)all cellswereplatedatadensityof0.5x105/insertandculturedtoconfluency(usuallywithin48h) beforetransepithelialresistance(TER)wasmeasureddailyforsevendays.Whencellswere usedforfurtherexperimentssuchasdextranflux,immunofluorescentstainingorimmuno- blotstheywereharvestedatsevendaysafterconfluency(9daysafterplating). StableSORBS2knock-out(KO)celllineswereobtainedbytransfectingcellswithpSpCas9 (BB)-2A-Puro(PX459)V2.0(62988;Addgene,Cambridge,MA;[27])containingSORBS2 sgRNA(S2Table)usingLipofectamine2000.Afterselectionfor48hin2μg/mlpuromycin (LifeTechnologies,Carlsbad,CA),dilutioncloninginto96-wellplateswasperformedto obtainsinglecellclones.ClonallineswereexpandedandtestedforSORBS2expression2–3 weeks(MDCKII)or5–6weeks(SKco15)aftertransfectionwithSORBS2antibodybyimmu- noblot.GenomicDNAwascollected(DNeasy;Qiagen,Hilden,Germany)fromcelllinesnega- tiveforSORBS2byimmunoblotandPCRamplification(Phusion,HFkit;NewEngland Biolabs,Ipswich,MA)oftheregionofinterestwasperformedusingspecificSORBS2primers (S2Table).PCRproductwasloadedtoa1%agarosegel(SeaKem1GTG1agarose;Lonza, Basel,Switzerland)andafterelectrophoresistheDNAbandswerevisualizedwithEthidium bromideandUVlight(Benchtop2UV™Transilluminator;UVP,Upland,CA),excisedandgel purified(QIAquick1,Qiagen)beforesendingtheDNAforsequencingtoACGTInc(Ger- mantown,MD).Sequencingprimersusedwerethesameasfortheamplification(S2Table). FulllengthSORBS2(pcDNA3-myc-ArgBP2α)wasagenerousgiftfromDr.Koh-Ichi NagataandwasclonedbyIn-Fusioncloningwithspecificprimers(S2Table)intopEGFP-C1 vector(Clontech#6084–1;PT3028-5).EGFP-SORBS2wastransientlyexpressedinMDCKII cellsaftertransfectionwithLipofectamine2000(LifeTechnologies).Cellswerefixedand stainedforimmunofluorescence72hoursposttransfection. FulllengthSORBS1(pcMV6-Entry-myc-DDK-SORBS1;RC224656(NM_006434))was obtainedfromOriGene(Rockville,MD).myc-DDK-SORBS1wastransfectedusingLipofecta- mine2000(LifeTechnologies)andexpressedtransientlyinHEK293Tet-Off1Advancedcells. CelllysateswereusedtotestspecificityoftheSORBS1antibody. SORBS1,2and3qRT-PCRandSORBS2PCRforisoformidentification TotalRNAwascollectedusingRNeasy(Qiagen)fromWTandSORBS2KOMDCKIIcells grownonculturedishesuntilconfluency.ReversetranscriptionwasperformedusingSuper- Script1VILO™kit(ThermoFisher,Waltham,MA).qRT-PCRwasperformedaspreviously described[28]usingprimersforcanineSORBS1,SORBS2,SORBS3andZO-1(SORBS1and SORBS3primers:S2Table,ZO-1primers:previouslypublished[28]). PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 3/24 SORBS2isacomponentoftheapicaljunctionalcomplex ForSORBS2isoformidentification,mRNAwascollectedfromWTSKco15andMDCKII cellsandreversetranscriptionwasperformedasdescribedabove.Primersweredesignedthat couldidentifyall,orspecificisoforms,ofSORBS2incanine-orhumancells(SORBS2primers, S2Table)andDNAwasamplified(Phusion,HFkit,NewEnglandBiolabs).ThePCRproducts wereloadedtoa1%agarosegel(SeaKem1GTG1agarose,Lonza)andseparatedbyelectro- phoresis.Ethidiumbromide-stainedDNAbandswerevisualizedbyUVimaging(MyECL imager,ThermoFisher). Antibodies AcustomRabbitanti-humanSORBS2C-terminal(7515)antibodywasmanufacturedby Covance(Madison,WI)againsttheimmunogenpeptidesequenceCSNKPQRPVFTHENIQ. Verificationoftheantibodywasperformedinamulti-stepeffort.Initialverificationwasper- formedbyimmunoblottinglysatesfromWTSKco15cellsandMDCKIIcells,comparing signalsforantibodypre-absorbedwiththeimmunogentothosefromuntreatedantibody.Sec- ondly,antibodyspecificitywastestedbyimmunoblotwithSORBS2KOcelllysatesversusWT lysatesandwithlysatesfromHEK293cellstransfectedwithhumanGFP-SORBS2.Mouse anti-ZO-1,ratanti-Ecadherin,mouseanti–α-actinin,mouse-anti-CIP4,mouseanti–γ-tubu- lin,rabbitanti–myosin2B,rabbitanti-GFP,rabbitanti-N-WASPandmouseanti-occludin weredescribedpreviously[28].Ratanti-ZO-1(R40.76)[29],mouseanti-myosin2B(3H2, ab684;Abcam,Cambridge,MA),mouseanti-myosin2A(ab55456;Abcam),rabbitanti-myo- sin2A(909801,BioLegend,SanDiego,CA),rabbitanti-cingulin(C532wasagenerousgift fromDr.SandraCiti,UniversityofGeneva,Geneva,Switzerland),mouseanti-vinculin(h- VIN-1,V9131;Sigma-Aldrich),mouseanti-afadin(AF6;BDTransductionLaboratories, FranklinLakes,NJ),mouseanti-p120catenin(BDTransductionLaboratories),rabbitanti- SORBS1(ProteinTech,Chicago,IL).Species-specificsecondaryantibodiesforimmunofluo- rescence(Cy2,Cy3,andCy5conjugated)aswellasF(ab’) -fragmentsusedforsuperresolution 2 microscopy(AlexaFluor1647-and594-conjugated)andimmunoblots(IR-labeled680/700 and790/800antibodies)werefromJacksonImmunoResearch(WestGrove,PA).Rhoda- mine–phalloidinwasfromLifeTechnologies. Immunofluorescencemicroscopy MDCKIIcellswereculturedonuncoatedandSKco15cellsoncollagen-coatedTranswellfilters (Corning),followedbyfixationineither1%paraformaldehyde(PFA)or100%ice-coldethanol asdescribedpreviously[28].FiltersweremountedwithMowiol(EMD,Billerica,MA)contain- ing1%n-propylgallate(Sigma-Aldrich).Frozentissuesectionsfrommouseliverwereobtained fromtheNationalHeart,Lung,andBloodInstitutePathologyCore;animalprocedureswere carriedoutinaccordancewiththeguidelinesoftheNationalHeart,Lung,andBloodInstitute AnimalCareandUseCommittee.Tissuesectionswerefixedin1%PFAandimmunofluores- cenceprocedurescarriedoutasdescribedabove.FixedsampleswereimagedonaZeiss(Thorn- wood,NY)710confocalmicroscope,using63×/NA1.4oilobjectiveor20xair,with488-,561-, and633-nmlaserlines,oraZeissLSM880AiryscaninSuperresolutionmodewitha63x1.4 NAobjective.RawdatawereprocessedusingAiryscanprocessingwith“autostrength”(Mean strength+/-S.D.=5.5+/-1.3)inZenBlacksoftwareversion2.3. SuperresolutionimagingwasperformedusingStimulatedEmissionDepletion(STED) microscopy.STEDimageswereobtainedusingacommercialLeicaSP8STED3Xsystem (LeicaMicrosystems,Mannheim,Germany),equippedwithawhitelightexcitationlaserand3 depletionlasers:592,660,and775nm.A100×/1.4NAoilimmersionobjectivelens(HCPL APOCS2,LeicaMicrosystems)wasusedforimaging.Imagestackswerecollectedas8bit, PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 4/24 SORBS2isacomponentoftheapicaljunctionalcomplex 1,024×1,024pixelimageswith25nmxandypixelsizesand0.2μmz-steps.Pinholewassetto 0.7AU,6lineaveragesandframeaccumulation=2(phalloidin)and4lineaveragesandframe accumulation=1(everythingelse).Rhodaminephalloidinwasimagedat568nm(emission bandwidth578–737nm,775STEDpulseddepletionlasersetat50%power),594-conjugatedF (ab’)fragmentswasimagedat594nm(emissionbandwidth604–737nm,775STEDpulsed depletionlasersetat20%power)and647-conjugatedF(ab’)fragmentsat647nm(emission bandwidth658–737nm,775STEDpulseddepletionlasersetat8%power);timegatingwasset toarangeof0.7–6.5ns(nanoseconds).STEDimagestacksweredeconvolvedusingHuygens software(ScientificVolumeImagingB.V.,Hilversum,TheNetherlands).ImageJFIJI(NIH, Bethesda,MD)wasusedtomakemaximumintensityZ-projectionsofthetotal2μmstacks imagedandtoperformlinescansoffluorescenceintensitytoconfirmthevisualobservations. Twentylinescans/fullimagefieldwereperformed,eachcenteredovercellcontacts.Lineswere 1000nminlengthandplacedwhereSORBS2couldbefoundonbothsidesofthecell-cell junction,regardlessofthefluorescenceintensityoneachside. Immunoblots ImmunoblotswereperformedwithMDCKIIorSKco15celllysates.Cellsweregrowneither onTranswellfilters(Corning)orin24-wellplates(Corning)forsevendaysafterconfluency. Onehundredμl4xSDS-samplebufferwasaddeddirectlytoeachwell,orfilterswereexcised andplaceddirectlyin100μl4xSDS-samplebuffer.Allsampleswerebrieflysonicatedbefore anequalvolumewaseitherfrozenat-20˚CuntiluseorloadedonaSDS-PAGEgel(4–12% Bis-Tris,1mmthickness;NuPage™,Invitrogen),transferredtonitrocellulosemembraneand stainedwithprimaryandsecondaryantibodiesdescribedabove.Proteinbandswerevisualized withLi-CorOdyssey(Li-CorBiosciences,Lincoln,NE). Tightjunctionbarrierfunctionandreassemblyassays;Transepithelial ElectricalResistance,dextranfluxandcalcium-switch TransepithelialElectricalResistance(TER)anddextranfluxwasperformedaspreviously described[30],howeverfluorescent-dextranconcentrationsinthewellundertheTranswell insertwerequantifiedusingSpectraMaxM3(MolecularDevices,Sunnyvale,CA)andexperi- mentalvalueswasdeterminedbyextrapolationfromthestandardcurveofknownfluorescent- dextranconcentrationsusinglinearregression(GraphPadPrism,SanDiego,CA).Calcium- switchwasperformedasdescribed[28].Statisticalanalysis(ttestsorANOVAasapplicable) wasperformedusingGraphPadPrismwithcorrectionsformultiplecomparisonsusingthe Sidak–Bonferronimethod. LatrunculinBwashout SKco15cellswereplatedatconfluentdensityoncollagen-coatedTranswellfilterinsertsand growntoconfluencyovernight.LatrunculinB(LatB;10μM),orDMSOascontrol,was addedtoboththetopinsertandthebottomwellandafter2hoursofincubationcellswere washedtwiceincompleteculturemediabeforeaddingfreshculturemediafortheindicated recoverytimes.Cellsforthe0minutesrecoverywerewashedimmediatelyinice-coldPBS andfixedin1%PFAinCSKbufferbeforeimmunostainingwithrhodaminephalloidinand mouseanti-ZO-1asdescribedabove.Cellswereimagedwithconfocalmicroscopyat20X magnification. PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 5/24 SORBS2isacomponentoftheapicaljunctionalcomplex Wound-healingassay Twotechniqueswereusedtomeasurewound-healing.First,weutilizedtheIncuCyte1Zoom (EssenBioscience,AnnArbor,MI)tomeasurewoundhealingina96-wellformat.MDCKII cells(WTandSORBS2KO)wereplatedatconfluentdensity(35,000cells/well)ona96-well ImageLockplate(suppliedbyEssenBioscience)thedaybeforetheexperiment.Thedayofthe experiment,awoundmakerdevice(EssenBioscience)wasusedtocreateawoundineachwell ofthe96-wellplate.Cellswerewashedtwiceincompletemediaandeithercompletemediaor completemediacontaining100ng/μlHGFwasadded.Woundhealingwastrackedbyimaging eachwellevery2hforupto48hwitha10xobjective.EssenBiosciencesoftwarewasusedto calculatetherelativewoundhealingineachwell.Inthesecondmethod,weculturedSKco15 cells(WTandtwodifferentSORBS2KOclones)on60mm-dishes.Awoundwascreatedman- uallyandcellswerephotographedatvarioustimepointsandwound-healingwasquantifiedin ImageJasdescribedpreviously[31]. Resultsanddiscussion DefiningSORBS2isoformsexpressedinhumanandcanineepithelial cells WewereunabletoidentifyareliablecommercialantibodyrecognizingSORBS2inepithelial cells,sowegeneratedacustomrabbitanti-SORBS2antibodytoaconservedregionbetween thefirstandsecondSH3domains.ThisdomainispresentinallSORBS2spliceformsandthus anantibodydirectedagainstthisregionwouldbeexpectedtoidentifyallhumanspliceforms, whichrangeinsizefrom71–134kDa(UniProt,[32]).Theresultingantibodyidentifiedthree distinctbands,e.g.atleastthreepotentialisoforms,ofapproximately70,75and80kDa,in immunoblottedlysatesfromhumanintestinalepithelialSKco15cells(Fig1A)andonedistinct bandincaninekidneyepithelialMDCKIIcellsaround75–80kDa(Fig1B).Afterutilizingthe CRISPR-Cas9systemtoknockoutSORBS2inSKco15andMDCKcells,allofthesebands wereabsent,consistentwiththeirbeingauthenticSORBS2isoforms(Fig1A(SKco15);Fig1B (MDCKII)).Asfurtherevidenceofantibodyvalidationanimmunofluorescencesignalwas observedatcellcontactsandactinstressfibersinWTMDCKIIcells,buttherewasnodetect- ablesignalinSORBS2KOcellsusingthesameconfocalmicroscopyfluorescentsensitivityset- tings(S1Fig,panelA). BecauseourSORBS2antibodyrecognizedseveraldifferentsizebandsinimmunoblot analysisandbothNCBIandUniProt[32]databasesreportedtheexistencemultiplesplice formsofSORBS2,wenextattemptedtoidentifytherelevantspliceformsexpressedin MDCKIIandSKco15cells.TwelvehumanSORBS2isoformsarereportedinUniProt[32] andaftereliminatingisoforms1,6,7and11asbeingtoolargeortoosmalltorepresentthe observedbandsweidentifiedbyimmunoblotting,wedesignedsixPCRprimerspairsthat coulddistinguishamongmRNAtranscriptsencodingtheremainingisoformsbasedonthe sizeofthePCRproducts(primersandPCRproductsizesaredescribedinS2Table).We confirmedthepresenceofmRNAforisoforms3and9inSKco15cellsbasedonthesizeof PCRproductsobtainedfromprimerpair5and6(uniquetoisoform3)andprimerpair2 (uniquetoisoform9)andobtainedweaksignalsforpotentialpresenceofmRNAencoding isoforms4and12withprimerpair2;(Fig1Cand1E).Isoform12hasthesameexpected sizeasisoform9withprimerpair4,whichmeansitcannotbeexcluded.Withtheprimers used,wedidnotobservePCRproductsofthesizeexpectedfromisoforms2,5,8and10in SKco15cells.IncontrasttothemanyhumanisoformspredictedforhumanSORBS2,Uni- ProtdescribestwocanineSORBS2isoformspredictedtobe134kDaand73kDa,onlythe PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 6/24 SORBS2isacomponentoftheapicaljunctionalcomplex Fig1.SORBS2isoformsexpressedinculturedhumanandcanineepithelialcells.(A)ImmunoblottedlysatesfromSKco15cellsrevealthreedistinct SORBS2proteinbandswithsizesbetweenapproximately65–85kDa.AfterCRISPRKOSORBS2proteinisnolongerdetectablebyimmunoblot.(B)One distinctSORBS2band,approximately75kDainsize,isvisibleinimmunoblottedlysatesfromMDCKIIcellsandafterCRISPRKOSORBS2proteinisno longerdetectable.(C)DNAgelfromPCRproductsgeneratedbyusingsixprimerpairstoidentifyvariousisoformsofhumanSORBS2.Primerpair1is showinga216bpproduct,confirmingisoform9.Primerpair2isshowingadistinctbandat185bpconsistentwithisoform9andaweakbandat401bp (couldbeisoforms3,4,5,12).Isoforms2and8canbeexcludedduetoalackofbandsat326and242bprespectively.Isoform2canagainbeexcluded duetothelackofa176bpproductwithprimerpair3.Primerpair4showsa457bp,confirmingpresenceofisoforms9and12.Primerpairs5and6both showthepresenceofisoform3at303bpand540bprespectively.(D)PCRproductsgeneratedbyusingspecificprimerstoidentifyvariousisoformsof canineSORBS2.Primerpair1shouldgivea900bpproductforisoformX23,whichisconfirmed.IsoformX25wouldhavebeen878bp,isoformX261109 bpandisoformX271177bprespectively,whichmeanstheseisoformscouldbeexcludedbysize.IsoformX25hasaverysimilarexpectedsizetoisoform X23,butwecouldexcludeitbasedonthenegativeresultobtainedwithprimerpair3.Primerpair2shouldgivea900bpproductforisoformsX23andX26 whichfurtherconfirmsthepresenceofisoformX23.Primerpair3shouldidentifyisoformsX25andX26at900bp,butthereisnoevidenceofadistinct productthatsize.WealreadyexcludedisoformsX25andX27andthereforethe850bpproductisthusisoformX23.Primerpairs5and6shouldrecognize allpotentialSORBS2isoforms(NCBImRNAisoformsX23,X25,X26,X27)at850bpand500bprespectively.(E)SchematicfigureofSORBS2isoforms identifiedinhumanSKco15cellsandcanineMDCKIIcells.ThesearetheonlyspliceformscompatiblewithPCRresultsandthebandsizesonthe immunoblots. https://doi.org/10.1371/journal.pone.0185448.g001 smallerofwhichmightbeexpressedinMDCKIIcellsbasedonthesizeofthebanddetected byimmunoblotting;severalmoreisoformsaredescribedintheNCBIdatabase.Bygenerat- ingPCRprimerstoidentifycanineSORBS2isoforms,intheobservedsizerangebetween 70–85kDareportedontheNCBIwebsiteandUniProt,weconfirmedexpressionofmRNA forisoformX23(NCBIreferencesequenceXM_014120127.1),whichcorrespondstopro- teinisoformX21(NCBIreferencesequenceXP_013975602.1)inMDCKIIcells(Fig1D and1E).Together,ourresultsaremostconsistentwithexpressionofspliceforms3,4,9 and12inSKco15cells.MDCKcellsexpressthecaninemRNAisoformX23,whichismost closelyhomologoustohumanisoforms9and12.Wenotethatallspliceformsexpressedin SKco15andMDCKIIepithelialcellscontainthecanonicalSoHodomainandthreeSH3 PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 7/24 SORBS2isacomponentoftheapicaljunctionalcomplex domains;whileotherspliceformsfoundinthedatabasesarelackingvariouscombinations ofdomains. SORBS2isco-localizedwithapicaljunctionalactin,andoverlapswith bothtight-andadherensjunctionproteinsinpolarizedepithelialcells TheBioIDresultssuggestedthatSORBS2wasproximaltothetightjunctionproteinZO-1[7], aswasconsistentwithlocalizationpreviouslyreportedinmurineepithelialNMuMGcells [19].WefirstverifiedtheinvivorelevanceoftheBioIDfindingbyconfocalmicroscopyof SORBS2inmurineliverandfoundthatSORBS2andZO-1partiallycolocalizeatTJsinbile canaliculiasshowninFig2A.ThispartialcolocalizationofSORBS2andZO-1wasalso observedinMDCKIIcellspolarizedonTranswellfilters(Fig2B)andSKco15cells.Z-stacks revealedoverlapbetweenZO-1andSORBS2,butZO-1extendsapicallyabove,andSORBS2 basallybelowtheregionofoverlap(Z-stack,Fig2B).SORBS2isalsolocalizedalongbasalactin stressfibersinMDCKIIcells(BasallocalizationinZ-axis:Fig2B,basallocalizationinX/Y axis:S1Fig,panelB). ToaskifSORBS2wasalsodistributedatadherensjunctions(AJ)weusedE-cadherin(E- cad)asanAJmarker[33].EventhoughSORBS2wasnotfoundinBioIDE-cadproteomic analysis[31,34](S1Table),weobservedthatSORBS2indeedco-localizeswiththemostapical E-cadsignal(Fig2C).Ourimmunofluorescentconfocalmicroscopydatathusindicatesthat SORBS2overlapswiththebasalregionoftheTJandtheapicalregionoftheAJinpolarized MDCKIIandSKco15cells,similartowhathasbeendescribedforthedistributionofafadin [35]. TofurtherdefinethespatialpositionofSORBS2inrelationtoTJ,AJandcytoskeletalpro- teins,weco-immunostainedpolarizedMDCKIIcellsforactin,occludin,non-musclemyosin IIB,E-cadherinandafadinandperformedSTEDsuperresolutionmicroscopy.Weobserved thatSORBS2waslocalizedmoredistalfromtheplasmamembranethanZO-1andoccludin, andunlikethoseproteinsithasanirregulardiscontinuouspattern(Fig3Aand3B).Inorder toapproximatethedistanceofSORBS2fromthemembranerelativetotheotherTJproteins, wemeasuredrelativefluorescenceintensityacrossthecell-celljunctionsbylinescanningat90 degreestotheplaneofcellcontacts.TheZO-1inopposingcellsissufficientlyclosethatitcan- notbevisuallyresolvedbysuperresolutionmethods;however,twopeaksofSORBS2signal areobservedapproximately250nmapart,eachatabout125nminsideofthecell-cellcontact (Fig3,rightpanels).SORBS2alsoappearstobeabsent,orlessabundant,attricellularjunc- tions(Fig3B,3Dand3E).SORBS2wasco-localizedwiththeveryapicalactinatcell-cellcon- tacts(Fig3C),howeveractinisalsodistributedinsmallpunctaovertheapicalcellsurfaceand alongthelateralmembranewhereasSORBS2immunofluorescencewasconfinedtotheperi- junctionalregion(Fig3C).ItappearsthatmyosinIIB,whichalsohasadiscontinuouspattern alongtheapicalcell-cellcontacts,isconcentratedintheareaswhereSORBS2islessabundant (Fig3D),thuslinescanshadtobeperformedatdifferentlocationsforSORBS2andmyosin IIB.Likeactin,butunlikeSORBS2,myosinIIBisalsolocalizedontheapicalcellsurface(Fig 3D).SORBS2isalsodistributeddistalfromthemembranerelativetothemostapicalE-cad signal(Fig3E).Theactin-bindingproteinafadin,whichinteractswithbothZO-1andadhe- rensjunctioncomponentslikenectin,partiallycolocalizedwithSORBS2(Fig3F).Incontrast tomyosinIIB,afadinandSORBS2appeartoco-concentrateindiscontinuousjunctionalspots, althoughtheirco-localizationisnotcomplete(Fig3F)[24]. TheSTEDimmunofluorescentanalysiswasdesignedforoptimalx-yresolution,andwedid notattemptZ-axisanalysis,sothemeasurementofthex-ydistributionsarenotmeantto implythattheseproteinsarepresentinexactlythesameapical-basalplanes.Inaddition,the PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 8/24 SORBS2isacomponentoftheapicaljunctionalcomplex Fig2.SORBS2partiallycolocalizeswithZO-1inmurinebilecanaliculiandwithZO-1andE-cadherin inpolarizedMDCKIIcells.(A)ConfocalimmunofluorescenceanalysisrevealsthatSORBS2islocalizedto bilecanalicularTJsinmurineliverandispartiallycolocalizedwithZO-1inX,YandZplanes(63xoilobjective wasused,scalebar:10μm).ImagesaremaximumintensityprojectionsofZ-stacks(totaldepth4.5μm). Mergedimagesshowthattheyoverlap,butthatZO-1alsoextendsmoreapically.(B)SORBS2iscolocalized withZO-1andtheapicalportionofE-cadherininpolarizedMDCKIIcellsculturedonTranswellfiltersfornine days(63xoilobjectiveused,scalebar:10μm).ImagesaremaximumintensityprojectionsofZ-stacks(total depthforX-Yimagesca4.5μm(toavoidthestrongsignalfrombasalactinstressfibers).SORBS2isalso faintlyvisibleasgreendotsatthebottomofthecells(crosssectionsofactinstressfibers)inZprojections, especiallyinpanelB.(fullstack:10μm). https://doi.org/10.1371/journal.pone.0185448.g002 positionoftheantibodyepitopesmaynotbecenteredontheproteinandthusincompletely reporttheproteinlocalization.Withthesecaveats,SORBS2appearstohaveauniqueirregular discontinuouslocalizationatthebicellularapicaljunctioncomplex(TJandAJ).SORBS2is oftenco-localizedwithapicaljunctionalactinandpartiallywithafadin.SORBS2isdistributed overlappingwith,butalsopartiallybasaltoZO-1andoccludin,butislackingattricellular junctionsandco-distributesonlywiththemostapicalE-cadsignal.Thislocalizationisdistinct fromtheotherTJandAJmarkersstudied. SORBS2isrecruitedtothesarcomere-likeperijunctionalactomyosin structurethatisinducedbyknock-downofZO-1andZO-2 TheevidenceaboveindicatesthatSORBS2isastructuralcomponentoftheperijunctional acto-myosinring.Wepreviouslyshowedthatdoubleknock-down(dKD)ofZO-1andZO-2 inMDCKIIcellsleadstoastrikingsarcomere-likeorganizationofnon-musclemyosinII PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 9/24 SORBS2isacomponentoftheapicaljunctionalcomplex PLOSONE|https://doi.org/10.1371/journal.pone.0185448 September29,2017 10/24

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