(SORBS2) is a component of the acto-myosin ring at the apical junctional complex in e

24 Pages·2017·24.52 MB·English

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