Frazzled can act through distinct molecular pathways in epithelial cells to regulate motility, apical

20 Pages·2017·28.78 MB·English

RESEARCHARTICLE
Frazzled can act through distinct molecular
pathways in epithelial cells to regulate
motility, apical constriction, and localisation of
E-Cadherin
SofiaGolenkina,VishalChaturvedi,RobertSaint¤,MichaelJ.Murray*
SchoolofBioSciences,UniversityofMelbourne,Melbourne,Victoria,Australia
¤ Currentaddress:FlindersUniversity,Adelaide,SouthAustralia,Australia
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*[email protected]
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Abstract
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NetrinreceptorsoftheDCC/NEO/UNC-40/Frazzledfamilyhavewellestablishedrolesin
cellmigrationandaxonguidancebutcanalsoregulateepithelialfeaturessuchasadhesion,
polarityandadherensjunction(AJ)stability.Previously,wehaveshownthatoverexpression
OPENACCESS ofDrosophilaFrazzled(Fra)intheperipodialepithelium(PE)inhibitswingdisceversionand
Citation:GolenkinaS,ChaturvediV,SaintR, alsogeneratescellularprotrusionstypicalofmotilecells.Here,wetestedwhetherthe
MurrayMJ(2018)Frazzledcanactthroughdistinct
molecularpathwaysbywhichFrainhibitseversionaredistinctfromthosedrivingmotility.
molecularpathwaysinepithelialcellstoregulate
Weshowthatindiscproper(DP)epithelialcellsFra,inadditiontoinducingF-Actinrichpro-
motility,apicalconstriction,andlocalisationofE-
Cadherin.PLoSONE13(3):e0194003.https://doi. trusions,canaffectlocalizationofAJcomponentsandcolumnarcellshape.Wethenshow
org/10.1371/journal.pone.0194003 thatthesephenotypeshavedifferentrequirementsforthethreeconservedFracytoplasmic
Editor:EdwardGiniger,NationalInstitutesof P-motifsandfordownstreamgenes.TheformationofprotrusionsrequiredtheP3motifof
Health,UNITEDSTATES Fra,aswellasintegrins(mysandmew),theRacpathway(Rac1,waveand,arpc3)and
Received:December21,2017 myosinregulatorylightchain(Sqh).Incontrast,apico-basalcellshapechange,whichwas
accompaniedbyincreasedmyosinphosphorylation,wascriticallydependentupontheP1
Accepted:February22,2018
motifandwaspromotedbyRhoGef2butinhibitedbyRac1.FraalsocausedalossofAJpro-
Published:March8,2018
teins(DE-CadandArm)frombasolateralregionsofepithelialcells.Thisphenotyperequired
Copyright:©2018Golenkinaetal.Thisisanopen
all3P-motifs,andwasdependentuponthepolarityfactorpar6.par6wasnotrequiredfor
accessarticledistributedunderthetermsofthe
protrusionsorcellshapechange,butwasrequiredtoblockeversionsuggestingthatcontrol
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and ofAJcomponentsmayunderlietheabilityofFratopromoteepithelialstability.Theresults
reproductioninanymedium,providedtheoriginal implythatmultiplemolecularpathwaysactdownstreamofFrainepithelialcells.
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Funding:ThisworkwassupportedbyAustralian
Introduction
ResearchCouncilDiscoveryProjectDP120104443
toRSandNationalHealthandMedicalResearch
Inrecentyears,ithasbecomeclearthatcellsurfacereceptorsthatmediatemotilityandguid-
CouncilAustraliaProjectGrantAPP1107123to
anceofmigratingcellsandaxonscanalsoplayaroleinepithelialmorphogenesisevents[1].A
MJM.SGwassupportedbyaInternational
caseinpointaretheNetrinreceptorsoftheDeletedinColorectalCarcinoma(DCC)/Neo-
PostgraduateResearchScholarship.Thefunders
hadnoroleinstudydesign,datacollectionand genin/UNC-40/Frazzledfamily[2–7].Netrinsareahighlyconservedfamilyofsecreted
PLOSONE|https://doi.org/10.1371/journal.pone.0194003 March8,2018 1/20
Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
analysis,decisiontopublish,orpreparationofthe proteins,thatcaneitherattractorrepelgrowingaxonsandmigratingcellsdependingon
manuscript. whichreceptorsareinvolved.DCC-familyreceptorsnormallymediateattractionbutcanalso
Competinginterests:Theauthorshavedeclared causerepulsionwhenpairedwithUNC5-familyreceptors[3,8,9].Duringchemoattraction
thatnocompetinginterestsexist. DCC-familyreceptorsactthroughSrcfamilykinasesandRhoGTPasestopromotecellprotru-
sionsbyregulatingtheF-Actincytoskeleton(reviewedin[10]).DCCcanalsoactasadepen-
dencereceptor,promotingapoptosisintheabsenceofitsligand[11].Furthermore,likeNotch
receptors,DCC-familyreceptorscanundergoectodomainsheddingandgamma-secretase
cleavage,allowingtheintracellulardomaintotranslocatetothenucleuswhereitcanactivate
transcription[12–14].
AlthoughDCC-familyreceptorsarebestknownfortheirrolesinneuronstheycanalsoreg-
ulateepithelialplasticityevents(reviewedin[10,15,16]).Forexample,Netrin-1andNeogenin
appeartoplayanadhesiveroleinmaintainingthestructureoftheproliferativeandinvasive
terminalendbudsduringmammaryglanddevelopment[17].InDrosophila,wehavefound
thatNetrinsandFrazzledregulatetwoepithelialevents:thedissociationoftheperipodialepi-
theliumofwingdiscsduringeversion[18]andtheformationofthemidgutepitheliumduring
embryonicdevelopment[19].Inbothcases,Frapromotesbothmotileandepithelialcharac-
teristics.InperipodialcellsFracaninduceextensiveprotrusions,butcanalsoinhibitthedisso-
ciation(i.e.partialEMT)oftheperipodialepithelium(unpublisheddata)andsubsequent
eversionofthewing[18].Similarly,inthemidgut,Fraisrequiredformidgutcellstomigrate,
andextendmotileprotrusions,butisalsoneededforapico-basalpolarizationandformation
ofanepithelium[19].
Thus,thequestionarises:whenFraispromotingepithelialphenotypes,isitactingthrough
thesamemolecularpathwaysthatmediatemigrationinmotilecells.Thisisadistinctpossibil-
itygivenrecentfindingsinahumanepithelialcellline.Inhumanintestinalcolorectalcancer
cellsNeogeninpromotesAJstabilitybyrecruitingandactivatingcomponentsoftheWAVE
F-ActinpolymerizationpathwaythroughRac1[20],asignalingpathwaythatplaysawell-
knownroleinaxonalextension.Arguingagainstthis,wehavepreviouslyfoundthatthetwo
phenotypesassociatedwithfraoverexpressionintheperipodialepitheliumhavedifferent
requirementsintermsoffraexpressionlevels.Forprotrusions,themorestronglyfrais
expressedthemoreprevalenttheprotrusions.Forblockingeversion,however,intermediate
levelsoffraweremosteffective[18].Theseobservationssuggestedthattheremaybedistinct
molecularpathwaysdrivingeachphenotype.Inthispaper,weprovidemoredirectevidence
formultiplepathwaysbyfirstlyestablishingarangeofphenotypesinepithelialcellsassociated
withFraoverexpression,andthenshowingthattheseareseparableintermsoftheirrequire-
mentforparticularregionsoftheFraprotein,orfordifferentdownstreamgenes.
ToassesstheimportanceofdifferentregionsofFrawefocussedonthethreehighlycon-
served,cytoplasmicP-motifsthatareacharacteristicofDCC-familyreceptors:P1,P2andP3
[4,21].Ofthethree,theP3motifappearsmostimportantforchemoattraction.DCCgain-of-
functionanalysisinXenopuslaevisspinalcordneuronsindicatedthattheP3motifisrequired
forgrowthconeattractiontoNetrin-1[22]whileinDrosophila,midlinecrossingofcommis-
suralneuronsdependsonlyontheP3motif[23].Invertebrates,theP3domainisrequiredfor
bindingofFAK,whichbecomesactivatedinresponsetoNetrin-1[24–26].Itisalsonecessary
forself-associationandinteractionwiththeRoboreceptor[27].InDrosophila,however,multi-
merizationofFradoesnotappeartodependontheP3motif[23]anditisunknownwhether
FAKbindingoccurs.TheP3motifalsointeractswiththeFERM-domainofMyosin-Xwhich
servestorecruitDCCtothetipsofneuritesgrowthcones,wherethereceptorregulatesMyo-
sin-X-mediatedformationandelongationofbasalfilopodia[28].TheP3motifofDrosophila
FrazzledisalsorequiredforasynergisticinteractionwithRho1leadingtoactivationofanother
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Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
memberofthemyosinfamily,non-musclemyosinII[29].FinallytheP3-motifisresponsible
forthetranscriptionalactivityoftheFraIntracellularmotif[14].
TherolesoftheP1andP2motifsaremoreenigmatic.InC.elegansanUNC-40gain-of-
functionstudyshowedthattheP1andP2motifs,butnottheP3,wasrequiredforexcessive
outgrowth,misguidance,branching,anddeformedcellbodiesofmechanosensoryandmotor
neurons[30].FurtheranalysisindicatedthatboththeP1andP2motifspromoteactinrear-
rangementsbuttheP1actsthroughtherecruitmentofUnc-34/Enabled,whereastheP2acts
viatheactivationofCed-10/Rac1andUnc-115(anactin-bindingprotein)[30].Drosophila
EnaandTrio,aRac/RhoGEF,actdownstreamofFrazzledduringattractivemidlineaxon
guidance,butspecificrequirementsofthePmotifshavenotbeeninvestigated[31].Interest-
ingly,expressionofafratransgenelackingtheP1motifcancauseaxonalprojectionerrors
leadingtospeculationthattheP1motifmayactivateothermolecularpathwaysthathavean
inhibitoryeffectonnormalFraactivity[32].TheP1motifalsointeractswiththeeIFsand
smallribosomalsubunitsinaxonsanddendrites[33]toregulatetranslation,andUnc5toreg-
ulategrowthconerepulsion[9].TheP2motifalsoharborsaWIRSmotifthatcanrecruitthe
WAVEregulatorycomplex[20].TheP2andP3motifshavealsobeenimplicatedinmicrotu-
buledynamicsduringaxonpathfinding:bothmotifsofDCCwerereportedtobinddirectlyto
TUBB3[34],aneuronalβ-tubulinisotypeIII,whichisknowntobeanessentialfactormediat-
ingaxonguidanceduringtheCNSdevelopment[35].
HereweshowthattheabilityofFratocreatemotileprotrusionsinepithelialcellsismost
dependentuponitsP3domainandactsthroughtheRac1RhoGTPase.Incontrast,induction
ofapicalconstriction,requirestheP1domainandactivatesRho1contractilepathways.Finally,
disruptionofnormalDE-CadlocalisationandeversionfailurerequirestheentireFraprotein
anddependsuponpar6.
Results
Fra-dependenteversionfailurerequiresfull-lengthFraandpar6
WebeganouranalysisoffraoverexpressionphenotypesbydeterminingwhichP-motifswere
requiredforeversionfailure.Todothiswecomparedtheeffectsofexpressingafull-length
UAS-fraFL-Myc transgenewithformslackingeithertheP1,P2orP3motifs(UAS-fraΔP1-Myc,
UAS-fraΔP2-Myc,UAS-fraΔP3-Myc)[23]inPEcells(Fig1A).Ubx-GAL4expressionoffull-
lengthfraFL-mycat25˚Cproduced~30%progenywitheversiondefectsorearlypupallethal-
ity:8.3%ofprogenyhadunevertedwings,9.2%displayedthoracicclefts,while13.3%were
lethal(n=120;Fig1B,Table1).OfthethreedeletiontransgenesonlyfraΔP2-mycproduced
eversionphenotypes,thoughthepenetrancewasmuchreducedto5%(n=396).Oftheadults
expressingfraΔP2-myc,0.8%offlieshadamissingwing,1.6%displayedthoracicdefects,and
2.5%werelethalatpupalstages.Incontrast,expressionoffraΔP1-mycandfraΔP3-mychadlit-
tletonoeffectoneversion(n=110,n=278respectively).
Next,wesoughttoidentifyothergenesactingdownstreamofFraduringeversion.Usinga
Ubx-GAL4,gal80tsdriverwescreened49genesforthosethat,whenknockeddowninlatelar-
valstages,couldsuppressfraover-expressionadulteversiondefects.Thesecrosseswereper-
formedat29˚CwhichisnotoptimalforFra-inducedeversiondefects[18],butwasrequired
formaximalRNAieffect.Thesegeneswerechosentoincludegenesimplicatedinnetrinsig-
naling,adhesionandcytoskeletalregulation,aswellasepithelialpolarity(S1Table).Unfortu-
nately,formostgenes,RNAiknockdowngeneratedextensivelarval/pupallethality,orstrongly
enhancedtheFraeversionfailurerate.Onlyknockdownofpar6,andfraitself,couldrepress
eversiondefects.Theproportionofnormalfliesincreasedfrom83%(Ubx>fraFL-myc)to94%
(Ubx>fraFL-myc,par6-IR)(p=0.026;Fig1C,Table2).Thisdegreeofrescuewascomparable
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Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
Fig1.Adulteversionfailurerequiresexpressionoffull-lengthFraandpar6.(A)Schematicofmyc-taggedfradeletiontransgenes[23].(B,C)Adulteversion
proportionswhentheindicatedtransgenesareexpressedusingtheUbx-GAL4peripodialdriverat25˚C(B)orUbx-GAL4,gal80tsat29˚C(C).(B)Expressionof
fraFLdisruptseversionin~30%ofdiscs.OnlyfraΔP2-mycisalsoabletoblockeversionbutismuchlessefficientthanfull-lengthfra.(C)Ubx>fraFL-mycand
Ubx>netA.IRdefectscanbelargelyrescuedbyco-expressionofeitherpar6.IRorfra.IR.Errorbarsshow95%CIfortheproportionofdiscswithnormaleversion
(byWilsonScoremethod).
https://doi.org/10.1371/journal.pone.0194003.g001
totheeffectsoffra.IRitselfwheretheproportionofnormalfliesincreasedto97%(Ubx>fra,
par6-IR)(p=0.012;Fig1C,Table2).
Thus,efficientdisruptionofeversionrequiresallthreeP-motifsandisdependentuponthe
polaritycomponentpar6.
EffectsofFra-overexpressioninepithelialcells
Next,wewishedtoestablisharangeofcellularphenotypesinepitheliaassociatedwithfra
overexpression.Wecreatedclonesofepithelialcellsoverexpressingfra,usingamosaicflip-out
GAL4approach.AswithUbx-GAL4,mosaicGAL4expressionofFraintheperipodialepithe-
liumproducedlongprotrusionsthatwererichinF-Actin(Fig2D’,arrows).Surprisingly,even
inregionsthatwerenotover-expressingfra,extensiveprotrusionswereobserved(Fig2D’,
arrowheads),aphenomenonneverseenincontrolmosaicdiscs(Fig2C’).Thus,fraoverex-
pressionelicitsstrongprotrusiveactivityand,surprisingly,appearstohaveanon-autonomous
effectonothercellsintheepithelium.
Ourmosaicanalysisalsorevealedseveralphenotypesindisc-proper(DP)cells,anepithelial
celltypethatdoesnotnormallyundergoEMT.Firstly,aswithPEcells,DPcellsextended
Table1. Adultwingdisceversiondefects.
Genotype Nodefects(%) Eversiondefects Earlylethality Total p-value
(%) (%) (n)
+;+;Ubx-GAL4/+ 100 0 0 214 -
fraFL-MYC/+;+;Ubx-GAL4/+ 69.2 17.5 13.3 120 <0.0001
fraΔP1-MYC/+;+;Ubx-GAL4/+ 100 0 0 110 1
fraΔP2-MYC/+;+;Ubx-GAL4/+ 95.1 2.4 2.5 396 <0.0001
fraΔP3-MYC/+;+;Ubx-GAL4/+ 99.7 0.3 0 278 1
Allcrossesperformedat25˚C.p-valuesshowFisher’sexacttestofnormalvsdisruptedproportionswithrespectto+;+;Ubx-GAL4/+controlgenotype.
https://doi.org/10.1371/journal.pone.0194003.t001
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Table2. par6RNAirescueseversiondefects.
Genotype Nodefects(%) Eversiondefects Earlylethality Total(n) p-value
(%) (%)
UAS-fraFL-myc/+;+;Ubx-GAL4,GAL80ts 83 13 4 75 -
/+
UAS-fraFL-myc/+;+;Ubx-GAL4,GAL80ts/ 97 0 3 60 0.012
UAS-fra.IR
UAS-fraFL-myc/+;+;Ubx-GAL4,GAL80ts/ 94 0 6 100 0.026
UAS-par6.IR
Allcrosseswereperformedat29˚C.p-valuesrepresentFisher’sexacttestvaluesofnormalprogenyvsabnormal(i.eeversiondefectsorearlypupallethality)compared
tocontrol.
https://doi.org/10.1371/journal.pone.0194003.t002
motileprotrusions,whichoccurredinbasalregionsofthecell(Fig2E).ClonesofDPcellsalso
exhibitedachangeincellshapewherebytheywereexpandedonthebasalside(Fig2G’and
2I’,reddouble-headedarrows).DPcellsalsoshowedachangeinAJcomponentdistribution.
Incontrolcells,DE-Cadwasstronglyexpressedatthezonulaadherens(ZA)(Fig2F’,double-
arrowheads),butwasalsofoundinpunctathatweredenselyaccumulatedinbasalregionsof
thecell(Fig2F’,arrowheads),andatlowerlevelsinthemiddleofthelateralmembrane(Fig
2F’,arrows).Infraexpressingclones,DE-Cadwasreducedinbasalregionsandalsoslightly
decreasedlaterally(Fig2F’and2G’,yellowarrowsandarrowheads).Asimilarreductionin
ArmexpressioninbasolateralregionsofDPcellswasalsoseen(Fig2H’and2I’).DE-Cadand
ArmlevelsattheZAdidnotappeartobealteredthoughitispossiblethattheintensityof
expressionatZAswouldobscuresubtlechanges.InPEcells,changesinDE-Cadexpression
alongtheapico-basalaxiscouldnotbedeterminedduetothesquamousmorphologyofthese
cells,butZAassociatedDE-Cadappearednormal(datanotshown).
Thus,overexpressionofFrainepithelialcellshasthreeeffects:extensionofprotrusions,an
apico-basalcellshapechange,andmislocalizationofAJcomponents.
Frainducesmotileprotrusions,whicharedependentupontheP3motif
Next,wewishedtodeterminewhichP-motifswererequiredforthesethreephenotypes.To
comparetheeffectsofdifferentfratransgenes,quantificationwasneeded,butthiswasdifficult
inmosaicdiscs,duetotherandomsizeandlocationofclones.Wethereforeusedtheptc-
GAL4driver,whichexpressesinaconsistentnarrowstripebetweentheanteriorandposterior
compartmentsintheDPepithelium(Fig3K).
First,wecomparedtheabilityofΔPtransgenestocreateprotrusionsinboththePEandDP
epitheliausingmosaicexpression.Protrusionswereclearlyvisualisedwithanti-Mycstaining
indicatingthatFra-Myclocalisedtothemembraneofthesefinecellularprocesses.Asprevi-
ouslyreported[23],alltransgenesexpressedatsimilarlevels.ProtrusionsinbothPEandDP
cellsweresubstantiallyreducedinptc>fraΔP3-mycdiscs(Fig3Eand3J),andslightlyreduced
inptc>fraΔP1-mycdiscs(Fig3Cand3H),whilethoseinptc>fraΔP2-mycwingdiscsappeared
similartocontrols(Fig3Dand3I).
Nextwequantifiedtheseobservationswiththeptcdriver.ptc-GAL4expressionoffraFL-
myc,inducedbasalprotrusionsinDPcells,whichemergedfromtheedgesoftheptcdomain
(Fig3L)andhadmeanlengthof8.08±0.2μm(SEM)(Fig3Land3P).Aswithmosaicclones,
protrusionsweresubstantiallyreducedinptc>fraΔP3-myc,discsdownto4.11±0.09μm
(p<0.0001;Fig3Oand3P)andsomewhatreducedinptc>fraΔP1-mycdiscsto6.69±0.24
(p=0.0046)(Fig3Mand3P),whilethoseinptc>fraΔP2-mycwingdiscswerenotsignificantly
differentfromcontrols(8.72±0.43;p>0.05)(Fig3Nand3P).Thus,theP3motifiscriticalfor
protrusions,whiletheP1motifplaysalesserrole,andtheP2motifisnotrequired.
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Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
Fig2.EffectsofclonaloverexpressionoffraonF-Actin,cellshapeandDE-Cadinepithelia.(A)Schematicofthirdinstarwingdisc.(B)CrosssectionalviewofA
showingthesquamousperipodialepithelium(PE)andthecolumnardiscproperepithelium(DP).Theapicalsurfacesfacethelumenwhilethebasalsurfacesface
outwardsandarecoveredinthebasementmembrane(BM,green).(C-I)Randomflipout-GAL4clonesexpressingGFP(green)only(C)orGFPandfra(D-I)stained
forF-Actin(C-D,grayscale),Fra(E),DE-Cad(F-G,grayscale),andArm(H-I,grayscale),withGFP(green).(C)IncontroldiscPEcells,F-actinismainlyenriched
nearthecell-celljunctionsinbothGFP-positiveandGFP-negativeclones.(D)Infra-overexpressingcells,F-actinfilamentsareoftenorganizedintothickextended
bundles(arrows).Innon-GFPwildtypecells,F-actinfilamentsarealsoarrangedinmultipleshorterprotrusions(arrowheads).Greendashedlines(C’,D’)indicate
thebordersofGFP-positiveclones.(E)ExpressionofFrainducesprotrusionsinDPcells(arrows).(F-I)InDPepithelialcells,overexpressionofFraaffectsDE-Cad
andArmdistributionandapico-basalcellshape.F’andH’showmagnifiedviewofboxedregionsinFandH.GandIgivecross-sectionalviewatdashedlinesinF
andH.InDPepithelialcells(F’,G’)DE-Cadlocalisestothezonulaadherens(ZA)(double-arrowheads),basal(arrowheads)andlateral(arrows)sides.Cells
overexpressingFra(green)havereducedDE-Cad(F’,G’)andArm(H’,I’)inbasal(yellowarrowheads)andlateral(yellowarrows)regionsandareexpandedbasally
(reddouble-headedarrows,G,I).Genotypes:C:hsFLP/+;Act5C:CD2:GAL4,UAS-GFP/+,D-I:hsFLP/+;Act5C:CD2:GAL4,UAS-GFP/UAS-fra.Scalebars10μm.
https://doi.org/10.1371/journal.pone.0194003.g002
Giventhebasallocationoftheseprotrusionswespeculatedthattheymightbeadheringto
thebasementmembrane(BM)viaintegrins.Consistentwiththis,RNAiknockdownofmys/
beta-PSandmew/alpha-PS1inptc>fraFL-mycdiscsreducedprotrusionlengthto5.2±0.2μm
and4.3±0.3μmrespectively(p<0.0001forboth).Inaddition,knockdownofRac1,itseffector
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Fig3.Overexpressionoffrainducesmotileprotrusions.MosaichsFLP;Act5C:CD2:GAL4,UAS-GFP(A-J)orptc-GAL4(K-O)wingdiscs
expressingfra-deletiontransgenesandstainedforMyc(grayscale).Afull-lengthFratransgeneinduceslongprotrusions(arrowheads)inbothDP
(B,L)andperipodialcells(G).FratransgeneslackingeithertheP1(C,H,M)orP2(D,I,N)motifselicitedprotrusionsofsimilarlengthtofull-
lengthfra,butfraΔP3-expressingcellsextendedmuchshorterprotrusions(E,J,O,arrowheads).(P)Quantificationoflengthofprotrusionsinptc-
GAL4wingdiscsexpressingtheindicatedfratransgene.DeletionoftheP1motifhasamildeffectonlength,whilelossoftheP3motifhalvesthe
lengthofprotrusions.KnockdownofothergenesmodifiesprotrusionlengthcausedbyfraFL-myc(seetextfordetails).Notethatexpressionlevels
ofthefourtransgenesappearedsimilar(immunostainingandimagingparameterswerekeptconsistentacrossgenotypes).Errorbarsshow
mean±SEMofmultiplediscs.Significancevaluesbasedontwo-tailedstudentst-test:nsnotsignificant,(cid:3)(cid:3)p-val<0.01,(cid:3)(cid:3)(cid:3)(cid:3)pval<0.0001.Scale
bars10μm.
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waveandtheArp2/3componentarpc3,aswellasthemyosin,Sqh,allsignificantlyreduced
protrusionlength(Fig3P).TheeffectsofRho1knockdownwerealsotestedbutcouldnotbe
quantifiedduetodrasticchangesinthemorphologyanddistributionofptc>GFPcells(see
below).AsanalternativewayofmodulatingRho1pathwayswetestedwhetherrhogef2could
influenceprotrusionsbutithadnoeffect.
TheresultssuggestthatFraactivatesatypicalaxon-guidancemotilitypathwayinDPcells,
inwhichRac1promotesF-Actin-richprotrusions,whichadheretotheBM.
Fracausesbasalexpansionindiscpropercells,whichisdependentupon
theP1motif
Aswithmosaicdiscs(Fig2Gand2I),ptc>fraFL-mycdiscsalsoexhibitedacellshapechange,
wherebythebasalsideofcellswasexpandedandtheapicalsidereduced(Fig4E).Inaddition,
therewasafurrowgeneratedalongthewholeptcstripe(Fig4D).Toquantifythecellshape
changewecalculatedtheratiobetweenbasalandapicalsidesofepithelialcellswithintheptc-
GAL4expressionstripe(Fig4P).Incontrolptc>GFPwingdiscs,GFP-positivecellshadeither
arectangularshapeorwereslightlyconstrictedonthebasalside(meanratio=0.88±0.04)
(Fig4A–4Cand4Q).Inptc>fraFL-myc,GFPcellstheapical/basalratiowassignificantly
increasedto1.63±0.06(p=0.0009;Fig4E,4Fand4Q).
WenexttestedwhichP-motifswererequiredfortheapico-basalcellshapechange,again
usingtheptc-GAL4driver.Inptc>fraΔP2-mycdiscstheapical-basalratiowas1.514±0.08sim-
ilartoptc>fraFL-mycdiscs(p=0.38,Fig4J–4Land4Q).ExpressionoffraΔP3-mycproduced
anintermediateeffectinwhichthebasal-apicalratiowasincreasedto1.31±0.045,whichwas
significantlyhigherthanincontrols(p=0.0002)butalsosignificantlylowerthanin
ptc>fraFL-mycwingdiscs(p=0.0048)(Fig4M–4Oand4Q),thoughnoapicalfurrowswere
detected.Incontrast,expressionoffraΔP1-mychadnoeffectonshapechange.Theratioof
0.95±0.06wasnotsignificantlydifferentfromtheptc>GFPcontrol(p>0.3;Fig4G–4Iand
4Q).
Thesechangesincellshapecouldpotentiallybeduetoeffectsoncontractility(e.g.eithera
decreaseinbasalcontractilityoranincreaseinapicalcontractility).Alternatively,thebasal
protrusionscouldcauseapicalfurrowsbyadheringtotheBMandgeneratingatractionforce
thatpullstheepitheliumdown.Insupportofthefirstmodelwefoundthatcell-shapechanges
weresuppressedbyknockdownofrhogef2(ratio=1.27±0.08;p=0.013,Fig4Q)andactually
enhancedbyknockdownofRac1(ratio=2.075±0.06;p=0.0012,Fig4Q).
ToshowthecontractionwasdependentuponRho1,weattemptedtorescuethephenotype
byco-expressionofUAS-Rho1.IR.However,ptc>fraFL-myc,Rho1.IRdiscsweretoohighlydis-
ruptedtoperformthequantification.Nevertheless,itwasclearthatGFP-expressingcellshad
reducedlevelsofDE-CadandlossofZAs,andhadbrokenfreeoftheepitheliumanddissemi-
natedthroughoutthelumenalspacebetweentheapicalsurfacesoftheDPandPE(Fig5).
Thus,inFra-overexpressingcells,Rho1appearscriticalformaintenanceofepithelialintegrity.
PLOSONE|https://doi.org/10.1371/journal.pone.0194003 March8,2018 8/20
Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
Fig4.OverexpressionoffrainducesbasalexpansionoftheDPepithelium.ptc>GFPwingdiscsstainedforGFP(green)andDE-Cad(grayscale).Reddashed
lines(B,E,H,K,N)indicatepositionofcrosssectionsdepictedin(C,F,I,L,O).(A)controldiscshowingtheexpressiondomainofptc-GAL4.(B,C)DPcellsare
roughlycolumnar:i.e.thelateralextentoftheptc-GAL4domaininapicalandbasalpartsofthecells(reddouble-headedarrows)issimilar.(D)ExpressionoffraFL-
myccreatesafoldthroughthedisc(D,F,arrow).(E-F).Theptc-GAL4domainiswiderinbasalregions.(G-I)ExpressionoffraΔP1-mycisunabletocreatetheapical
foldorchangeinaspectratio.(J-L)fraΔP2-mycisaseffectiveasfull-lengthFraingeneratingfoldsandshapechange.(M-O)fraΔP3-mycaffectstheshapechangeto
anintermediatedegree,butdidnotcreatefolds.(P)Schematicshowingmethodforquantification.(Q)Quantificationofbasal-apicalratioinptc-GAL4wingdiscs
expressingtheindicatedfratransgenes.(seetextandfordetails).Errorbarsshowmean±SEMofmultiplediscs.Significancevaluesbasedontwo-tailedstudentst-
test:nsnotsignificant,(cid:3)(cid:3)p-val<0.01,(cid:3)(cid:3)(cid:3)(cid:3)pval<0.0001.Scalebars10μm.
https://doi.org/10.1371/journal.pone.0194003.g004
PLOSONE|https://doi.org/10.1371/journal.pone.0194003 March8,2018 9/20
Frazzledcanregulatemotility,cellshape,andE-Cadlocalisationinepithelialcells
Fig5.RNAiknockdownofRho1infra-expressingcellsdisruptsepithelialintegrity.(A-E)ptc>GFP,fraFL-myc,Rho1.IRdiscstainedforDE-Cad(grayscale)with
GFP(green).(A)SectionofthediscattheleveloftheapicalsurfacesoftheDPandPEepithelia.(B)CloseupoftheboxedregioninA.CellsintheGFP-expressing
domaindonothavethenormalZAcell-celljunctionalstainingandlevelsofDE-Cadarereduced(B’,arrow).(C)Cross-sectionatthepositionofthedashedlineinB.
GFPexpressingcellsshowastrongreductioninDE-Cadlevels(C’,arrows).(D)MorebasalviewofthesamediscshowingGFPexpressingcellshavedisseminated
awayfromthecentralptc-GAL4stripe(arrows).(E)CrosssectionatthepositionofthedashedlineinD.GFPcellshavespreadoutfromthecentralGFPstripearea
(arrowhead)anddisseminatedinthespacebetweentheapicalsurfacesofthePEandDPepithelia(arrows).Scalebars50μm(A,D),20μm(C,E).
https://doi.org/10.1371/journal.pone.0194003.g005
TofurthertesttheideathatFra-overexpressionwasactivatinganapicalcontractileRho1--
pathwayweutilisedtheSqh1Pantibody[36],whichdetectsanactive,phosphorylatedformof
themyosinSpaghettiSquash(Sqh)(Fig6).Wefirsttestedtheantibodyonwingdiscsinwhich
afurrowwasinducedusingaUAS-rhogef2transgene,whichisknowntoactivateRho1-path-
ways.Sinceptc>rhogef2discswereverystronglyfoldedwetemporallyrestrictedtheperiodof
expressionusingaptc-GAL4,gal80tsdriver.Expressionofrhogef2for4hrsat29˚Ccauseda
mildfurrowwhichwasclearlyenrichedforSqh1P(Fig6D’and6E’).Inptc>fraFL-mycdiscsa
similar,thoughmilder,increaseinSqh1Plevelsinthevicinityofthefurrowwasseen(Fig6G’,
6H’and6I’).
TheresultsareconsistentwithamodelinwhichFraactivatesaRho1-contractilepathway
whichcausesapicalconstrictionandachangeintheapico-basalratio,andthisismediated
PLOSONE|https://doi.org/10.1371/journal.pone.0194003 March8,2018 10/20