Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal

27 Pages·2016·4.73 MB·English

RESEARCHARTICLE
Cdc42 and k-Ras Control Endothelial
Tubulogenesis through Apical Membrane and
Cytoskeletal Polarization: Novel Stimulatory
Roles for GTPase Effectors, the Small
GTPases, Rac2 and Rap1b, and Inhibitory
Influence of Arhgap31 and Rasa1
PieterR.Norden1,DaeJoongKim1,DavidM.Barry2,OndineB.Cleaver2,George
E.Davis1*
1 DepartmentofMedicalPharmacologyandPhysiology,UniversityofMissouriSchoolofMedicine,Dalton
CardiovascularResearchCenter,Columbia,MO,UnitedStatesofAmerica,2 DepartmentofMolecular
Biology,UTSouthwesternMedicalCenter,Dallas,TX,UnitedStatesofAmerica
*[email protected]
OPENACCESS
Citation:NordenPR,KimDJ,BarryDM,Cleaver Abstract
OB,DavisGE(2016)Cdc42andk-RasControl
EndothelialTubulogenesisthroughApicalMembrane
Acriticalandunderstudiedpropertyofendothelialcellsistheirabilitytoformlumensand
andCytoskeletalPolarization:NovelStimulatory
RolesforGTPaseEffectors,theSmallGTPases, tubenetworks.Althoughconsiderableinformationhasbeenobtainedconcerningthese
Rac2andRap1b,andInhibitoryInfluenceof issues,includingtheroleofCdc42andRac1andtheireffectorssuchasPak2,Pak4,
Arhgap31andRasa1.PLoSONE11(1):e0147758.
Par6b,andco-regulatorssuchasintegrins,MT1-MMPandPar3;manykeyquestions
doi:10.1371/journal.pone.0147758
remainthatarenecessarytoelucidatemolecularandsignalingrequirementsforthisfunda-
Editor:PontusAspenstrom,KarolinskaInstitutet,
mentalprocess.Inthiswork,weidentifynewsmallGTPaseregulatorsofECtubulogenesis
SWEDEN
includingk-Ras,Rac2andRap1bthatactinconjunctionwithCdc42aswellasthekey
Received:September24,2015 downstreameffectors,IQGAP1,MRCKβ,beta-Pix,GIT1,andRasip1(whichcanassem-
Accepted:January7,2016 bleintomultiproteincomplexeswithkeyregulatorsincludingα2β1integrinandMT1-
Published:January26,2016 MMP).Inaddition,weidentifythenegativeregulators,Arhgap31(byinactivatingCdc42
andRac)andRasa1(byinactivatingk-Ras)andthepositiveregulator,Arhgap29(byinac-
Copyright:©2016Nordenetal.Thisisanopen
accessarticledistributedunderthetermsofthe tivatingRhoA)whichplayamajorfunctionalroleduringtheECtubulogenicprocess.
CreativeCommonsAttributionLicense,whichpermits HumanECsiRNAsuppressionormouseknockoutofRasip1leadstoidenticalphenotypes
unrestricteduse,distribution,andreproductioninany
whereECsformextensivecordnetworks,butcannotgeneratelumensortubes.Essential
medium,providedtheoriginalauthorandsourceare
rolesforthesemoleculesduringECtubulogenesisinclude;i)establishmentofasymmetric
credited.
ECcytoskeletalpolarization(subapicaldistributionofacetylatedtubulinandbasalmem-
DataAvailabilityStatement:Allrelevantdataare
branedistributionofF-actin);andii)directedmembranetraffickingofpinocyticvacuolesor
withinthepaperanditsSupportingInformationfiles.
otherintracellularvesiclesalongacetylatedtubulintrackstothedevelopingapicalmem-
Funding:ThisworkwassupportedbyNational
branesurface.Cdc42co-localizessubapicallywithacetylatedtubulin,whileRac1andk-
InstitutesofHealth,NationalHeartLungandBlood
Institute,HL126518(GD,OC)andHL128584(GD). Rasstronglylabelvacuole/vesiclemembraneswhichaccumulateandfusetogetherina
Thefundershadnoroleinstudydesign,data polarized,perinuclearmanner.Weobservepolarizedapicalmembraneandsubapical
collectionandanalysis,decisiontopublish,or
accumulationofkeyGTPasesandeffectorsregulatingEClumenformationincluding
preparationofthemanuscript.
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NovelRegulatorsofECTubulogenesis
CompetingInterests:Theauthorshavedeclared Cdc42,Rac1,Rac2,k-Ras,Rap1b,activatedc-RafandRasip1tocontrolECtubenetwork
thatnocompetinginterestsexist. assembly.Overall,thisworkdefinesnovelkeyregulatorsandtheirfunctionalrolesduring
humanECtubulogenesis.
Introduction
Inrecentyears,considerableprogresshasbeenmadetowardourunderstandingofvascular
morphogenesis,includingthesubjectofthismanuscript,whichaddresseshowendothelialcells
formtubenetworkswithdefinedlumens[1–6].Previousworkhasshownthecriticalimpor-
tanceofintegrins,membrane-typematrixmetalloproteinases(MT1-MMP),RhoGTPases,
particularlyCdc42andRac1,smallGTPaseregulatorssuchasRasip1,kinasecascadesinvolv-
ingPKCepsilon(PKCε),Srcfamilymembers,Pak2,Pak4,Raf,MekandErk,andboththe
actinandmicrotubulecytoskeletons[3–5,7–13].OtherinterestingEClumenregulatorsare
proteinssuchasthecerebralcavernousmalformation(CCM)proteins,CCM1,CCM2,
CCM2L,andCCM3,aswellasthepolarityproteins,Par6b,Par3andjunctionaladhesion
receptorswithaffinityforPar3includingJamB,JamCandVE-cadherin[4,8,14–19].An
importantfuturedirectionofthisworkistofurtherunderstandhowECsbecomepolarized
duringlumenformation[20].Anothercriticalissueishowdefinedgrowthfactorsworkincon-
junctionwiththeextracellularmatrixtodirectECtubulogenicsignaling(throughtheabove
keymolecularregulators).Recently,wehavedescribedthatfivegrowthfactorstogetherare
abletostimulatehumanECtubulogenesisin3Dcollagenorfibrinmatricesunderserum-free
definedconditionsandtheyare;stemcellfactor(SCF),interleukin-3(IL-3),stromal-derived
factor-1α(SDF-1α),fibroblastgrowthfactor-2(FGF-2)andinsulin[21,22].Howsignaling
throughthiscombinationofgrowthfactorsandactivatedreceptorsleadstoEClumenand
tubeformationisacriticalandfundamentalquestionthatremainstobeanswered.
TheroleofpolarityregulatorshasbeendemonstratedduringEClumenformation(i.e.
Cdc42,Par6b,Par3)[8,14],buthowthiscontributestothedevelopmentofanECapicalmem-
branesurfaceandpolarizedcytoskeletalapparatusisnotwellunderstood.Inparticular,which
membranetraffickingeventsarenecessarytodevelopthespecializedECapicalmembrane
surfaceofECtubesduringtheirformationandfollowingtubematurationeventsincluding
muralcellrecruitmentandtheexposureofECstoflowforces?Manyyearsago,weandothers
demonstratedthatintracellularvacuoles/vesiclesappeartobenecessaryfortherapidlumen
formationabilityofECswhentheyareexposedtoa3Dmatrixenvironment[7,23–27].Fur-
thermore,weshowedthatCdc42andRac1werenecessaryfortheabilityofECstoformintra-
cellularvacuolesandsubsequentlumens[7].Also,weinitiallydemonstratedthatthemajority
ofthevacuolesobservedwerepinocyticinnatureandthatboththeactinandmicrotubulecyto-
skeletonswerenecessaryfortheirformation[23].Inaddition,weshowedthatotherintracellu-
larcompartmentsinECs,namelyWeibel-Paladebodies,wereobservedtofusewithvacuoles
duringtheirtransittotheapicaldomaininthatalargeproportionofvacuolescontainedhigh
amountsofvonWillebrandFactor[23].Ourlaboratoryhasnowidentifiedmanyregulatorsof
EClumenandtubeassemblyasmentionedabove[3,4,28],buthowthesemoleculescontrol
suchcriticaleventsincludingvacuoleformation,vacuolefusionwithpolarizedtargetingofves-
iclestothedevelopingapicalmembrane,howthecytoskeletonismodifiedandpolarizedto
specificallydirectvacuoles/vesiclestotheapicalsurfaceandthenhowthisprocessiscoordi-
natedwithMT1-MMP-dependentproteolysistocreateECtubenetworksthatareprepared
forthepolarizedrecruitmentofpericytestotheabluminaltubesurfaceremainslargely
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NovelRegulatorsofECTubulogenesis
unanswered.Thus,therearemanyfundamentalcellbiologicalquestionsthatstillneedtobe
investigatedwithregardtohowECformtubestructuresduringvascularmorphogenesis.
WerecentlydemonstratedthatEClumenformationin3Dmatricesresultsinpartdueto
theestablishmentofasymmetriccytoskeletalpolarizationwithF-actinexpressedinabasal
fashionandwithmodifiedtubulinsincludingacetylatedanddetyrosinatedtubulinlocalizedin
asubapicaldomaintosupportthedevelopingapicalmembranesurface[29].Keyplus-end
microtubuleregulatoryproteins,EB1,p150gluedandClasp1,controlEClumenformation
throughthesubapicalpolarizationandexpressionlevelsofthesemodifiedtubulins[29].In
parttheyacttogethertonegativelyregulatethetubulindeacetylases,HDAC6andSirt2.siRNA
suppressionofthesedeacetylasessinglyorincombination,ledtoincreasedEClumenforma-
tion,whileincreasedexpressionofHDAC6andSirt2interferedwithlumenformation[29].In
addition,disruptionofmicrotubuleswithcolchicineorotheragentssuchasthechemothera-
peuticdrug,vinblastine,causedECtubedisassemblyandcollapseandimportantly,thereis
alsorapidlossoftubulinacetylationandactivationofRhoA.Thus,tubulinmodificationsare
majorregulatorsofEClumenformation,butalsolumenandtubemaintenanceviasupportof
theapicalmembranedomain[29,30].Akeypointisthatregulationoftheactincytoskeletonis
alsocrucialforEClumenformation,inthatactinrearrangementsdownstreamofintegrin-,
Cdc42-andRac-dependentsignalingarenecessarytoformpinocyticintracellularvacuoles
leadingtolumenandtubeformation[3,7,23,26,27].Chemicaldisruptionofeitheractinor
tubulinpolymerization(whenaddedfromthebeginningoftheassay)completelyblocksEC
tubulogenesis[23].PolarizedF-actinatthebasalECsurfaceduringlumenformation[29]and
EC-ECjunctionsoncetubeshavebeguntoassembleandstabilizeisalsocriticaltothisprocess
[12].Thus,themechanismsthatunderliehowECsformtubenetworkscriticallyinvolvethe
ECactinandmicrotubulecytoskeletonswhichneedtobeintricatelycoordinatedduringeach
stageoftheprocess.
Here,inthisnewstudy,wehavecharacterizedthenovelstimulatoryroleofsmallGTPases,
keydownstreameffectors,andnegativeregulatorsincludingGTPaseactivatingproteins
(GAPs)duringEClumenandtubeformation.Ourstudieshaveidentifiedimportantnewroles
forCdc42,Rac2,k-RasandRap1bandsiRNAsuppressionofCdc42incombinationwiththese
otherthreeGTPasescausesprofoundinhibitionofECtubulogenesis.Furthermore,wehave
identifiedArhgap31andRasa1asGAPsthatinterferewithCdc42,Rac,andk-Ras,andwhich
markedlyblockthelumenformationprocess.Incontrast,Arhgap29,aRho-specificGAP,does
theoppositeandactuallystimulatesECtubeassemblythroughitsRhoA-inhibitoryactivity.
Additionally,weidentifyanovelrolefordownstreameffectorsoftheseGTPasesincluding
IQGAP1,MRCKβ,beta-Pix,GIT1,andRasip1duringthisprocess.Finally,weinvestigateEC
polarizationduringlumenformationanddemonstratetheapicaltargetingofsmallGTPases
throughmembranetraffickingeventsalongacetylatedtubulin-enrichedmicrotubuletracks,as
wellastheapicalmembranetargetingofkeydownstreamregulatorsincludingRasip1andc-
Raf.
MaterialsandMethods
Reagents
Stemcellfactor(SCF),stromalcell-derivedfactor1alpha(SDF-1α),andinterleukin-3(IL-3)
wereobtainedfromR&Dsystems(Minneapolis,MN).TubacinwasobtainedfromTOCRIS
Bioscience(Bristol,UnitedKingdom).Ascorbicacid,12-O-tetradecanoyl-phorbol-13-acetate
(TPA),andantibodiesagainstArhgap31,acetylatedtubulin,α-tubulin,andphospho-C-Raf
Tyr341wereobtainedfromSigma-Aldrich(St.Louis,MO).Recombinantfibroblastgrowth
factor2(FGF-2)andantibodiesagainstRac2,detyrosinatedtubulin,andβ-actinwasobtained
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fromEMDMillipore.AntibodiesagainstMRCKβ,k-Ras,Rasip1,GIT1,andMT1-MMPwere
obtainedfromAbcam(Cambridge,MA).AntibodiesagainstIQGAP1,ROCK1,andIntegrin
α2wereobtainedfromBDBiosciences(SanJose,CA).AntibodiesagainstRap1B,alpha-Pix,
beta-Pix,Cdc42,phospho-p44/42MAPK(ERK1/2)Thr202/Tyr204,ERK1/2,phospho-PAK2
Ser141,PAK2,phospho-PAK4Ser474,PAK4,phospho-B-RafThr401,phospho-B-RafSer445,
B-Raf,phospho-C-RafSer388,C-Raf,phospho-SrcY416,Src,phospho-p38MAPKThr180/
Tyr182,p38MAPK,andphospho-TyrwereobtainedfromCellSignalingTechnologies(Dan-
vers,MA).AnantibodyagainstRasa1wasobtainedfromEpitomics(Burlingame,CA).Anti-
bodiesagainstRhoAandRac1wereobtainedfromCytoskeleton(Denver,CO).Anantibody
againstArhgap29wasfromBethylLaboratories(Montgomery,TX).Anantibodyagainst
1
GAPDHwaspurchasedfromResearchDiagnosticsInc(Flanders,NJ).Alexafluor 488and
1 1
Alexafluor 633antibodies,andAlexafluor 488and633phalloidinwerefromMolecular
Probes(Eugene,OR).WTPKCεadenoviruswaspurchasedfromSevenHillsBioreagents
(Cincinnati,OH)andWTCSKandDNCSKadenoviruseswerepurchasedfromCellBiolabs
(SanDiego,CA).GFP,GFP-Cdc42,GFP-Rac1,GFP-RhoA,GFP-N17Rac1,GFP-N19RhoA,
GFP-N17Cdc42andGFP-V12Rac1adenovirusesweregeneratedaspreviouslydescribed,as
wellasDN-Pak4adenovirus,andMT1-ΔCWTandMT1-ΔCEAadenoviruses.
Vasculogenictubeassemblyassays
HumanumbilicalveinendothelialcellswereobtainedfromLonza(Walkersville,MD)and
werecultured(passage3–6)asdescribedpreviously[31].ECswerethensuspendedat2x106
cells/mLin2.5mg/mLcollagentypeImatricesandassayswereperformedaspreviously
described[21,32].Briefly,SCF,IL-3,SDF-1α,andFGF-2wereaddedat200ng/mLintocolla-
gentypeI.Cultureswerefedwithmediacontainingreducedserumsupplement(RSII),ascor-
bicacid,andFGF-2at40ng/mL.Cultureswereallowedtoassembleintocapillarynetworks
overaperiodof0–120hrwhencultureswerefixedorcollectedforfurtherprocessing.Samples
werefixedin2%paraformaldehydeor3%glutaraldehydeinPBS.Culturesfixedinparaformal-
dehydewerethenstainedforfluorescentmicroscopyimaging,whereasculturesfixedinglutar-
aldehydewerestainedin0.1%toluidinebluein30%methanol.Additionallyunfixedcollagen
gelswerelysedtoexamineproteinexpressionattheindicatedtimepointsusingstandardwest-
ernblottingtechniques.RecombinantadenovirusinfectionofECswasperformedaspreviously
described[31].
ECsiRNAsuppression
siRNAsuppressionprotocolsusingthesiRNAlistbelowwereperformedaspreviously
described[31].Thecellswereallowedtorecoverfor48hrandtransfectionwasrepeated.The
cellswerethenallowedtorecoverovernightbeforebeingharvestedforusein3Dassays.
siRNAsfromAmbionareasfollows:
Control(AM4637)SilencerSelectNegativeControl#2
Cdc42(s2765)5’-UGGUGCUGUUGGUAAAACA-3’
Rac1(s11711)5’-CUACUGUCUUUGACAAUUA-3’
Rac2(s11714)5’-CCUCUUUUGGAACAACAUA-3’
RhoA(s758)5’-CACAGUGUUUGAGAACUAU-3’
k-Ras(s7939)5’-CUAUGGUCCUAGUAGGAAA-3’
Rap1b(s224515)5’-AGAUUCUUCGAGUUAAAGA-3’
Pak2(s10024)5’-CAGAGGUGGUUACACGGAA-3’
Rasip1(s29763)5’-CGAGCUGUUCAAAUCCGAA-3’
Alpha-Pix(s16948)5’-GUAAAAGCCCUAAAACGAU-3’
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NovelRegulatorsofECTubulogenesis
Beta-Pix(s18122)5’-CAACGACAGGAAUGACAAU-3’
GIT1(s26306)5’-CCUUGAUCAUCGACAUUCU-3’
Rock1(s12097)5’-GGUUAGAACAAGAGGUAAA-3’
Arhgap31(s33202)5’-GGACAGAUCUCUACAUAGA-3’
Rasa1(s11820)5’-CAUAGAUCACUAUCGAAAA-3’
Arhgap29(s485)5’-GACCAAGGCUAAAACGAAU-3’
StealthsiRNAsfromInvitrogenareasfollows:
Pak4(NM_001014834_stealth_749)5’-UGCUUGCGCAGGUCCAUCUUCUUGA-3’
IQGAP1(NM_003870_stealth_421)5’-GCCUCCACUUUAGACACACUGAUAA-3’
MRCKβ(NM_006035_stealth691)5’-UAAAUCACCACCCACAUAGUAAUCC-3’
GenerationofS-epitopetaggedCherry,Cherry-fusionproteins,and
AcGFP-Rasip1adenoviruses
Cdc42,Rac1,Rac2,RhoA,k-Ras,andRap1bwereamplifiedfromcDNAobtainedfromMis-
souriS&TcDNAResourceCenter(Rolla,MO)andhumanRasip1andPKCεwereamplified
fromcDNAobtainedfromOpenBiosystems(OpenBiosystems,GEDharmacon,Lafayette,
CO),andstandardrestrictiondigestcloningforindividualGTPasesandPKCεinto
pmCherry-C1plasmidandRasip1intopAcGFP-C1(Clontech,MountainView,CA)using
EcoRI-HFandBamHI-HF,XhoIandBamHI-HF,andEcoRI-HFandXbaIrestrictionenzymes
respectively(NewEnglandBiolabs).AmplifiedS-Cherry,S-Ch-GTPase,S-Ch-PKCε,and
AcGFP-Rasip1constructsweresubclonedintopShuttle-CMVexpressionplasmidusing
NotI-HF,XbaI,XhoI,KpnI-HFandSalI-HFrestrictionenzymes,respectively(NewEngland
Biolabs).Recombinantadenoviralvectorswerethengenerated[33]andpropagatedasprevi-
ouslydescribed[7].ThePCRprimersusedarelistedbelowwiththeupstreamfirstfollowedby
thedownstreamprimer.
Cdc425’AGGAATTCTATGCAGACAATTAAGTGTGTTG-3’
5’-AGGGATCCTTAGAATATACAGCACTTCCTTTT-3’
Rac15’-AGGAATTCTATGCAGGCCATCAAGTGTGTGGTG-3’
5’-AGGGATCCTTACAACAGCAGGCATTTTCTCTTC-3’
Rac25’-AGGAATTCTATGCAGGCCATCAAGTGTGTGGTG-3’
5’AGGGATCCCTAGAGGAGGCTGCAGGCGCGCTTC-3’
RhoA5’-AGGAATTCTATGGCTGCCATCCGGAAGAAACTG-3’
5’-AGGGATCCTCACAAGACAAGGCACCCAG-3’
k-Ras5’-AGCTCGAGCTATGACTGAATATAAACTTGTGGTAG-3’
5’-AGGGATCCTTACATAATTACACACTTTGTCTTTG-3’
Rap1b5’-AGGAATTCTATGCGTGAGTATAAGCTAGTCG-3’
5’-AGGGATCCTTAAAGCAGCTGACATGATGAC-3’
Rasip15’-AGGAATTCTATGCTGTCTGGTGAACGGAAGGAGG-3’
5’-AGGTCGACTCAAGGAGACGTGGCCACGGGAGGCCCATG-3’
PKCε5’-AGCTCGAGCTATGGTAGTGTTCAATGGCCTTC-3'
5'-AGGGATCCTCAGGGCATCAGGTCTTCACCAAAG-3'
PrimersusedforcloningintothepAdCMVShuttleplasmidareasfollows.Thefirsttwo
primers(S-CherryandAcGFP)areupstreamprimerswhilethefollowingonesaredownstream
primers.
S-Cherry5’-AGGCGGCCGCACCATGGCAAAAGAAACCGCTGCTGCGAAATTTGAACGC
CAGCACATGGACTCGATGGTGAGCAAGGGCGAGGAG-3’
AcGFP5’-AGGGTACCACCATGGTGAGCAAGGGCGCCGAGCTGTTCAC-3’
Cdc425’-AGTCTAGATTAGAATATACAGCACTTCCTTTT-3’
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NovelRegulatorsofECTubulogenesis
Rac15’-AGTCTAGATTACAACAGCAGGCATTTTCTCTTC-3’
Rac25’-AGTCTAGACTAGAGGAGGCTGCAGGCGCGCTTC-3’
RhoA5’-AGTCTAGATCACAAGACAAGGCACCCAG-3’
k-Ras5’-AGCTCGAGTTACATAATTACACACTTTGTCTTTG-3’
Rap1b5’-AGTCTAGATTAAAGCAGCTGACATGATGAC-3’
PKCε5’-AGTCTAGATCAGGGCATCAGGTCTTCACCAAAG-3'
Rasip15’-AGGTCGACTCAAGGAGACGTGGCCACGGGAGGCCCATG-3’
ECvasculogenesispull-downassay
PulldownassaysusingS-epitopetaggedmCherryGTPasefusionproteinexpressingadenovi-
ruseswereperformedsimilarlytothatpreviouslydescribed[31].ECvasculogenesisassays
weresetupin3.75mg/mLcollagentypeIgelsusingadenovirusinfectedECsandextractedat
theindicatedtimepoints.3Dcultureswereplacedinlysisbufferconsistingof1%TritonX-
100,10mMTris-base(pH7.5),150mMNaCl,1mMDTTand5mMMgCl ,or1mMMgCl
2 2
and1mMCaCl ,CompleteEDTA-freeproteaseinhibitorcocktailtablets(RocheDiagnostics,
2
Indianapolis,IN),collagenase(150μg/μLhigh-purity;Sigma-Aldrich),and100μMGTPγS
(Calbiochem).Lysateswereincubatedina37°Cwaterbathfor15minutestoaidincollagen
digestionandwereclarifiedbycentrifugationat16,000gfor20minutesat4°C.Supernatants
werethenincubatedwithS-proteinagarosebeads(Novagen,EMDMillipore,Billerica,MA)
equilibratedwithwashingbuffer(respectivelysisbuffercontaining0.1%TritonX-100)for1
hourat4°Conarockingplate.Beadswerethenwashed4timeswithwashingbufferbefore
boundproteinwaselutedwith1.5Xsodiumdodecylsulfatesamplebuffercontaining7.5%β-
Mercaptoethanol.BoundGTPase-associatedproteinsweredetectedbywesternblotanalysis.
Invitrocultureimmunofluorescentstaining,microscopicimaging,and
analysis
Foranalysisof3Dcultures,immunostainingwascarriedoutaspreviouslydescribed.Immu-
nostainedcultureswereimagedusingaconfocalmicroscope(LeicaTC5SP5)connectedtoa
multiphotonsystem(Leica,BuffaloGrove,IL)usingexcitationwavelengthsof488nmand543
nmor488nmand633nmsequentially.High-resolutionimageswerecapturedusinga63X
waterimmersionobjective(NA1.2)utilizingLeicaApplicationSuite(LAS)software.Toluidine
bluestainedcultureswereimagedusinglightmicroscopyoninvertedmicroscopes(Eclipse
TE2000-E;Nikon,Melville,NYwithPhotometricsCoolSNAPHQ2camera,Tucson,AZ,and
OlympusCKX41withOlympusDP70camera,CenterValley,PA).Photographswereanalyzed
usingMetamorphsoftware(MolecularDevices,Sunnyvale,CA)bytracingvesselareaand
lumenarea.Time-lapsevideomicroscopywasperformedusinglightmicroscopyanda20X
objectivewithafluorescentinvertedmicroscope(DMI6000B,Leica)overa72hrperiod.
Immunofluorescentandimmunocytochemicalstainingofembryonic
tissues
FemalemiceexpressingtheRosa26yellowfluorescentprotein(YFP)reporterwerematedwith
malemiceexpressingCadherin5-CreERT2[12,34].Pregnantfemaleswereinducedwith
tamoxifen(2mg/40gMouse)atE12andE13,andthenembryosdissectedatE14.Headdermis
positiveforYFPwasisolated,fixedin4%PFA/PBSfor1hrat4°CthenwashedinPBS.Primary
antibodyincubationswerecarriedoutat4°CO/N(diluted1:300forGFP,1:100forRasip1),
slideswerewashedinPBS,andthenincubatedinsecondaryantibodyfor4hrsatRT(diluted
1:500).SlideswerewashedinPBSincubatedwithDAPIandmounted.Imageswereobtained
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NovelRegulatorsofECTubulogenesis
usingaLSM710MetaZeissconfocal.Antibodiesusedinclude:anti-GFP(Aves/GFP-1020),
Rasip1(NovusBiologicals/NB300-967),andAlexaFluor555donkeyanti-goat(Invitrogen/
A21432,Donkeyanti-chicken488(JacksonImmunoCat#703-545-155).Rasip1+/-and
Rasip1-/-embryoswerefixedin4%PFA/PBSandstoredin75%ethanol.Embryoswerepro-
cessedandsectionedasdescribed.Sectionswereincubatedwithprimaryantibody(diluted
1:100PECAM,1:100Endomucin)overnightat4°C,andafterwashing,wereincubatedwith
Donkeyanti-RatHRPsecondaryantibody(diluted1:100).TheDABreactionwasperformed
usingaperoxidasesubstratekit(Vector).TheslideswereimagedusingaNeoLumarstereomi-
croscope(Zeiss).Antibodiesusedinclude:Goatanti-ratIgG(SantaCruz/A10549),PECAM
(BDBiociences/553370),Endomucin(SantaCruz/sc-65495).
Statisticalanalysis
StatisticalanalysiswascompletedusingMicrosoftExcel.Statisticalsignificancewassetatmini-
mumwithP<0.05.Studentt-testswereusedwhenanalyzingtwogroupswithinindividual
experiments(withaminimumn=10).
EthicsStatement
AllanimalstudieswereperformedinaccordancewithUTSouthwesternMedicalCenterInsti-
tutionalAnimalCareandUseCommittee(IACUC)approvedprotocolAPN2008–0310,
approvaldateSeptember25,2014.Micearehousedinamodernair-conditionedfacilityand
aremaintainedaccordingtoNIHguidelines.Inaddition,thefacilitiesaresupervisedbyfull
timeveterinariansandtechnicalstaffandarefullyaccreditedbytheAmericanAssociationfor
AccreditationofLaboratoryAnimalCare.MicewererapidlyeuthanizedbyCO gasand
2
asphyxiationwhichminimizespainanddiscomfortandthismethodisconsistentwiththerec-
ommendationsoftheAVMAPanelofEuthanasia.
Results
IdentificationofnewsmallGTPaseregulatorsofECtubulogenesis
Inthisnewstudy,weperformedabroadsmallGTPasescreenusingsiRNAsuppressionwhere
weidentifiedseveralnewregulatorsaswellaseffectorsoftheseGTPases(Fig1andseelater
on).Wedemonstrateakeyrolefork-Ras,Rac2andRap1bduringthisprocess(Fig1andS1
Fig).Forcomparison,siRNAsdirectedtoCdc42andRac1alsoinhibitECtubeformation,
whilesiRNAsuppressionofRhoAhadnoinfluence(Fig1).Wehavepreviouslyreportedsimi-
larfindingswithregardtoCdc42,Rac1andRhoA[8].Toaddressthesequestionsusingadif-
ferentexperimentalstrategy,weincreasedexpressionoftheseGTPasesusingwild-type
proteinsthatwerefusedontheirN-terminuswithmCherryandanS-epitopetag(forbiochem-
icalpulldownassays).IncreasingexpressionofCdc42,k-Ras,andRap1ballsignificantly
enhancedEClumenformation,whileothersdidnot(S1Fig).UsingcombinationsofsiRNAs,
wedemonstratedthatknockdownofCdc42withk-Ras,Rac2,andRap1b,appeartohavethe
greatestblockinginfluence(Fig1Cand1D)comparedtoCdc42knockdownalone.Combined
knockdownofk-RaswithRac1,Rac2orRap1bdidnotblockinasignificantmannercompared
tok-Rasalone(notshown),suggestingthepossibilityofsignalingoverlapdownstreamofthese
GTPases.Overall,thisworksuggeststhatCdc42-dependentsignalingincombinationwith
eitherk-Ras,Rac2andRap1b,appearstobeparticularlycriticalforECstoformlumensand
tubesina3Dmatrixenvironment.
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NovelRegulatorsofECTubulogenesis
Fig1.IdentificationofkeysmallGTPasesthatcontrolECtubulogenesisin3Dmatrices.(A)IndividualECculturesweretransfectedwithcontrolsiRNA
orwithsiRNAsdirectedtoCdc42,Rac1,Rac2,RhoA,k-Ras,andRap1b,andthenweresuspendedin3Dcollagenmatricesfor72hrusingtheFactor-
inducedmodel.Dataarereportedasthemeantotalvesselareaperhigh-powerfield(HPF)±standarddeviation(SD)(n=15,p<0.01).Asteriskindicates
significancecomparedtocontrolcultures.Fixedcultureswerefixed,stainedandphotographed.Barequals25μm(B).(B)LysatesgeneratedfromsiRNA
transfectedculturesin(A)wereusedinWesternblotstoassessspecificproteinknockdownversuscontrol.(C,D)IndividualECculturesweretransfected
withsiRNAtargetingCdc42,combinationsofCdc42withRac1,Rac2,k-Ras,andRap1BsiRNAs,oracontrolsiRNA.ECtubulogenesisassayswere
performedwiththecellstheFactor-inducedmodelandafter72hr,cultureswerefixed,photographed(D)andquantitated(C).Dataarereportedasthemean
vesselarea±SD(n=15;p<0.01).AsteriskindicatessignificancecomparedtocontrolwhilethesquareindicatessignificancecomparedtoCdc42siRNA
treatment.Barequals25μm.
doi:10.1371/journal.pone.0147758.g001
FunctionallyinterchangeableFactor-andPhorbolester-inducedhuman
EClumenandtubeformationmodels
Ourstudieshaveutilizedtwohighlydefinedandrelatedsystemswhichhaveallowedusto
investigatethehumanEClumenformationprocess.Oneofthemutilizesdefinedgrowthfac-
tors(Factor-inducedmodel)thatdriveECtubulogenesis,whiletheotherdependsonthe
additionofphorbolester[21,23,31].TocharacterizethisnewFactor-inducedsystemforcom-
parisonwithpreviousstudies[9,29],wehaveperformeddetailedWesternblotstoassesssig-
nalingpathwaysandrequirementsduringthetubulogenicprocess(Fig2).UsingtheFactor-
inducedmodel,lumenformationismarkedlystimulatedbyPKCε,dominantnegativeCsk(to
activateSrckinases)aswellasacytoplasmictaildeletedwildtypeMT1-MMPconstruct(Fig
2C),allofwhichmimicwhatwereportedinpastwork[9,14].Furthermore,wedemonstrate
thatincreasedexpressionofCsk,toblockSrcactivation,additionoftheSrcinhibitor,PP2(but
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NovelRegulatorsofECTubulogenesis
Fig2.SignalingeventsthatcharacterizeECtubulogenesisovertimein3Dcollagenmatrices.(A)ECssuspendedin3Dcollagenmatricesusingthe
Factor-inducedmodelwerelysedattheindicatedtimepointsforWesternBlotanalysistoassessexpressionandsignalingoftheindicatedmoleculesorwere
fixedandphotographedwhileECsareassemblingintotubesovertime.Barequals50μm.(B)Thesametimecourseswereanalyzedforphosphotyrosine
(pTyr)-containingproteinsandincreasedlevelsofpTyrbandsat225,150,130,120,100,76,68,60,45,40,36,and30kDawereidentifiedovertime
correlatingwiththeECtubulogenicprocess.(C)ECswereinfectedwiththeindicatedrecombinantadenovirusesandthenalumenassaywasperformed
over72hr.Upperpanel-ECswereinfectedwithmCherryorPKCεadenoviruses,thenculturedin3DcollagengelsusingtheFactor-inducedmodel,fixedand
photographed.Barequals50μm.Lowerpanel-ECswereinfectedwithadenovirusescarryingGFP,Csk,DNCsk,PKCε,tail-deletedcatalyticallyactiveWT
MT1-MMP(MT1-ΔCTwt)oradominantnegativeMT1-MMPconstruct(MT1-ΔCTEA),thensuspendedin3Dcollagenmatricestoassesstheirabilitytoform
lumens.Dataarereportedasthemeanvesselarea±SDperHPF(n=12;p<0.01).AsterisksindicatesignificancecomparedtocontrolGFPcultures.
doi:10.1371/journal.pone.0147758.g002
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NovelRegulatorsofECTubulogenesis
notPP3,itsinactivecontrol)(notshown)orexpressionofadominantnegativeinhibitorof
MT1-MMP(cytoplasmictaildeletedcombinedwithaninactivatingmutation)alldramatically
interferewithlumenformationjustlikewepreviouslyobserved(Fig2C)[9,14],suggesting
thatourtwomodelsystemsappeartobefunctionallyinterchangeable.Afinalpointisthatwe
previouslyreportedthatincreasedtubulinacetylationanddetyrosinationaccompanylumen
formationandarenecessarytostabilizethedevelopingECapicalmembraneusingthephorbol
estermodel[29].Here,weshowthesameincreasesintubulinacetylationanddetyrosination
duringlumenformationusingtheFactorsystem(Fig2),andfurthermore,additionofthe
HDAC6inhibitor,tubacin,whichstronglyincreasestubulinacetylation,leadstomarked
increasesinlumenformation(S1Fig).
DifferentialsignalingeventsduringFactor-inducedEClumenandtube
formation
DuringthetimecourseoflumenformationusingtheFactor-inducedmodel,weobserve
increasesintheexpressionofbothα2β1integrinandMT1-MMP,aswellasincreasedphos-
phorylationofSrc,Pak4,B-Raf,c-Raf,andErk,whilelevelsofp38Mapkinasephosphorylation
remainlowcomparedtocontrols(Fig2A).WealsoperformedWesternblotsovertimeto
assesswhetherchangesinproteinsthataretyrosinephosphorylatedaredifferentiallyregulated
duringlumenformation.Weobserveincreasedtyrosinephosphorylationofbandsduringthis
processat225,150,130,120,100,78,68,60,45,40,36,and30kDa(Fig2B).ECtubulogenesis
isSrcfamily-andreceptortyrosinekinase-dependentthroughFactorandextracellularmatrix
signalingeventsandworkisongoingtoidentifythesetyrosinephosphorylatedbandsthatare
inducedduringthisprocess.
SmallGTPasetargetingandpolarizationtoapicalmembranesandthe
microtubulecytoskeletonenrichedinacetylatedtubulinduringEClumen
formation
Oneofthekeystepsinlumenformationiscreationofauniqueapicalsurface.Weareputting
forthaconsiderableeffortinourlaboratorytodefinehowtheapicalsurfaceformsandstabi-
lizesduringEClumenformation.Forexample,werecentlyshowedthatthemodifiedtubulins,
acetylatedanddetyrosinatedtubulinaresubapicallypolarizedduringlumenformation(Fig3)
andthisdependsonthemicrotubuleplus-endregulators,EB1,p150gluedandClasp1[29].
Blockadeofthesemoleculesresultsininterferencewithlumenformationandapicalpolariza-
tion.Bycontrast,F-actinasvisualizedusingphalloidin,ispolarizedinabasallocation.Here,
wehaveimagedthesubcellularlocalizationofsmallGTPases(usingfluorescentfusionpro-
teins)thatdirectlyinfluencethisprocess.Cdc42,Rac1,Rac2,k-Ras,andRap1ballshowtarget-
ingabilitytotheapicalsurfaceincomparisontothebasaldistributionofF-actin(Fig3andS1
Fig).Inaddition,akeyactivatedeffectordownstreamoftheseGTPasesisphospho-c-Raf[9,
14],whichalsoshowsapicaltargetingduringtheseevents(S1Fig).
Furthermore,wehaveexaminedtherelationshipbetweenCdc42andRac1andtheappear-
anceoftheseproteinsintheapicaldomainalongwithacetylatedtubulin,whichisstrongly
polarizedsubapically(Fig3).Cdc42concentratesinasubapicaldistributionthatalsoshows
focalco-localizationwithacetylatedtubulin(Fig3).Bycontrast,althoughacetylatedtubulinis
subapicallydistributed,GFP(control)orRhoAdonotdisplayapicallocalization.Wealsoper-
formedexperimentsbyexpressingconstitutivelyactiveGFP-V12Rac1(whichenhanceslumen
formation)[7],whichstimulatedtheappearanceofacetylatedanddetyrosinatedtubulinand
promotedlocalizationofacetylatedtubulinsubapically(Fig3andS2Fig).Expressionofdomi-
nantnegativeCdc42,Rac1,andPak4constructs(whichblocklumenformation)[7,8]reduced
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