Epha Kinase Activation Regulates HGF-Induced Epithelial Branching

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Introduction Hui Miao, mouse kidneydevelopment,membraneprotrusionsoccur (Sutherland etal.,1996;Ribeiro2002).Similarly,in tension ofcellprocessesatthetipinvadingepithelialbud and Bingcheng Wang epithelial branching morphogenesis EphA kinaseactivation regulates HGF-induced HGF andephrin-A1reducedsproutingofcellprotrusions, branching morphogenesisincollagengel.Cotreatment with hepatocyte growth factor/scatterfactor(HGF)–induced Darby caninekidney(MDCK)cellsnegatively regulates sophila a stepthatisconservedduringevolution.Forexample, of cellprotrusionsfromthebasolateralsideepithelialbud, of branchingmorphogenesisischaracterizedbytheextension (Affolter etal.,2003).Atthesubcellularlevel,initiation oforganospecificproximal anddistalstruct differentiation branch outgrowth,reiterationofbranchingprocess,and bud formation,initiationofbranchingmorphogenesis, Theyincludeformationofanorgananlage,primary discerned. on tissuesandspecies,aconservedseriesofeventscanbe gland.Althoughtheexactprocessvariesdepending mammary including thekidney,lung,prostate,andsalivary opmental processthatgivesrisetomanyepithelialorgans Epithelial branchingmorphogenesisisafundamentaldevel- ephrin-A1 suppressedHGF-inducedMDCKcellscattering. actions betweenEphsandephrinsinvivo, immobilized a newlydeveloped assay thatsimulatesthelocalizedinter- collapse andretraction ofpreexistingcellprotrusions.In addit an earlystepinbranching morphogenesis.Moreover, 2 1  Key words: EphA2; ephrin-A1;RhoGTPases; kidney;MAPK email: [email protected] Cleveland, OH44109.Tel.:(216) 778-4256.Fax:(216)778-4321. Research, R421MetroHealthMedical Center,2500MetroHealthDrive, Address correspondencetoBingcheng Wang,RammelkampCenterfor http://www.jcb.org/cgi/doi/10.1083/jcb.200304018 The Journal ofCellBiology

School ofMedicine, Yale University, New Haven, CT06520 Rammelkamp CenterforResearch, MetroHealth ofPharmacology Campus,andDepartment andIreland CancerCenter, Case Western Reserve University SchoolofMedicine, Cleveland, OH44109

TheRockefeller University Press, 0021-9525

Article

that activation ofendogenousEphAkinasesinMadin- in epithelialcellsvitroandvivo. Ourresultsshow kinasesandtheirephrinligandsarewidelyexp ph ion ofephrin-A1afterHGFstimulationresultedin trachealbranchingmorphogenesisinvolvestheex-

1 ChristianH.Nickel, , Volume 162, Number 7,September 29,20031281–1292 1 2 /2003/09/1281/12 $8.00 Lloyd G.Cantley, ressed Dro- ures 2 LeslieA.Bruggeman, extension offinemembraneprocesses thattakeplacewithin extension epithelial cellsincollagengelsischaracterizedbyinitial process. HGF-inducedbranchingmorphogenesisofMDCK simulates someaspectsofinvivoepithelialmorphogenesis et al.,1993;Soriano1995;Pohl2000),which Weidner three-dimensional matrix(Montesanoetal.,1991a; morphogenesis ofseveraltypesepithelialcellsgrownin a al., 1995).Invitro,HGF/c-Metsignalinginducesbranching kinase (RTK)c-Met(BorosandMiller,1995;Brinkmann et in virtuallyeverytissueofthebodythroughareceptortyr is apotentmitogen,motogen,andmorphogen,functions factor/scatter factor(HGF),amesenchymallyderivedfactor, regul and Rosenblum,2002). (Daviesetal.,1995;Fisher2001;Pisc organogenesis metanephric mesenchyme,anearlystepduringkidney on thetipofauretericbud(UB)thatisinvadinginto protein protrusion was independentofthemitogen-activated kinase activity; however, theephrin-A1effectoncell Ephrin-A1 inhibitedbasalERK1/2mitogen-activated protein UB, uretericbud. receptor tyrosinekinases;SOIL,scattering ontoimmobilizedligands; factor; PAK,p21-activatedkinase;ROCK, Rho-associatedkinase;RTK, Abbreviations usedinthispaper:HGF, hepatocytegrowthfactor/scatter HGF-induced MDCKbranching morphogenesis. a majormechanism by which EphAkinaseactivation inhibits withc-MetsignalingtoRhoGTPasesinterfering represents inhibitory effectsofephrin-A1. These data suggestthat Rho-associated kinase(Y27632)substantiallynegatedthe ﬁbers. Moreover, dominant-negativeof RhoAorinhibitor activation was retained, leadingtothepreservation ofstress activation ofRac1andp21-activated kinase,whereas RhoA Cytokines andtheirreceptorsareamongthecritical ators ofbranchingmorphogenesis.Hepatocytegrowth kinase pathway. Ephrin-A1suppressedHGF-ind 1 LauraN.Bennardo, 1

brought toyouby provided byPubMedCentral osine uced 1281 ione CORE The Journal of Cell Biology 1282 that RhoGTPases alsocontributetoepithelial morphogen- sis involvessimilar cellularprocesses,ithas beensuggested and Hall,2002).Asepithelial cellbranchingmorphogene- (Luo, 2000;daSilvaandDotti, 2002;Etienne-Manneville many ofwhichconvergeon the RhofamilysmallGTPases guidance cuesstimulateanumber ofsignalingpathways, guidance ofbothattractive and repulsivefactors.These navigates topredeterminedinnervationsitesunderthe neurites polarizestobecometheaxon,whosegrowthcone neurotrophic factorstimulationofneurons.Onethe protrusions isanalogoustotheextensionofneuritesafter sano etal.,1991b). elaborated intocellstrandsandtubularstructures(Monte- hours oftreatmentwithHGF,andwhicharethenfurther represent meansfromsixrandomlyselectedfields, of cellprotrusions.Cellswithmembraneprocesseslongerthanthe diametersofcellbodiesafter24hincubationwerecount Materials andmethods.Imageswerecollectedattheindicatedtimesonadigitalcameramountedaninvertedmicroscope.(D) MDCK cellswereculturedincollagengelandstimulatedwith20ng/mlHGFthepresenceof1 immunoblot usinganti-phosphotyrosineandanti-EphA2.(C)Cotreatmentwithephrin-A1inhibitsHGF-inducedbranchingmorphogenes stimulation. Lysatesfromcellsstimulatedwithephrin-A1–Fcfortheindicatedtimeswereprecipitatedandan were detectedusingFITC-conjugatedgoatanti–humanFc.Bar,20 (A) ExpressionofEphAkinasesinMDCKcells.Cellswereincubatedwithephrin-A1–Fc(EA1-Fc)orFcfor1hat4 Figure 1. Morphologically, theinvasivegrowthofmembrane The JournalofCellBiology

Activation ofendogenousEphAkinasesbyephrin-A1inhibitsHGF-inducedMDCKcellprotrusionsandbranchingmorphogenesis.

Volume 162,Number7,2003 SD. m. (B)InductionoftyrosinephosphorylationEphAbyephrin-A1–Fc 2000), andsynapse formation(Dalvaetal., 2000).Ephre- Vanderhaeghen, 1998;HolderandKlein,1999; Wilkinson, gration, compartmentboundary formation(Flanaganand known tobeinvolvedinaxon guidance,neuralcrestcellmi- the cellinterior.Innervous system,Ephsandephrinsare teractions, bothreceptorsand ligandscantransmitsignalsto cell–cell contactsignaling.Unique toEphkinase–ephrinin- Wilkinson, 2001).Thus,Eph–ephrininteractionsmediate (ephrin-B;EphNomenclatureCommittee,1997; domain dylinositol lipidmoiety(ephrin-A)oratransmembrane are membraneanchoredthrougheitheraglycosylphosphati- of vertebrateRTKs.LigandsforEphkinases,calledephrins, mental evidenceisstilllacking. esis (LubarskyandKrasnow,2003).However,theexperi- With 16members,EphRTKsrepresentthelargestfamily g/ml ephrin-A1–FcorFcasdescribedin C. Theligand-bound rec ed. Values Quantitation alyzed by eptors is.

The Journal of Cell Biology contact regions (Fig.1A).Incontrast,nostaining wasob- MDCK cellshighly expressedEphAreceptors atcell–cell to mostEphAkinases(Wilkinson, 2001),showedthat cells hereafter).Stainingwith ephrin-A1–Fc,whichbinds 8 cells(NelsonandVeshnock, 1986;referredtoasMDCK iments, weusedthepreviously characterizedMDCKclone man IgG1heavychain(Fc).In thisandallfollowingexper- using recombinantephrin-A1dimerizedbyfusiontohu- EphA kinases,immunofluorescenceanalysiswasperformed 1991b; Pohletal.,2000).Todetermineexpressionof epithelial branchingmorphogenesis(Montesanoetal., are commonlyusedasaninvitromodelsystemtostudy MDCK cellsarederivedfromthecaninerenaltubuleand HGF-induced branchingmorphogenesis Stimulation ofMDCKcellswithephrin-A1antagonizes epithelial organogenesisinvivo. suggesting thatEph–ephrininteractionsmayalsoregulate cells thatareactivelyundergoingbranchingmorphogenesis, nases, EphA2,ispreferentiallyexpressedonUBepithelial ing RhofamilysmallGTPases.Finally,oneoftheEphAki- epithelial branchingmorphogenesisbydifferentiallyregulat- shows thatEphAkinasesnegativelyregulateHGF-induced membrane protrusions.Cellularandbiochemicalevidence HGF treatmentcausedcollapseandretractionofpreexisting collagen gel.Moreover,additionofephrin-A1–Fcafter protrusions andsubsequentbranchingmorphogenesisin cells byephrin-A1inhibitedHGF-inducedsproutingofcell genesis. StimulationofendogenousEphAkinasesinMDCK that EphAactivationregulatesepithelialbranchingmorpho- epithelial organogenesisremainsunclear.Here,wereport al., 2001).However,theroleofEph–ephrininteractionsin epithelial tissues(LindbergandHunter,1990;Coulthardet EphA1 andEphA2kinasesarehighlyexpressedinnumerous tro and ephrinsarewidelyexpressedinothercelltypesvi- development (AdamsandKlein,2000;Chengetal.,2002). ceptors andtheirligandsalsoplayimportantrolesinvascular counted. Valuesrepresentmeansfromsixrandomlyselectedfields, recorded ond2and3.Bar,40 the growthmediumcontainingfreshHGF,andcellswereculturedforanadditional24h(d3).Branchingmorphogenesisrespo by 20ng/mlHGFincollagengelond1.After24hoftreatment(d2),ephrin-A1–Fcwasaddedtoafinalconcentration1 Figure 2. Results In additiontonervousandvascularsystems,Ephkinases and invivo(TuziGullick,1994).Forexample, Ephrin-A1 causescollapseofpreexistingcellprotrusionsinducedbyHGFinMDCKcells.

m. (B)QuantitativeanalysisofdatainA.Cellprotrusionslongerthanthediameterscellbodieswere SD. rin-A1–Fc, butnotFccontrol. membrane protrusionwasconsistentlyobservedwitheph- Fig. 1D;atwo-tothreefoldreductionofHGF-induced tubule formation.Aquantitativeanalysiswaspresentedin tenuated extensionofcellprotrusions,leadingtoretarded tree (Fig.1C).Additionofephrin-A1–Fcdramaticallyat- rated andelongatedtoformanorganizedtubularbranching ing gel.Byd5,thesetubularstructureswerefurtherelabo- thicker cellprotrusionsinvadingdeeperintothesurround- HGF. 2dlater,cellsbegantoorganizeintostrandswith protrusions tookplaceduringovernightstimulationwith al., 1991b;SantosandNigam,1993).Outgrowthofcell phogenetic responsewasinducedbyHGF(Montesanoet formed cysts(Wangetal.,1994;Fig.1C).Acomplexmor- ously, incontrolmedium,MDCKcellscollagenmatrix presence ofephrin-A1–Fc,HGF,orboth.Asreportedprevi- phogenesis, MDCKcellswereseededincollagengelthe phorylation ofthereceptors. A1, whichwasindicatedbytheincreasedtyrosinephos- MDCK cellscanberapidlyactivatedbyitsligandephrin- served withFccontrol.Fig.1BshowsthatEphAkinasesin regulating epithelial morphogenesis. a novelcrosstalk mechanismbetweenEphA andc-Metin traction ofpreexistingcelloutgrowth. Theseresultsrevealed ter HGFstimulation,ephrin-A1–Fc causedcollapseandre- A1–Fc reducedinitiationofcell protrusions;whengivenaf- Therefore, whengivenatthe sametimeasHGF,ephrin- membrane processesaswell preventionofnewoutgrowth. rin-A1–Fc causedcollapseandretractionofthepreexisting were examined24hlater.Fig.2showstheadditionofeph- Fc alone)wasthenaddedtogetherwithfreshHGF.Cells HGF for24htoinducecellprotrusions.Ephrin-A1–Fc(or MDCK cellswereseededincollagengelandstimulatedwith how ephrin-A1stimulationaffectedpreexistingprocesses. new cellprotrusionsinducedbyHGF.Next,weexamined EphA activationcansignificantlyattenuatethesproutingof The resultsfromthepreviousparagraphdemonstratethat preexisting cellprotrusions EphA ligationcausescollapseandretractionof To testtheeffectofEphAactivationonbranchingmor- Regulation ofepithelialmorphogenesisbyEphA| (A)Membraneprotrusionswereinduced Miaoetal. .5 nse was g/ml to 1283 The Journal of Cell Biology 1284 to agradientofsolublechemotacticorinsolublecontact- manner (Fig.3,BandC). icantly inhibitedMDCKscatteringinadensity-dependent coated surface.Incontrast,immobilizedephrin-A1–Fcsignif- off theedgeofcoverslipsandontouncoatedorFc- data). InthepresenceofHGF,MDCKcellsreadilyscattered and rarelyscatteredontothecoateddishes(unpublished layer onthecoverslipsduringcourseofexperiments, the absenceofHGF,MDCKcellsgrewasacompactmono- scattered cellandtheimmobilizedephrin-A1onplate.In calized engagementbetweenEphAsattheleadingedgeof onto thecoatedsurfacebytreatmentwithHGF,allowinglo- cells weretheninducedtoscatterofftheedgeofcoverslips coated withdifferentdensitiesofFcorephrin-A1–Fc.The on coverslips,andweretransferredontodishesthatpre- In thisassay,MDCKcellswereculturedtonearconfluency a scatteringontoimmobilizedligand(SOIL)assay(Fig.3A). presenting cell.Tosimulatethislocalizedcontact,wedevised but notitstrailingcellbody,comesincontactwithanephrin- activated invivowhentheleadingedgeofamigratingcell, cells toscatter.WereasonedthatEphkinasesarelikelybe A prerequisiteforbranchingmorphogenesisistheability and directionalcellmigration Ephrin-A1 inhibitsHGF-inducedMDCKcellscattering Cell migrationinvivoisfrequentlydirectionalresponse The JournalofCellBiology

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represent means Cells migratingthrough thefilterwerecountedfrom sixhighpowerfields.Values lower chambercontaining20ng/ml HGFand1 added totheupperchamberof Transwellandallowedtomigratetowardthe sides oftheTranswellwerethen coated with10 cell migration.100ngFNwasdried ontheundersideofTranswellfilter,andboth scattering coating with2 scatt (B) Immobilizedephrin-A1–Fc(butnotFc)inhibitsHGF-induced MDCKcell coverslips, theringsofcellsthathadscatteredoffcoverslipswere photographed. with 20ng/mlHGF,cellswerefixedandstainedcrystalviolet. Afterremoving 6-well dishesprecoatedwitheitherFcorephrin-A1–Fc.After24–48 hofstimulation to reachnearconfluenceoncoverslipsin24-wellplatesandwere transferredonto cell migration. Figure 3. ering inadensity-dependentmanner. (C)Highresolutionimageshowsthat compared withFccontrol.(D)Ephrin-A1 inhibitsHGF-induceddirectional Ephrin-A1 inhibitsHGF-inducedMDCKcellscatteringanddirectional (A)SchematicillustrationofSOILassay.MDCKcellswerecultured g/cm SD. 2 ephrin-A1–Fcsignificantlyinhibited HGF-inducedcell induced byHGFwereinhibitedephrin-A1. dom scatteringanddirectionalmigrationofMDCKcells rin-A1–Fc inthelowerchamber(Fig.3D).Thus,bothran- tion, whichwaspotentlyinhibitedbytheinclusionofeph- the lowerchamberinducedvigorousdirectionalcellmigra- MDCK cellsexhibitedlittlemigration.AdditionofHGFto migration. Fig.3DshowsthatintheabsenceofHGF, used topresentHGFinagradientinducedirectionalcell tility, amodifiedBoydenchambercellmigrationassaywas gate howEphAactivationcanmodulatedirectionalcellmo- ment ofcellswhenHGFispresenteduniformly.Toinvesti- effects ofEphAkinaseactivationonchemokineticmove- SOIL assaydescribedinthepreviousparagraphmeasures factors(LauffenburgerandHorwitz,1996).The attractant cating thattheinteractionsmaytakeplacefurtherdownstream phosphorylation betweenthekinases(unpublisheddata),indi- between c-MetandEphAkinases,norwastherecrosstyrosine immunoprecipitation assaysdidnotdetectphysicalassociation trusions andcausesthecollapseofpreexistingprocesses.Co- ephrin-A1 inhibitsHGF-inducedsproutingofnewcellpro- Next, weinvestigatedmolecularmechanismsunderlyinghow membrane protrusions is notresponsibleforsuppressionofHGF-induced Inhibition ofERK1/2MAPKbyEphAactivation g/ml ephrin-A1–FcorFcovernight. g/ml collagen.MDCKcellswere The Journal of Cell Biology by immunoblotwith antibodyagainstRaf1. replenished withHGF.Branchingmorphogenesis wasrecorded24hlater. Bar,10 replenished membrane protrusions.Transiently transfectedMDCKcellswerestimulatedwithHGFfor24h;ephrin-A1–Fc wasthenaddedtomedi 24 h later,GFP-positivecellswere recorded.Bar,10 24 transiently transfectedwithplasmids encodingRaf1-BXBand/orGFPwereseededincollagengel inopticaltissueculture96-wel immunoblot. (B)Dominant-active Raf1(BXB)doesnotblocktheinhibitoryeffectofephrin-A1onbranching morphogenesis.MDCKc presence of20ng/mlHGFforthe indicated timesandlysed.Immunoprecipitatestotalcelllysatewere analyzedfortheindi activ means fromatleasteightrandomly selectedfields Figure 4. MDCK cellsweretransfectedwithdominant-activeRaf1 bition ofHGF-stimulatedcellprotrusion. by ephrin-A1treatmentmaynotberesponsiblefortheinhi- dicate thatthemildsuppressionofERK1/2MAPKactivity tory effectat30-minand20-htimepoints.Theseresultsin- as thatobservedafterHGFalone,withonlyaminorinhibi- and HGF,MAPKactivitywasstimulatedtoasimilardegree early timepoints.Incellsco-stimulatedwithephrin-A1–Fc basal ERK1/2MAPKactivation(Fig.4A). stimulated MDCKcellsrevealedsustainedsuppressionof al., 2001).ExaminationofMAPKactivityfromephrin-A1– in severaldifferentcelltypes,includingepithelialcells(Miaoet ephrin-A1 potentlyinhibitstheRas-MAPKsignalingcascade shown thatstimulationofendogenousEphAkinaseswith esis invitro(Karihalooetal.,2001).Previously,wehave tial componentofHGF-mediatedrenalepithelialmorphogen- 1998), andactivationofERK1ERK2MAPKisanessen- MEK activation(RoyalandPark,1995;PotempaRidley, lial cell–celljunctionsandcellmigrationareknowntorequire from receptoractivation.HGF-inducedbreakdownofepithe- To furtherinvestigatetheroleofMAPKinthisprocess, HGF treatmentstimulatesMAPKactivity,particularlyat ation inhibitsbasalERK1/2MAPK activitiesinMDCKcells.Cellswerestimulatedwith1 EphA kinasesnegativelyregulateHGF-inducedcellprotrusions through anERK1/2MAPK-independentpathway.

SD.(D)Dominant-activeRaf1(BXB) doesnotpreventephrin-A1–inducedcollapseof m. (C)Quantitativeanalysisofbranching morphogenesisresultsinB.Valuesrepresent morphogenesis viaRaf/MAPK-independentmechanisms. negatively regulatesthecellprotrusionstepofbranching fectants (Fig.4D).Together,thesedatasuggestthatEphA the retractionofpreexistingcellprotrusionsinbothtrans- equally inhibitedbyephrin-A1.Similarly,ephrin-A1caused sprouting ofcellprotrusionsinresponsetoHGF;bothwere or cotransfectedwithGFPandBXBbothexhibitedtypical shown inFig.4(BandC),cellstransfectedwithGFPalone the inhibitoryeffectsofephrin-A1oncellprotrusions.As active, bypassMAPKinhibitionbyephrin-A1,andnegate EphA activation,BXBwouldkeepERK1/2constitutively tributes totheinhibitionofbranchingmorphogenesisby subject tocollagengelassay.IfsuppressionofMAPKcon- (BXB) togetherwithaGFPexpressionvectorastracer,and ganization. WewonderedwhetherRhoGTPasesrepresent small GTPasesarekeyregulatorsofactincytoskeletalreor- skeleton undergoesextensivereorganization.Rhofamily During cellmigrationandmorphogenesis,theactincyto- stress fiberscausedbyHGF stress fiberassembly,andinhibitsthelossofactin EphA activationenhancescorticalcytoskeletonand Regulation ofepithelialmorphogenesisbyEphA| m. (E)Theexpression oftransfectedRaf1-BXBwas detected g/ml Fcorephrin-A1–Fcintheabsence or cated proteinsby Miaoetal. (A)EphA l plates. ells um 1285 The Journal of Cell Biology at aconstantexposuretime.Bar,20 stained usingTexasred–conjugatedphalloidin.Imagesweretaken HGF orbothfortheindicatedtimes.Theactincytoskeletonwas changes inMDCKcells. Figure 5. 1286 step inthesprouting ofneurites(daSilvaand Dotti,2002), of thecortical cytoskeleton hasbeensuggested asacritical for stabilizingthecellmembrane; breakingtheconstraints ter overnightcotreatment.Cortical actinisakeystructure cells, andpreservedsignificant levelsofstressfibersevenaf- duced theinitiallossofcortical cytoskeletoninperipheral cells. InthepresenceofHGF, ephrin-A1significantlyre- sistent corticalactinbundlesalongtheedgeofperipheral actin stressfibers.Incontrast,ephrin-A1–Fcinducedper- podia. Treatmentfor2horlongerresultedinthelossof ery cells.Thelatterwascorrelatedwithextensionoflamelli- (Fig. 5)anddisassemblyofcorticalcytoskeletonsinperiph- stress fiberassemblyintheinnercellsofanMDCKcolony sembly werebiphasic.Earlyaftertreatment,HGFpromotes sis revealedthattheeffectsofHGFonactinstressfiberas- ephrin-A1–Fc, HGF,orboth.Immunofluorescenceanaly- skeletal changesaftershort-andlong-termtreatmentwith c-Met RTKs.Tothisend,wefirstanalyzedtheactincyto- a pointofconvergenceforthecrosstalkbetweenEphAand The JournalofCellBiology Ephrin-A1 andHGFcausedistinctactincytoskeletal Cellswerestimulatedwithephrin-A1–Fcor

Volume 162,Number7,2003 m.

with thosetreatedHGFalone(Fig.5). in cellstreatedwithbothHGFandephrin-A1compared sponsible forthesignificantpreservationofactinstressfibers of RhoA.TherelativeincreaseinRhoAactivitymaybere- tilted thebalanceamongdifferentGTPaseactivitiesinfavor HGF onRac1whilemaintainingRhoAactivity,ephrin-A1 ent effectsonRac1.Bysuppressingthestimulatoryof both HGFandephrin-A1activateRhoA,theyexertdiffer- ment (Fig.6C).Together,thesedatasuggestthat,although which wasnotsignificantlyalteredbyephrin-A1cotreat- erately stimulatedbyHGFtreatmentalone,thepatternof vented Rac1activation(Fig.6C).Cdc42activitywasmod- GTP-loading andcotreatmentwithephrin-A1–Fcpre- PAK activationstatus,HGFtreatmentstimulatedRac1 the presenceofephrin-A1(Fig.6B).Consistentwith tantly, theHGF-inducedPAKactivationwassuppressedin fect wasobservedwithephrin-A1–Fctreatment.Impor- treated withHGF(Fig.6B).Incontrast,nosignificantef- vealed adramaticincreaseinPAKphosphorylationcells a previouslycharacterizedantibody(Shamahetal.,2001)re- downstream effectorofRac1andCdc42.Immunoblotwith sured theactivationstatusofp21-activatedkinase(PAK),a enne-Manneville andHall,2002).Therefore,wenextmea- cesses controlledbyRhoGTPases(Shamahetal.,2001;Eti- RhoA andRac1activitiesthatdeterminesbiologicalpro- account fortheeffectsofephrin-A1. treatment alone.Therefore,RhoAactivationalonecouldnot RhoA activationlevelandkineticsweresimilartoHGF ephrin-A1. IncellstreatedwithbothHGFandephrin-A1, stimulated theRhoAactivity,eventoagreaterdegreethan induce dissolutionofstressfibers,HGFalsopersistently A1 stimulation(Fig.6A).Surprisingly,despiteitsabilityto served amoderateincreaseinRhoAactivitiesuponephrin- Using aGST-Rhotekinpull-downassay,weconsistentlyob- RhoA isakeyregulatorofactinstressfibers(Hall,1998). activation byHGF Ephrin-A1 preservesRhoAandsuppressesRac1 the activatedc-Met. the lossofcorticalcytoskeletonandstressfibersinducedby phogenesis (Fig.5).Insum,EphAactivationcouldprevent membrane protrusionsduringepithelialbranchingmor- and isalsolikelytobeacriticalstepintheformationof genesis, farexceeding HGFtreatmentalone(Fig. 7,AandB). Y27632 andHGF resultedinprecociousbranching morpho- membrane processes(Fig.7, AandB).Cotreatmentwith the epithelialcellmembrane andpreventingsproutingof gesting thatbasalactivityofROCK isimportantforstabilizing collagen gels,albeittoalessextent thanHGFtreatment,sug- ingly, Y27632byitselfinduced outgrowthofcellprocessesin inhibitor Y27632anddominant-negativeRhoA(19N).Strik- genesis, wetestedtheeffectsofRho-associatedkinase(ROCK) crease inRhoactivitytheregulationofbranchingmorpho- To directlyassessthefunctionalsignificanceofrelativein- by EphAkinases the negativeregulationofbranchingmorphogenesis Inhibition ofRhoA-dependentsignalingattenuates Recent evidenceshowsthatitisthebalancebetween The Journal of Cell Biology lapse ofpreexistingcellprotrusionsbyephrin-A1(Fig.7C), (Fig. 7,AandB).Similarly,Y27632largelypreventedthecol- trusions becameresistanttotheinhibitoryeffectofephrin-A1 Moreover, inthepresenceofY27632,HGF-inducedcellpro- Figure 6. analyzed byimmunoblotwithanti-Rac1oranti-Cdc42.Representativeresultsfromthreemoreindependentexperimentsareshow (but notCdc42)activationbyHGFisinhibitedephrin-A1.ActiveRac1/Cdc42wasprecipitatedusingGST-PAK-CD.Theprecipita were analyzedfortheactivationofPAKbyimmunoblotusingapreviouslydescribedphospho-PAKantibody(Shamahetal.,2001). with anti-RhoA.Quantitativeanalysisisshownintherightpanel.(B)Ephrin-A1suppressesactivationofPAKbyHGF.Total on HGF-inducedRhoAactivity.ActivewasprecipitatedusingGST-Rhotekin.TheGSTpull-downmaterialswereanalyzedbyimm Ephrin-A1 differentiallyregulatesHGF-inducedactivationofRhoGTPasesinMDCKcells.

characterized MDCKcellsthatinduciblyexpressdominant- signaling toinducethecollapsiveresponse. indicating thatEphAkinasesrequireRho/ROCK-mediated To furtherconfirmtheseobservations,weusedthepreviously fields corded. Valuesrepresentmeansfrom sixrandomlyselected HGF andY27632.Branchingmorphogenesis responsewasre- A1–Fc wasthenaddedtothegrowth mediumcontainingfresh lagen gelbyHGFinthepresenceofY27632for24h.Ephrin- membrane processes.Cellprotrusionswereinducedincol- (C) Y27632inhibitsephrin-A1–inducedcollapseofpreexisting ues representmeansfromsixrandomlyselectedfields (B) QuantitationofbranchingmorphogenesisresultsinA.Val- genesis assaywasperformedintheabsenceorpresenceof10 tion ofbranchingmorphogenesis.MDCKmorpho- (A) Y27632significantlyattenuatesephrin-A1–inducedinhibi- protrusions andattenuatestheinhibitoryeffectofephrin-A1. Figure 7. Regulation ofepithelialmorphogenesisbyEphA| M Y27632.Cellimagesweretaken24hlater.Bar,40 SD. Inhibition ofROCKenhancesHGF-inducedcell (A)Ephrin-A1hasnosignificantinfluence Miaoetal. celllysates (C) Rac1 tes were unoblot n. SD. m. 1287

The Journal of Cell Biology 1288 branching morphogenesis ofmousemurine inner medullary- In additiontoMDCK cells,wealsofoundpotent inhibitionof branching morphogenesis expressed onaUBthatisundergoing One oftheEphAkinases,EphA2, isselectively hibit negative RhoA(19N),ephrin-A1treatmentcouldstillin- morphogenesis. InthepresenceofY27632ordominant- tivation inhibitsthemembraneprotrusionstepofbranching unlikely tobetheonlymechanismthroughwhichEphAac- noted thatdifferentialregulationofRhoGTPaseactivities is step ofbranchingmorphogenesis.However,itshouldbe EphA kinaseactivationinhibitscellextension,theinitiation of RhoAactivationrepresentsakeymechanismbywhich can leadtounintendedtargetsandconsequences. subject totightspatialcontrol;inwhichcaseglobalactivation proper Rac1function.Alternatively,activationmaybe suggested isthatGTP–GDPexchangemayberequiredfor tems (Etienne-MannevilleandHall,2001).Oneexplanation of dominant-activeRac1havealsobeenreportedinothersys- predicted stimulatoryeffects(Fig.8).Suchunexpected caused adecreasedbranchingmorphogenesis,insteadofthe sions (Fig.8).Intriguingly,dominant-activeRac1(12V)also Rac1 (17N)completelyabolishedsproutingofcellprotru- to bethecase,asinducedexpressionofdominant-negative (17N) isexpectedtoinhibittheprocess.Thisindeedturnsout branchingmorphogenesis,dominant-negativeRac1 induced ment. IfRac1signalingplaysasignificantroleinHGF- MDCK cells,whichwaslargelynegatedbyephrin-A1cotreat- icantly inhibitedthebranchingmorphogenesis. the inducibleexpressionofdominant-activeRhoA(14V)signif- gated byexpressionofRhoA-19Nmutant(Fig.8).Bycontrast, over, theinhibitoryeffectofephrin-A1–Fcwassubstantiallyne- inducibly expressingdominant-negativeRhoA(19N).More- hanced branchingmorphogenesiswasobservedinMDCKcells and Nelson,1998).ConsistentwiththeeffectsofY27632,en- nant-negative Rac1(17N),ordominant-active(12V;Jou negative RhoA(19N),dominant-activeRhoA(14V),domi- resent meansfromsixrandomlyselectedfields, longer thanthediametersofcellbodieswerecounted.Valuesrep- presence ofephrin-A1–FcorFc,cellswithmembraneprocesses in Materialsandmethods.Aftera24-hstimulationwithHGFthe 2 d.Branchingmorphogenesisassayswereconductedasdescribed was inducedbyremovingtetracyclinefromtheculturemediumfor inant-negative anddominant-activeRhoARac1inMDCKcells hibitory effectofephrin-A1oncellprotrusions. Figure 8. yet unidentifiedmechanisms may alsoplayaminorrole. In sum,thesedatasuggestthattiltingthebalanceinfavor C-Met activationbyHGFledtorobustRac1signalingin The JournalofCellBiology 20% ofthemorphogeneticeffectsHGF.Other Dominant-negative RhoAsubstantiallyabolishesthein-

Volume 162,Number7,2003 Expressionofdom- SD. which mayaccountforthelackofrenalphenotypesin EphA1, EphA2,EphA3,andEphA4(unpublisheddata), that fetalkidneysexpressmultipleEphAkinases,including (line KST085;Mitchelletal.,2001).Immunoblotrevealed tential regulatoryroleinrenalorganogenesis. morphogenesis suggeststhatEphA2ispositionedtoplayapo- EphA2 inUBepithelialcellsundergoingactivebranching culture (unpublisheddata).Thepreferentialexpressionof rons derivedfrommetanephricmesenchymeafterlongerorgan also notdetectedintubularepithelialcellsofdevelopingneph- sion ofPax-2(compareFig.9BwithD).EphA2was in metanephricmesenchymethatwasmarkedbytheexpres- (Fig. 9D).Veryloworundetectableexpressionwasobserved UB cells(Fig.9B)thatweremarkedbycytokeratinexpression for low attachment,photographed(Fig.9,AandC),stained dissected fromheterozygousembryos,culturedovernighttoal- ize theexpressionofendogenousEphA2.E12.5kidneyswere Discussion sion inKST085 EphA2 neys usingthepreviouslydescribed this end,weexaminedexpressionofEphA2inembryonickid- nephric mesenchyme(Saxen,1987;VainioandLin,2002).To peated branchingofaUBuponreciprocalinductionbymeta- early fetalkidneydevelopment,whichischaracterizedbyre- investigated potentialinvivorelevanceofourfindingsduring of renalbranchingmorphogenesis(Pohletal.,2000).Next,we lished data).Bothcelltypesarewidelyusedasinvitromodels collecting ductcellsinthree-dimensionalcollagengel(unpub- EphA2 kidneys. Figure 9. cells invitroandvivo.However,theirroleepithelial Eph kinasesandephrinsarewidelydistributedinepithelial stain allnuclei(D). Bar,100 metanephric mesenchyme, respectively.DAPI(blue) wasusedto keratin (green)andanti-Pax-2(red) tomarkUBepithelialcellsand tively, kidneyswerefixedwithmethanol andstainedwithanti-cyto- tographed (AandC)processed forX-galstaining(B).Alterna-

-gal activity(Fig.9B).EphA2wasselectivelyexpressedin

-null mice.Nevertheless,the lac

E12.5kidneysweredissectedfrom theheterozygous

Z knockoutembryos.Theywere cultured overnight,pho-

EphA2 isselectivelyexpressedon UBsofembryonic

EphA2

- lac Z miceenablesustopreciselylocal- m. EphA2 -gal reportergeneexpres- - lac Z knockoutmice The Journal of Cell Biology vious datademonstrate thatendogenousEphA kinasesme- Ras-MAPK signaling pathway(Furgeetal., 2000). Ourpre- proteins; manyoftheseproteins arecapableofactivatingthe phorylation ofthereceptorand therecruitmentofvarious the bindingtoitsreceptorc-Met, whichtriggersautophos- and spaceofbranchingmorphogenesis. also operateinvivo,andmay helpdeterminethedirection gels. Conceivably,suchnegativeguidancemechanismscan lapse ofmembraneprocessesinthree-dimensionalcollagen cell migrationintwo-dimensionalassays,andcausedcol- EphA activationinhibitedMDCKscatteringanddirectional lial–stromal interactions.Theinvitroresultshereshowthat cell–cell contactsincludingepithelial–epithelialandepithe- cise spatialpatterns,whichwillencounterdifferenttypesof volves migrationandreorganizationofepithelialcellsinpre- necessitate cell–cellcontact.Epithelialorganogenesisin- membrane anchored,Eph–ephrininteractionandsignaling ithelial cellsofembryonickidneys.Becauseephrinsare the EphAkinases(EphA2)isselectivelyexpressedinUBep- mined, althoughourpreliminaryanalysesrevealthatoneof and causedcollapseofexistingones. inhibited HGF-inducedextensionofnewcellprotrusions, that activationofendogenousEphAkinasesinMDCKcells mechanisms ofbranchingmorphogenesis.Here,wefound vestigate cellularprocessesandtheunderlyingmolecular 2000). Epithelialcellsofkidneyoriginhavebeenusedtoin- inductive mesenchyme(Daviesetal.,1995;Tepass of membraneprocessesfromUBepithelialcellsfacingthe Lin, 2002).Microscopically,anearlyeventistheextension (Saxen, 1987;PiscioneandRosenblum,2002;Vainio mesenchymethatgeneratefilteringnephrons metanephric tually formthecollectingsystem,anddifferentiationof migration, proliferation,andbranchingofUBsthateven- on UBcells.Theinitialinductionisfollowedbyrepetitive creted bymetanephricmesenchymeandactsonRetreceptor the glial-derivedneurotrophicfactor(GDNF).GDNFisse- epithelial cellsintometanephricmesenchymeinresponseto velopment, theprocessisinitiatedbyinvasionofUB gans suchaskidney,lung,breast,andprostate.Inrenalde- logical processunderlyingthedevelopmentofmanyvitalor- of thesefindings. branching morphogenesis,suggestingtheinvivorelevance pressed onaUBepitheliumthatisundergoingactive Finally, oneoftheEphAkinases,EphA2,isselectivelyex- HGF-induced branchingmorphogenesisbyEphAkinases. ERK1/2 MAPK,playsanimportantroleinregulating Rac1 activitiesinfavorofRhoA,butnottheinhibition demonstrate thatatiltinthebalancebetweenRhoAand lapse andretractionofpreexistingprotrusions.Wefurther Ephrin-A1 stimulationafterHGFtreatmentinducedcol- cell protrusions,anearlystepinbranchingmorphogenesis. ment withHGFandephrin-A1reducedthesproutingof phogenesis inthree-dimensionalcollagenmatrix.Cotreat- negatively regulatesHGF-inducedepithelialbranchingmor- for thefirsttimethatactivationofendogenousEphAkinases well-characterized MDCKcellmodelsystem,weshowhere cell biologyremainsincompletelyunderstood.Usingthe HGF-induced branchingmorphogenesis isinitiatedby The invivorelevanceofourfindingsremainstobedeter- Epithelial branchingmorphogenesisisafundamentalbio- of neuritesfrom neuronsuponneurotrophic factorstimula- brane protrusions inMDCKcellsisanalogous tosprouting 2001). Morphologically,HGF-induced formationofmem- ing tothecollapseofaxonal growthcones(Shamahetal., Rac1/Cdc42 inactivationupon EphAkinaseligation,lead- ephexin. Moreover,ephexin mediates RhoAactivationand associate withaguaninenucleotide exchangefactorcalled ing, Shamahetal.(2001)found thatEphAkinasesdirectly collapse (Wahletal.,2000).Usingyeasttwo-hybridscreen- rin-A5 causedactivationofRhoAandinducedgrowthcone Yamaguchi, 2002).Stimulationofganglionaxonswitheph- 2001; Penzesetal.,ShamahIrieand dritic spinemorphogenesis(Wahletal.,2000;Ethell in therepulsiveguidanceofaxonalgrowthconesandden- a relativeincreaseinRac1activity. promoted eitherbyarelativedecreaseinRhoAactivityor morphogenesis. Thus,epithelialbranchingmorphogenesis is RhoA (19N),ephrin-A1lostmostofitsinhibitoryeffectson tivity waspreventedeitherbyY27632ordominant-negative ing whileleavingRhoAintheactivestate.Whenac- morphogenetic effectsofHGFbysuppressingRac1signal- its morphogeneticeffects.Third,ephrin-A1attenuatedthe lates Rac1signaling,anddominant-negativeblocked signaling inepithelialmorphogenesis,HGFpotentlystimu- favor ofRac1.Second,supportinganimportantroleRac1 naling toROCK,Y27632isexpectedtiltthebalancein could potentlysynergizewithHGF.ByinhibitingRhoAsig- sufficient toinduceextensionofmembraneprocesses,and epithelial morphogenesis.First,Y27632treatmentalonewas tween differentmembersofRhoGTPasescriticallyregulates support thisnotion,anddemonstratethatthebalancebe- as keyregulatorsofbranchingmorphogenesis.Ourresults skeleton. Forthisreason,RhoGTPaseshavebeensuspected much ofwhichinvolvesdynamicregulationactincyto- cess formation,cellshapechange,andreorganization, morphogenesis entailshighlycoordinatedmembranepro- formation (Kaibuchietal.,1999;Ridley,1999).Branching other downstreameffecterofRho,Dia,inducesstressfiber and regulatescellcontractility.ROCKtogetherwithan- tor ROCK,stimulatesmyosinlightchainphosphorylation, Nobes andHall,1995).RhoAactsviaitsdownstreameffec- mation offilopodia(Ridleyetal.,1992;Kozma1995; of lamellipodia,andactivationCdc42stimulatesthefor- merization atthecellperiphery,resultinginformation and cellshape.Rac1activationleadstolocalizedactinpoly- are involvedinregulatingepithelialmorphogenesis. nisms thataremoresensitivetoregulationbyEphAkinases via Raf/MAPK-independentmechanisms;othermecha- sis nases negativelyregulateepithelialbranchingmorphogene- branching morphogenesis.ThesedatasuggestthatEphAki- with dominant-activeRaf1,ephrin-A1–Fcstillinhibited MAPK. Moreover,inMDCKcellstransientlytransfected enough tooverridethestimulatoryeffectofHGFon inhibitory effectofephrin-A1–FconMAPKwasnotstrong basal ERK1/2MAPKactivityinMDCKcells.However,the activation ofendogenousEphAkinasescanalsoinhibitthe ferent celltypes(Miaoetal.,2001).Here,weshowthat diate down-regulationofRas-MAPKsignalinginseveraldif- Eph kinaseregulationofRhoGTPaseshasbeenreported The RhofamilysmallGTPasescontrolactincytoskeleton Regulation ofepithelialmorphogenesisbyEphA|

Miaoetal. 1289 The Journal of Cell Biology 1290 7.4, and5 Cells weredetached usingtrypsin-EDTAandsuspended ataconcentration For eachmlofthegel,800 collagen gels Branching morphogenesisassays inthree-dimensional chased fromR&DSystems.Y27632 was obtainedfromCalbiochem. tained fromMolecularProbes,Inc. RecombinanthumanHGFwaspur- ImmunoResearch Laboratories.Texasred–conjugatedphalloidinwasob- and FITC-conjugatedgoatanti–humanFcwerepurchasedfromJackson and anti-Cdc42werepurchasedfromBDBiosciences.HumanFcfragment School, Boston,MA)(Shamahetal.,2001).Mousemonoclonalanti-Rac1 anti-p-PAK wasprovidedbyDr.MichaelE.Greenberg(HarvardMedical andanti-Raf1werefromSantaCruzBiotechnology,Inc.Polyclonal p-ERK anti-RhoA, mousemonoclonalanti-phosphotyrosine(PY99),andanti- Pandey etal.,1994).Rabbitpolyclonalanti-EphA2antibody,anti-ERKand Ephrin-A1–Fc wasproducedasdescribedpreviously(Davisetal.,1994; Antibodies andreagents and Nelson,1998). duction ofgeneexpressionwasperformedasdescribedpreviously(Jou James Nelson(StanfordUniversitySchoolofMedicine,Stanford,CA).In- RhoA(14V), RhoA(19N),Rac1(12V),andRac1(17N)weregiftsofDr.W. 100U/mlpenicillin-streptomycin.MDCKcellsinduciblyexpressing and collecting ductcellsweregrowninDMEsupplementedwith10%FBS MDCK clone8(NelsonandVeshnock,1986)murineinnermedullary- Cell culture concert controlepithelialorganogenesisinvitroandvivo. rin-As inorchestratingdiverseenvironmentalcuesthat gation isrequiredtoassessthevariousrolesofEphAsandeph- bers andinhibitingbranchingmorphogenesis.Furtherinvesti- for theeffectsofEphAactivationinpreservingactinstressfi- tively. ArelativeincreaseinRhoAactivityislargelyresponsible tively andpositivelyregulatebranchingmorphogenesis,respec- point ofcellsignalingbyEphAandc-MetRTKsthatnega- our datademonstratethatRhoGTPasesaretheconverging in thewell-characterizedMDCKmodelsystems.Moreover, EphA kinasesinregulatingepithelialbranchingmorphogenesis adhesion (unpublisheddata). MDCK cellcolonies,possiblyreflectingenhancedcell–cell with theacquisitionofamorecompactmorphology 2000). Theweakenedcell–matrixadhesionwasassociated with whatwehavereportedearlierinPC-3cells(Miaoetal., stimulated MDCKcelladhesiontofibronectin,consistent EphA kinasesresultedinadecreasedbasalaswellHGF- vice versa.Wehaveobservedthatephrin-A1bindingto instance fromadispersedpatterntoanaggregatedone,or cell–matrix adhesioncanregulatethisphenotypechange,for tion ofbranchedtubularstructures.Alteredcell–celland of Rac1activationbyHGFinMDCKcells. identify theEphAeffectorproteinsthatmediatesuppression in thenervoussystem,furtherinvestigationisnecessaryto RhoA activation.Becauseephexinispreferentiallyexpressed tivation ofRac1signalingwhilesimultaneouslymaintaining thermore, ephrin-A1wasabletosuppressHGF-inducedac- atively regulatedepithelialbranchingmorphogenesis.Fur- similar torepulsiveguidanceofaxons,EphAactivationneg- one oftheneuritesbecomesanaxon.Here,wefoundthat tion (daSilvaandDotti,2002).Afterneuronalpolarization, was mixedwith100 Materials andmethods In summary,thereportheredescribesanovelfunctionof Cell dissociationisanearlyphaseofHGF-inducedforma- The JournalofCellBiology l 1MNa 2 l 10 HCO 3 DME/F12(GIBCOBRL),10 . 0.1NNaOHwas usedtoneutralizethepH. l 2.5mg/mltypeIcollagen(BDBiosciences)

Volume 162,Number7,2003 l 1MHepes,pH (CoolSNAP cf; verted microscope(modelDMIRE2;Leica)equippedwithadigitalcamera changed everyday.Branchingmorphogenesiswasrecordedusinganin- thesda, MD). Scion ImagesoftwarefromtheNational InstitutesofHealth(NIH;Be- al., 2000).Forquantitativeanalysis, banddensitiesweremeasuredusing immunoprecipitation andimmunoblot asdescribedpreviously(Miaoet ence of20ng/mlHGFfortheindicatedtimes,andthenwereprocessed The lysatewasclarifiedat14,000rpm4 hibitor cocktail(Sigma-Aldrich),andproteaseinhibitorsonicefor5min. or 1 adjustment ofgrowthincollagengel,20ng/mlrecombinanthumanHGF were gentlywipedoffwithQ-tips were fixedandstainedwithcrystalviolet.Cellsretainedontheupperside of 2–5 cated densitiesinPBSovernightat4 Ephrin-A1–Fc orFcwascoatedon6-wellclustercultureplatesattheindi- SOIL assay with 2 Cells platedoncoverslipswerechilleddowniceandthenincubated Immunofluorescence grown attheair–liquid interfaceon0.45- et al.,2001).Heterozygous E12.5kidneysweredissected andimmediately EphA2 Kidney organcultureandstaining Cells werestimulatedwith1 Stimulation, immunoprecipitation,andimmunoblot Nonidet P-40,100mMNaCl,10%glycerol,5MgCl cold PBSandlysedwithabuffercontaining50mMTris-HCl,pH7.4,1% After stimulation,subconfluentMDCKcellswerewashedoncewithice- GST pull-downassay assay asdescribedabove. detached withtrypsin-EDTAandprocessedforbranchingmorphogenesis Plus transfectionreagents(GIBCOBRL).24haftertransfection,cellswere Raf1-BXB togetherwiththatencodingGFP(3:1)usingLipofectAMINE™ About 40%confluentMDCKcellsweretransfectedwithplasmidencoding Transient transfection The Transwell(Costar)wascoatedbyspotting100ngFNin10 Cell migrationassay slides usingvector-shieldmountingmediumcontainingDAPI. loidin wasused.Afterthewashcycles,coverslipsweremountedon Fc for30min.Foractincytoskeletonanalysis,Texasred–conjugatedphal- 1 hatRTfollowedbyincubationwithFITC-conjugatedgoatanti–human min. Cellswerethenblockedwith2%goatserumand3%BSAinPBSfor were washedwithice-coldPBSandfixed3.7%PFAonicefor20 A1–Fc werephotographed. and theringsofcellsthathadscatteredontoimmobilizedFcorephrin- PFA for20minandstainedwithcrystalviolet.Coverslipswereremoved, placed ineachwell.After24–48hofculture,cellswerefixedwith3.7% were gentlypresseddownwithpipettetips.Uptothreecoverslips control mediumorsupplementedwith20ng/mlHGF.Coverslips cell-face-up ontothecoatedoruncoated6-welldishescontaining1.5ml plates. Whencellsreachednearconfluence,coverslipsweretransferred were detachedwithtrypsin-EDTAandplatedoncoverslipsin24-well in serum-freemediumcontaining0.1%BSA.Cellsuspension100 to 1ml.100- counted fromsixhighpowerfields. taining 20ng/mlHGFand1 was addedtotheupperchamberofTranswell.Thesamemediumcon- sides oftheTranswellwerethencoatedwith10 onto theundersideoffiltermembrane,andallowingtoair-dry.Both the beadsweresuspendedinSDS-PAGEsamplebuffer. plates andallowedtogelfor30minat37 with serum-freemediumandsuspendedinaconcentrationof10 MDCK cellsweredetachedwithtrypsin-EDTAandwashedthreetimes et al.,1999)at4 was incubatedwithGST-Rhotekin–orGST-PAK-CD–coupledbeads(Ren lower chamber.Afterovernightincubationat37 knockoutmice(lineKST085)were describedpreviously(Mitchell g/ml ephrin-A1–Fcwasaddedasindicated.Themedium g/ml ephrin-A1–FcorFcfor1h.Attheendofincubation,cells 10 4 cells/mlincollagengel,afterthevolumehasbeenadjusted

l aliquotsofthecellsuspensionweredispensedin96-well Photometrics C for30min.Afterwashingthreetimeswithlysisbuffer, ) . g/ml ephrin-A1–FcorFcwasaddedtothe g/ml ephrin-A1–Fcintheabsenceorpres- ® . Cellsmigratingthroughthefilterwere

C andwashedwithPBS.MDCKcells

C for5min.Clarifiedcelllysate C beforeaddingmedium.After

m Transwellsat37

g/ml collagenovernight.

C, cellsonbothsides

, phosphatasein-

6 C, 100%

cells/ml

l PBS

The Journal of Cell Biology Eph NomenclatureCommittee. 1997.Unifiednomenclature forEphfamilyrecep- Ethell, I.M.,F.Irie,M.S.Kalo,J.R.Couchman, E.B.Pasquale,andY.Yamaguchi. Davis, S.,N.W.Gale,T.H.Aldrich,P.C.Maisonpierre,V.Lhotak,T.Pawson,M. Davies, J.,M.Lyon,J.Gallagher,andD.Garrod.1995.Sulphatedproteoglycan is Dalva, M.B.,M.A.Takasu,M.Z.Lin,S.M.Shamah,L.Hu,N.W.Gale,andM.E. da Silva,J.S.,andC.G.Dotti.2002.Breakingtheneuronalsphere:regulation of Coulthard, M.G.,J.D.Lickliter,N.Subanesan,K.Chen,G.C.Webb,A.J.Lowry, in afixative(60mMNa nM PGE Na stained overnightat30 glutaldehyde, and0.02%NP-40for30min),washedwithPBStwice, K DME/F12 (Hepesand Cho, E.A.,L.T.Patterson,W.T.Brookhiser,S.Mah,C.Kintner,andG.R. Cheng, N.,D.M.Brantley,andJ.Chen.2002.TheephrinsEphreceptorsin Brinkmann, V.,H.Foroutan,M.Sachs,K.M.Weidner,andW.Birchmeier.1995. Boros, P.,andC.M.Miller.1995.Hepatocytegrowthfactor:amultifunctionalcy- Affolter, M.,S.Bellusci,N.Itoh,B.Shilo,J.P.Thiery,andZ.Werb.2003.Tube Adams, R.H.,andR.Klein.2000.Ephreceptorsephrinligands.Essentialme- References Accepted: 13August2003 Submitted: 3April2003 Cantley bytheNIH(DK54911). Army (DAMD17-99-1-9019), theNIH(CA96533,CA92259),andtoL.G. fusion proteinpreparation. Rhotekin pull-downassayandtoNancyLiufortechnologicalassistancein ases. WearegratefultoDr.MarthaKonieczkonskiforadviceintheGST- W.JamesNelsonforMDCK cellsinduciblyexpressingmutantRhoGTP- Dr. We thankDr.MichaelE.Greenbergforanti-phospho-PAKantibody,and humidity, and5%CO on adigitalcamera. Laboratories) wasadded,andtheimagesofwholekidneywerecollected with PBSTover3h,adropofDAPI-containingmountingsolution(Vector X–conjugated anti–rabbitantibodiesinBSfor2hat37 overnight, andthenincubatedwithFITC-conjugatedanti-mouseRed- mouse anti-pan-cytokeratin(Sigma-Aldrich)inBSfor2hat37 bated with3 solution (BS;2%BSAand1%goatseruminPBST).Thekidneyswereincu- washed withPBS/0.1%Tween20(PBST),andblockedfor3hinblocking In brief,culturedkidneyswerefixedwith100%methanolfor15min, performed asdescribedpreviously,withmodifications(Choetal.,1998). tomycin, 1 4 Fe(CN) For X-galstaining,afterovernightculturekidneyswerefixedfor30min This workissupportedbygrantstoB.WangfromDepartmentofthe Staining withantibodiesagainstPax-2andcytokeratinwasessentially 2 HPO tors andtheirligands, theephrins. 31:1001–1013. 2001. Ephb/syndecan-2signalingindendritic spinemorphogenesis. Science. tyrosine kinasesthatrequiremembraneattachment orclusteringforactivity. Goldfarb, andG.D.Yancopoulos.1994. 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L -glutamine) with45mMNaHCO 2 2 , inserum-freeIdealExplantMediaconsistingof HPO 2 C withX-galinastainingsolution(60mM PO 4 4 , 40mMNaH , 2mMMgCl Development. Trends Cardiovasc.Med. Development. Cell. Cell. 103:945–956. 90:403–404. Nat. Rev.Neurosci. 13:75–85. J. CellBiol. 2 125:803–812. PO 2 , 1mMK 121:1507–1517. 4 , 1%formaldehyde,0.2% 131:1573–1586. 3 10:183–188. , penicillin-strep- 3 C. Afterwashing Fe(CN) 3:694–704. Growth Fac- C, washed 3 , 1mM Neuron. 3 g/ml Dev. , 70 Piscione, T.D.,and N.D.Rosenblum.2002.Themolecular controlofrenal Penzes, P.,R.C.Johnson,R.Sattler,X. 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