usrt n yokltlatnognzr xrsino a of Expression organizer. RPTP actin in Src mutant cytoskeletal major cortactin phosphomimetic a a cortactin, and of substrate phosphorylation RPTP tyrosine Mechanistically, appropriate for engagement. E-cadherin of isine nvriyo uesad t ui,Bibn 02 Australia. 4072, Brisbane Lucia, St. Queensland, of University Bioscience, [email protected]) 3M5, M4N ON Toronto, Avenue, Toronto, Bayview of Canada. 2075 University Institute, The Research Immunology, Sunnybrook of Maaløes Department Denmark. Ole address: N, Copenhagen, *Present Copenhagen of 2200 University 5, Sciences, Vej Health of Faculty Sciences, France. 13, Cedex Ba CNRS, 7216 at Truffi cortactin Marta of phosphorylation c-Src-mediated to engagement RPTP ARTICLE RESEARCH 2420 2014 February 10 Accepted 2013; May 2 Received § 3 1 Me 2010; the Tepass, with and interaction (Harris its cytoskeleton and actin E-cadherin with of associated binding homophilic is through adhesion the which intercellular mediate of They pathologies. dysfunction multiple organization, tissue determinants of fundamental are junctions epithelial Cadherin-based INTRODUCTION c-Src Cortactin, Tyrosine phosphatase, junctions, Adherens E-cadherin, WORDS: KEY c-Src. through intercellular phosphorylation to tyrosine engagement cortactin at E-cadherin RPTP homophilic linking that by accumulation signaling indicate mediated E-cadherin findings These and contacts. F-actin rescues effects These RPTP for density. role a F-actin with correlate peri-junctional E-cadherin defective and RPTP by manifested RPTP accumulation E-cadherin. as of integrity, Loss to junction contact, culture. adherens proximity cyst three-dimensional cell–cell for in and molecular of architecture adhesion cadherin-dependent in time appropriate for is the required at it junctions where or receptor adherens identity the that specific epithelial report their RPTP we about Here, tyrosine PTP known junctions. epithelial is Protein at little integrity. functions but adhesion junctional kinases, cadherin-based signaling, to control and tyrosine contribute also that activates (PTPs) tissue Src, phosphatases phosphorylation. E-cadherin as of tyrosine of such by determinants binding regulation fundamental Homophilic to subject are organization, junctions Epithelial ABSTRACT fSnaoe 4SineDie4 1 ee ,175 Singapore. 117557 2, Level S1A 4, Drive Science 14 Singapore, of lh .Yap S. Alpha uhr o orsodne([email protected]; correspondence for Authors Biomedical of Department and Centre Innovation and Research Biotech Universite 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,22–42doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ` ´ rsn drs:Cnr o iIaigSine,Ntoa University National Sciences, BioImaging for Centre address: Present ai ieo,Sron ai Cite Paris Sorbonne Diderot, Paris ˆ a a ietLmrk ae74,3 u He Rue 35 7042, Case Lamarck, timent 1 ,Ve hmngn name gene (human 2 2 iiino oeua elBooy nttt o Molecular for Institute Biology, Cell Molecular of Division otoseihla deesjntos ikn E-cadherin linking junctions, adherens epithelial controls ulem .Gomez A. Guillermo , rnqeDubreuil ´ronique a nclua c-r ciaina sites at activation (c)-Src cellular in ´ pgntc n elFt,UMR Fate, Cell and Epigenetics , PTPRA 1,§ a dpee el partially cells -depleted unLiang Xuan , 2 ´ le a ` n a Sap Jan and eBin -50 Paris F-75205 Brion, ne srcutdto recruited is ) otoscadherin- controls ` ee l,2006). al., et ge a srequired is a impairs 2 ahleVacaresse Nathalie , a 1,3,§ is 05.A h aetm,cntttvl cievrl()Scalso (v)-Src viral active constitutively time, al., same et the Takahashi At 1998; significantly al., 2005). actin et and al., activity (Calautti adhesion organization (c)-Src et cytoskeleton homophilic distribution, cellular (McLachlan E-cadherin junctional perturbs of regulate interface and Inhibition cell–cell transmission properties. signal the binding in homophilic participate at 2007), to response E-cadherin in 2008). activated of Nelson, are family 2007; (Src SFKs) family Yap, Src kinases; the and of kinases McLachlan to tyrosine accumulated 1985; Locally contribute al., protein proteins, et 2005; Yap, involving Maher G and (Braga turnover those small and remodeling assembly, as junction and such phosphorylation pathways, tyrosine signaling Diverse Adr ta. 06 seoe l,21;Fcse l,1996; RPTP al., phosphatase SFKs. et junctional control Fuchs to in 2010; shown junctions been al., PTPs has epithelial et PTPs Espejo of cell-contact-associated 2006; function Mu for al., and et role integrity (Anders studies the a epithelial some controlling Although the reported disrupts Src 2011). Yap, E-cadherin-activated in have treatment and impairs (McLachlan of pervanadate activity interestingly, signaling Inhibition role and, by (PTP) integrity. a junctions activity to junction phosphatase point intercellular PTP tyrosine also kinase of on data maintenance protein discrete effects Recent negative of 2005). for or number (Roskoski, positive a activity with of residues, phosphorylation tyrosine through to regulation as so force. locale, mechanical cellular or contact such this signals, cell–cell received as at locally by affected regulators are SFKs necessary SFK how understand it the makes identify junctions cadherin-based to complexity at both The functions 2007). SFK can al., et of (McLachlan signaling strength signal SFK SFK on depending the Clearly, function, E-cadherin affect contact 1987). negatively and positively impairing Nelson, 1991; contacts, and al., et Volberg Warren cell–cell 1993; al., et of (Behrens extension and formation integrity the disrupts hog t r-ciaigatvt TeprWlse l,2010; al., et (Tremper-Wells activity Src-activating its through RPTP to of related al., behaviors (Labbe et cell ways affect complex Ye in to oncogenicity 2005; recognized al., now are et PTPs (Bodrikov 2008). neurons at cell in neuronal various molecules with transduction function 1999; adhesion to al., and force 2003), et al., Su et Wichert and 2006; von integrin- al., motility et as (Jiang linkages cell integrin–cytoskeleton such spreading, processes, dependent adhesion-mediated fibroblastic is al., in and RPTP et cells integrity. epithelial their Su for in 1999; required junctions al., intercellular to et localizes (Ponniah 1999), c-Src of regulator physiological le ta. 99 ht ta. 07,nn fteidentified the of none 2007), al., et Sheth 1999; al., et ¨ller ee erpr httetasebaercpo rti tyrosine protein receptor transmembrane the that report we Here, to subject are kinases Src mechanisms, regulatory other Among a a nuefbols rnfrain presumably transformation, fibroblast induce can 3, ,Fben Nigon Fabienne *, a hmngn name gene (human a saraykont participate to known already is ´ ta. 02.Overexpression 2012). al., et 1 PTPRA iwPn Han Ping Siew , ,awell-established a ), 2, ` ,

Journal of Cell Science ersnaieo he needn xeiet sshown; is experiments independent three of representative 10 bar: Scale RPTP Venus-tagged expressing cells A431 10 of bar: microscopy Scale video establishment; time-lapse contact using cell–cell by RPTP followed and was (PH) formation contrast contact Intercellular contacts. cell–cell atr facmlto fRPTP striking a of revealed This accumulation lines. of cell between epithelial protein pattern various fusion in a 2002) expressed RPTP we human context, epithelial the n eil-soitdmmrn rti- VM3 eaiecnrl;adwti admGPCer uinpoen aaso h mean the show Data protein. fusion GFP–Cherry tandem a within and control); negative (VAMP3, protein-3 membrane RPTP vesicle-associated of A and shown. distribution are the sections illustrates optical Single arrow) microscopy. the confocal of by analyzed direction and the RPTP purple) (both (following ZO-1 contacts (D) individual or E-cadherin of (C) for immunolabeled were (green) RPTP Venus-tagged hn ta. 92.Although 1992). al., et Zheng ARTICLE RESEARCH i.1 RPTP 1. Fig. RPTP of localization intracellular E-cadherin the to investigating proximity start molecular in To is and cells, epithelial RPTP RESULTS e nih noterlvneo eetrPP o h rprisof epithelia. properties neoplastic the for and provides PTPs normal receptor activation of relevance c-Src the E-cadherin-dependent into insight new of the mediator in function a RPTP biological Identifying as investigated. Krndija cell been its 2011; hardly 2013), al., has al., context et epithelial et Huang Meyer 2010; 2000; al., al., et et (Ardini invasiveness or RPTP that indicate a a n -ahrn()o O1()a ucin ( junctions at (D) ZO-1 or (C) E-cadherin and oaie ocdei-ae neclua ucin in junctions intercellular cadherin-based to localizes a m .()FE fiinymaueet nCc- el ewe -ahrnand E-cadherin between cells Caco-2 in measurements efficiency FRET (E) m. oaie ttecdei-ae neclua ucin neihla el,weei si ls rxmt oE-cadherin. to proximity close in is it where cells, epithelial in junctions intercellular cadherin-based the at localizes a n h loecn rti eu Ngie al., et (Nagai Venus protein fluorescent the and a a nmnlyrclue fCc-,A3 n C7cls cl as 20 bars: Scale cells. MCF7 and A431 Caco-2, of cultures monolayer in a locnrlcrioatmrgnssand/ tumorigenesis carcinoma control also can a Vnseiloecneiae eeaqie vr 0mnts n eetdtm-onsaeson rohas RPTP Arrowheads, shown. are time-points selected and minutes, 10 every acquired were images epifluorescence –Venus nvitro in a m and .(,)Clclzto nlsso RPTP of analysis Colocalization (C,D) m. tcl–eljunctions cell–cell at nvivo in n 5 3 w needn xeiet)wsaaye ycluaino ero’ orlto ofiin (r). coefficient correlation Pearson’s of calculation by analyzed was experiments) independent two 23, , observations 5cl–elcnat.**** contacts. cell–cell 75 a in a aea ebae(ero’ orlto ofiin r coefficient RPTP correlation contrast, RPTP (Pearson’s membrane that ZO- lateral to found respect with We detected 1. was localization different a whereas ouigo deesjntos akdb E-cadherin 1D). (Fig. by ZO-1 by marked marked complexes, junctions, RPTP junctions, tight between overlap junctional on Extensive adherens and with 1C), (Fig. on colocalization focusing its investigated cells 1B; local 1). (Fig. at Movie two interface material remained cell–cell contact supplementary the subsequently from along of it accumulating that sites, stages protrusions contact and another, early lamellipodial one the touched when by from contacts RPTP sites formation, cell–cell that adhesion of noticed intercellular formation localization, We this of imaging. of process kinetics time-lapse the the document followed to we order In 1A). (Fig. ocaatrz h pteillclzto fRPTP of localization epithelial the characterize To P , a .01 s o significant. not ns, 0.0001; ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal ihEcdei n O1 ao2clsepesn RPTP expressing cells Caco-2 ZO-1. and E-cadherin with m a .()Tm-as nlsso RPTP of analysis Time-lapse (B) m. b n -ahrn()o RPTP or (C) E-cadherin and ctnn(oiiecnrl;Ecdei n RPTP and E-cadherin control); (positive -catenin a n O1ceryocpe itntprin of portions distinct occupied clearly ZO-1 and a ooaie ihEcdei tthe at E-cadherin with colocalized a n -ahrnwsobserved, was E-cadherin and a n O1() vra between Overlap (D). ZO-1 and a uigteetbihetof establishment the during a a erie to recruited was A eeto of Detection (A) 6 a ... a s.e.m., E-cadherin ; 5 z a ai view -axis .) by 0.7); a Phase- . a –Venus a nearly in 2421 ,we

Journal of Cell Science nrytase FE)btenbt oeue nlv Caco-2 E-cadherin/ live in the molecules using both between cells, resonance (FRET) fluorescence transfer measured energy we Subsequently, complexes. xeiet nCc- el eeldwa 10 relative ( (130% significant weak revealed but cells control) Caco-2 to Co-immunoprecipitation another. in one experiments to proximity molecular in r correlation the (Pearson’s membrane the ARTICLE RESEARCH 2422 RPTP 2. Fig. E- between that to similar was that and cadherin junctions RPTP between at efficiency E-cadherin FRET and a al., et revealed (Ferrari analysis respectively This controls, 2012). negative and positive as pairs within E-cadherin of enrichment shown) anti-RPTP not data experiments; ncdw (RPTP knockdown cuuae tahrn ucin,wihaecaatrzdb the E-cadherin. by of characterized are presence which junctions, adherens at accumulates uliaesandwt AI(ht) cl a:10 bar: Scale (white). DAPI RPTP with 20 stained and are bar: nuclei control Scale both (white). DAPI in with lumina stained multiple n are with Nuclei or (purple). F-actin morphology for normal immunofluorescence with by obtained were lumina multiple with RPTP 10 of Immunofluorescence (B) RPTP experiments). of independent quantification Densitometric cells. RNAi-treated and utr naMtie arx aaso h mean the show Data matrix. Matrigel a in culture 5 ete netgtdwehrRPTP whether investigated then We 0) E al tgs(4husad7 or)o ytfraini ohcnrladRPTP and control both in formation cyst of hours) 72 and hours (24 stages Early (E) 100). m .()Cssfre fe w ek fcluei arglmti.Rpeettv ofclpcue fcsswt omlmrhlg snl central (single morphology normal with cysts of pictures confocal Representative matrix. Matrigel a in culture of weeks two after formed Cysts (C) m. z ai iw.Teedt eelta RPTP that reveal data These views. -axis a a muecmlxsa oprdwt oto IgG control with compared as complexes immune elto niisCc- opoeei,adrslsi bratpstoigo h pclcmateti Dcultures. 3D in compartment apical the of positioning aberrant in results and morphogenesis, Caco-2 inhibits depletion b ctnn(i.1E). (Fig. -catenin a hN1adsRA)o oto cr hN)Cc- el eegnrtdb etvrlifcin A etr ltaayi fRPTP of analysis blot Western (A) infection. lentiviral by generated were cells Caco-2 shRNA) (ctrl control or shRNA2) and shRNA1 b ctnnadE-cadherin/VAMP3 and -catenin P 5 .2 nnn independent nine in 0.027 a 5 n -ahrnmgtbe might E-cadherin and 6 .) sas hw by shown also as 0.3), m ...(he needn experiments, independent (three s.e.m. .()Qatfcto fcsswt eta igeaia ufc rwt utpeaia ufcsatr7 or of hours 72 after surfaces apical multiple with or surface apical single central a with cysts of Quantification (F) m. a a a eesnraie to normalized levels gen ncnrladRPTP and control in (green) specifically a kokonclsi hw stemean the as shown is cells -knockdown a h w ol fRPTP RPTP of residual shRNA. of levels pools control non-targeting two a RPTP The expressing cells of had We Caco-2 knockdown cells that control 2008). cells lentiviral-induced Caco-2 polarized al., stable of pools et highly undergone between (Jaffe formation assemble of cells. cyst junctions cells compared epithelial consisting Caco-2 epithelial by matrix, structures interconnected by Matrigel ordered three- a typical generated into in cysts, of cultivated structures formation When the (3D) for required dimensional is it and whether whether assess Halbleib To 2009). 2005; RPTP Takeichi, and (Gumbiner, Nishimura 2006; morphogenesis Nelson, and tissue epithelial of integrity determinants important are in junctions Adherens compartment apical the cultures of 3D positioning aberrant in results RPTP a tblnadcmae ihcnrli hw stemean the as shown is control with compared and -tubulin a a n a a ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal elto opoie ao2mrhgnss and morphogenesis, Caco-2 compromises depletion 5 kokoncls uliaesandwt AI(ht) cl bar: Scale (white). DAPI with stained are Nuclei cells. -knockdown kokoncls muotiigfratnwspromd(purple); performed was actin for Immunostaining cells. -knockdown sivle neihla raiain eexamined we organization, epithelial in involved is 0) * 100). P , .5 ** 0.05; a P rti Fg AB.Cs omto was formation Cyst 2A,B). (Fig. protein , a 6 .1 *** 0.01; kokonclsrtie different retained cells -knockdown ...(he needn experiments, independent (three s.e.m. P , 0.001. m .()Qatfcto fcysts of Quantification (D) m. 6 ...(five s.e.m. tbeRPTP Stable a ue)or lumen) ncontrol in a and a

Journal of Cell Science oprmn ihrsett h rwn cyst. growing the that apical to the and respect of cells, positioning with aberrant Caco-2 compartment from in results probably morphogenesis effect cyst this defective to leads RPTP contrast, By progressively panels). should upper lumen 2E, the (Fig. where at structure, open maintained the was of region stage apical center two-cell single the the the from grew, cyst cysts surface a nascent As the cells, onward. apical of control middle precursor the in in expected, appeared F-actin-marked As points seeding). placed time after early symmetrically at hours examined 72 also and RPTP was (24 cysts of of effect development the the lumina, multiple in resulted h ecnaeo yt ihmlil pclsrae was that RPTP surfaces found that apical conclude we We multiple 2F). culture, (Fig. with (22%) RPTP of controls cysts in higher hours of multiple 72 significantly or percentage single after multiple with the grew, cysts surfaces cysts Counting as panels). apical structure and, lower same the cyst, 2E, within (Fig. detected nascent respect were the with patches membrane surface of apical apical center their the of shift to lateral a displayed often 53% to 37% from lumina multiple with 2C,D). (Fig. cysts of population pnn nvrospstoswti h yt Quantification cyst. lumina the multiple RPTP within with that positions abnormal, various revealed appeared in often opening RPTP control more cysts, of majority single-lumen cysts the developed central Although efficiently a F-actin. of cells by emergence revealed through culture, as of lumen, days 14 after assessed ARTICLE RESEARCH it n vrg ie nest fEcdei tiiga the the RPTP at 4C, staining measured Fig. E-cadherin in we shown of As intensity contacts. alterations, pixel cell–cell such average discontinuous reducing and a quantify width cells, with To interface, control cell–cell distribution. in lateral staining the throughout linear and Contrasting membrane. RPTP sharp suggesting cell 4A), the the (Fig. at with E-cadherin distribution of it protein level although in total defects panels), the left affect RPTP 4B, not (Fig. did overall staining E-cadherin by reduced perturbed cells Caco-2 siRNA knockdown confluent transient of of Transfection effect RPTP the of analyzed subsequently We monolayers cell epithelial RPTP cells control time, this At RPTP contrast, 3A). By (Fig. aggregation. hours RPTP calcium-dependent 4 underwent for or shaking al., with et Control (Nagafuchi is adhesion that 1987). process E-cadherin a homophilic suspension, by in promoted aggregate we to possibility, cells this Caco-2 RPTP address isolated structures of to knockdown step whether 3D first determined a As E-cadherin-based aberrant of junctions. functions adherens and of establishment the regulating appearance in RPTP RPTP the from of resulting that colocalization speculated the Given aggregation RPTP eue iecmae ihcnrl Fg B.W conclude We suspension. in 3B). aggregation (Fig. intercellular controls RPTP of with aggregates that formed compared or size isolated remain reduced to tended cells depleted ogi nih notemcaimb hc RPTP which by mechanism the into insight gain To a a a sncsayfrteitgiyo elcl otcsin contacts cell–cell of integrity the for necessary is intercellular cadherin-based impairs depletion eesidcdabodnn fEcdei staining E-cadherin of broadening a induced levels a a njntoa opooyi ooae culture. monolayer in morphology junctional on ncdw opoie E-cadherin-mediated compromises knockdown a a elto ih elc oefrRPTP for role a reflect might depletion elto infcnl nrae the increased significantly depletion , a a dpee el eeincubated were cells -depleted 0 Fg A.RPTP 4A). (Fig. 90% kokoncls(5)ta in than (55%) cells -knockdown a a ihEcdei,we E-cadherin, with fetdteaiiyof ability the affected a a ncdw was knockdown a ncdw on knockdown dpee cells -depleted a a a knockdown a a eesin levels depletion depletion -depleted a a - hN)ageae nteasne(ET)o rsne(+Ca presence or (+EDTA) absence the in aggregated against shRNA) shRNA Phase- expressing (A) cells RPTP minutes. Caco-2 human 90 transduced with after of incubated formation images EDTA, aggregate contrast of for presence analyzed in and isolated shaking were cells Caco-2 depleted hwdrdcdFatndniya otcs(i.4) Both mouse RPTP 4D). siRNA-resistant by (Fig. expressing by RPTP caused rescued contacts distribution be could F- at F-actin depletion and of RPTP density E-cadherin ring cells, F-actin aberrant intense between reduced also an interface showed showed the was at cells junctions, RPTP actin control adherens Whereas by the panels). perturbed at in E-cadherin greatly signal discontinuous with more linked a by staining replaced RPTP was continuous the cells along that control cell–cell measured revealed in at also signal, contacts, E-cadherin intensity of intercellular staining distribution a average The and contacts. in staining E-cadherin reduction broader significant significantly with associated i.3 RPTP 3. Fig. ucin n h neligatncytoskeleton. intercellular actin E-cadherin-based underlying the of and organization junctions RPTP and Fig. that integrity material indicate data the (supplementary our MCF7 together, Taken were line S1). defects cell junctional carcinoma Similar breast 2006). RPTP al., when et observed suggesting Otani 4E), 2011; Fig. RPTP Yap, panels; with of right compared 4B, loss straight (Fig. that cells less control and of wavy those more appeared junctions RPTP following boundaries; intercellular RPTP of knockdown by impaired be to not seemed junctions eeaaye) * analyzed). were 20 bar: Scale bevdb tiigwt O1(i.4,rgtpnl) However, panels). right 4B, (Fig. RPTP ZO-1 with staining by observed mean otatn ihteeosrain,teitgiyo tight of integrity the observations, these with Contrasting 6 a a a ...(he needn xeiet,i aho hc iefields five which of each in experiments, independent (three s.e.m. dpee el.Tepr-ucinlatncytoskeleton, actin peri-junctional The cells. -depleted ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal Fg 7C). (Fig. elto eue ucinllnaiya h apical the at linearity junctional reduced depletion a a m B uniiaino grgt ie aaso the show Data size. aggregate of Quantification (B) m (RPTP srqie o elaggregation. cell for required is P , .5 ** 0.05; a a eue ucinltnin(caha and (McLachlan tension junctional reduces hN1 hN2 ranntreigsRA(ctrl shRNA non-targeting a or shRNA2) shRNA1, a a nce onb sn iN nthe in siRNA using by down knocked was P , 0.01. a ncdw Fg B middle 4B, (Fig. knockdown a oto n RPTP and Control ncdw,cell–cell knockdown, a kokoncells -knockdown a srqie for required is ++ fcalcium. of ) a 2423 - a ,as a

Journal of Cell Science non-significant. linearity. of loss 10 showing bar: junctions Scale arrows, E-cadherin; of distribution eaiet h oto treidpnettasetos o aho which of each for transfections, independent (three . mean control the the show to by Data relative assessed staining. linearity ZO-1 junctional of F-actin the analysis peri-junctional of the Quantification E-cadherin of (E) of Quantification intensity. spread (D) the junctions. and across junctions, signal along signal E-cadherin of RNAi- and control in panel) (right Single ZO-1 cells. or treated panel) (left (middle E-cadherin F-actin for panel), Immunofluorescence (B) experiments). independent to normalized iN) n nlzda 2husatrtaseto.()Wsenblot Western RPTP (A) of transfection. after RPTP hours of 72 analysis at analyzed and siRNA), RPTP against monolayers. cell EERHARTICLE RESEARCH 2424 RPTP 4. Fig. 0jntoswr nlzd.* analyzed). were junctions 30 a mdl ae)adEcdei rgtpnl expression panel) (right E-cadherin and panel) (middle a a m a .()Qatfcto fteitniyadheterogeneity and intensity the of Quantification (C) m. tblni hw stemean the as shown is -tubulin sncsayfrteitgiyo ucin nepithelial in junctions of integrity the for necessary is (RPTP a ao2clswr rninl rnfce ihsiRNA with transfected transiently were cells Caco-2 nbt odtos(etpnl.Dnioercquantification Densitometric panel). (left conditions both in z ofclpcue r hw.Arwed,abnormal Arrowheads, shown. are pictures confocal a iN)o ihsrmldcnrlRA(ctrl RNA control scrambled with or siRNA) P , .5 ** 0.05; P , 6 .1 *** 0.01; 6 ...(tlatthree least (at s.e.m. ...adaeexpressed are and s.e.m. P , .0;ns, 0.001; Kvc ta. 02) el i o pedo PDL-coated on spread not did cells 2002b), al., et (Kovacs 99 ona ta. 99 hn n hloa,2001; Shalloway, and Zheng al., 1999; et RPTP (Su al., settings of extent other et an depletion in Ponniah by seen 5A,B, pY419-Src, that Fig. 1999; to of in comparable fraction the (50–60%) the shown with reduced correlates As loop significantly which state. of activation against phosphorylation antibody an active c-Src, an checked (pY419-Src), in used 419 residue we we 2008). purpose, tyrosine contacts, al., this phosphorylated cell–cell et For cells. Vacaresse at epithelial 2005; present RPTP al., is whether Zheng et 1999; c-Src Bodrikov al., et Because 2000; Ponniah multiple 1999; al., inhibitory in al., their et et dephosphorylate SFKs (Su to of residue ability C-terminal its activator to physiological owing RPTP contexts, established al., Indeed, 2011). et Yap, an and (Burns to McLachlan is 1999; PTPs signaling al., structures certain and et Su of activity these 1994; ability kinase the of modulate reflect positively might maintenance inhibition phosphorylation PTP the tyrosine upon Decreased is for 2011). Yap, activity and and cell– (McLachlan PTP at content junctions, that phosphotyrosine balanced cell indicates a keeping vanadate for and required using assembly Broad-spectrum inhibition the complexes. PTP both adhesive cadherin-based for of crucial stability is junctions phosphorylation epithelial Tyrosine at phosphorylation tyrosine controls RPTP ciaini pteilcls epoeddt etteinvolvement the RPTP test to proceeded of we cells, epithelial in activation RPTP unknown. that yet demonstrated as findings remains our however, process Because 2003); this Kovacs, of and mechanism molecular Yap the 2007; other al., and et (McLachlan c-Src SFKs activates adhesion cell–cell Cadherin-dependent RPTP total RPTP of and distribution control the between compared phosphorylation tyrosine then protein We 2005). Roskoski, xeto pedn fcnrladRPTP and the control compared we of spreading, spreading cellular of cadherin-based the extent in from role absent a was it RPTP RPTP whether whereas of Ecad–Fc, edge observed leading on We spreading minutes. 90 cells for Ecad–Fc with RPTP coated substrata to 2003). of Kovacs, contact that cell–cell and from during (Yap exchanged ligation are that homophilic signals to E-cadherin juxtacrine us other of allowed approach effect of This the moiety 1996). Fc isolate al., the et to (Adams fused (Ecad–Fc) E-cadherin consistingIgG human ligand of recombinant ectodomain a the using at of cells E-cadherin isolated activated of homophilically surface we the end, this To adhesion. RPTP phosphorylation. RPTP tyrosine transient consequently, that promote where, reveal contacts, – intercellular findings at These activation 5E,F). c-Src (Fig. by RPTP c-Src Y419-phosphorylated contacts of intercellular accompanied in fraction at the was decrease reduced knockdown local phosphorylation a tyrosine contacts junctional cell–cell at RPTP phosphorylation tyrosine following revealed in decrease immunostaining local a Anti-phosphotyrosine cells. depleted nEa–ccae ufcso ufcscae ihtenon- the poly- with coated ligand surfaces adhesive or signaling surfaces Ecad–Fc-coated on oto n RPTP and Control a a a a srqie o oi r ciiyi pteilcls and cells, epithelial in activity Src tonic for required is ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal srqie o -ahrnmdae r activation Src E-cadherin-mediated for required is otiue oScatvt neihla el,particularly cells, epithelial in activity Src to contributes pcfclyacmltn ttelaigeg fcontrol of edge leading the at accumulating specifically a neryrsosst -ahrndpnetcell E-cadherin-dependent to responses early in a a cuuaina cielaigegsmgthave might edges leading active at accumulation a elto ih fetiskns ciiyin activity kinase its affect might depletion ncdw Fg CD.Ti eraein decrease This 5C,D). (Fig. knockdown a kokonclswr loe oadhere to allowed were cells -knockdown a kokoncls(i.6) Wondering 6A). (Fig. cells -knockdown D lsn PL.A expected As (PDL). -lysine a a kokoncells -knockdown sesnilfrSrc for essential is a slkl to likely is a a a a -

Journal of Cell Science b 5)i pedn nEa–co RPTP of Ecad–Fc on decrease spreading the change in fibronectin not 35%) on did (by but cells integrins, blocking fibronectin-binding Caco-2 additional of reflecting of S2C), Fig. spreading material (supplementary the impaired cRGD fet uigsraigo cdF,w sdteintegrin- the used integrin-dependent we by Ecad–Fc, an interference (cRGD), on GPenGRGDSPCA out peptide spreading blocking rule Wichert (von during To vitronectin effects 2003). on spreading al., cells et of edge leading the EERHARTICLE RESEARCH el rnfce ihtels fetv hN2(i.6) We 6C). (Fig. shRNA2 RPTP effective shRNA that less showed effective the previously most with RPTP of transfected the that of cells with to area transfected relative (RPTP cells surface reduced for significantly total controls was the cells because knockdown defect, spreading RPTP Strikingly, E- Ecad–Fc. an by inhibited S2A,B). Fig. was on material to it (supplementary Spreading antibody as cadherin-blocking 6B). adhesion cadherin-dependent, (Fig. upon was lamellipodia the Ecad–Fc prominent spreading by revealed of as rapid E-cadherin, appearance recombinant in with coated engaged substrate but surfaces, r ncnrlclsrvae ciecSca h E-cadherin-driven the at c-Src pY419- active revealed for cells immunostaining control in cadherin, Src initial recombinant after to minutes al., 90 RPTP adhesion At et of activation. effect Src (McLachlan E-cadherin-mediated the on SFKs monitored of therefore We activation 2007). the stimulates substrate material supplementary 6C; (Fig. S2E). controls Fig. of that with compared ete tde h novmn fRPTP of involvement the studied then We rvossuishv hw htahso oE-cadherin to adhesion that shown have studies Previous a hN1,wt ednytwrsardcdae for area reduced a towards tendency a with shRNA1), a a elto a soitdwt a with associated was depletion soitswith associates a a kokoncells -knockdown a a nsraigon spreading in v v b b a 3 3 knockdown antagonist. nernat integrin a nefrn hN2cue ednytwrsdecreased provide E-cadherin. towards observations of RPTP downstream effectively our tendency that together, less Taken a evidence the 6D,E). whereas (Fig. caused staining controls, shRNA2 lamellipodia by of revealed cadherin-based interfering that as at with compromised, staining compared was sites pY419-Src these decreased at c-Src activate el ie tcaatrzn neato atesadsubstrates and partners interaction RPTP characterizing for at the aimed tyrosine observed an cells We requires in 2009). cortactin phenotype al., of et presence this E-cadherin (Ren cortactin junctional of of affects phosphorylation rescue depletion Ren and 2004; Cortactin al., accumulation, 2009). et E-cadherin- it (Helwani al., Src that for E-cadherin-activated et and of reorganization, target necessary actin a signaling is and is formation the multiple that contact cortactin shown mediated the have protein we studies, of actin-binding previous In which RPTP junctions. mediates epithelial c-Src determine by triggered to events wished We phosphorylation tyrosine cortactin RPTP aelpda ycnrs,teaiiyo RPTP of ability the contrast, By lamellipodia. olwdb nipopoyoieimnbotn,revealed immunoblotting, RPTP in phosphorylation immunoprecipitation, anti-phosphotyrosine tyrosine cortactin reduced Cortactin by cortactin. followed of phosphorylation cortactin between interaction functional RPTP the and investigate to us led efrtdtrie hte RPTP whether determined first We a ** w xeiet sson 0 ucin eeaaye.* analyzed. were junctions 200 shown; the is of mean experiments intensities the two of as ratio represented the signals, show two Data junctions. along intensity 10 with transfected RPTP transiently against c-Src cells siRNA activated Caco-2 and in (green) purple) c-Src (pY419-Src, total for Immunostaining which (E) of each for experiments, ucin.Dt hwtemean the along show intensity Data signal junctions. phosphotyrosine of Quantification (D) control in RPTP (pY) stable phosphotyrosine and total for Immunofluorescence (C) -r ees aaso h mean the total show to Data relative levels. levels c-Src pY419-Src of quantification Densitometric (B) RPTP n oa -r cSc nttlcl xrcso ao2cells Caco-2 of RPTP extracts human cell against total shRNAs in expressing (c-Src) c-Src total and epithelial the at phosphorylation junctions. tyrosine controls and cells, RPTP 5. Fig. ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal euae ahrnbsdjntosb affecting by junctions cadherin-based regulates P a , m a .()Qatfcto frltv Y1-r esscScsignal c-Src versus pY419-Src relative of Quantification (F) m. splmnaymtra i.S) hs observations These S3). Fig. material (supplementary .1 *** 0.01; teihla junctions. epithelial at a hN2 ranntreigsRA(tlshRNA). (ctrl shRNA non-targeting a or shRNA2) A etr ltaayi fatvtdcSc(pY419-Src) c-Src activated of analysis blot Western (A) P a , srqie o oi r ciiyi epithelial in activity Src tonic for required is a 0.001. a kokoncls cl a:10 bar: Scale cells. -knockdown srqie o h ciaino Src of activation the for required is a rwt cabe oto N.Saebar: Scale RNA. control scrambled with or nvitro in 6 § uldw sa rmCaco-2 from assay pull-down ...(he independent (three s.e.m. 0jntoswr analyzed). were junctions 20 6 a ...(he experiments). (three s.e.m. 6 elto fet tyrosine affects depletion ... ersnaieof representative a s.e.m.; a dpnetcnrlof control -dependent a (RPTP a hN1clsto cells shRNA1 a m a m. -knockdown hN1or shRNA1 P , 2425 0.05;

Journal of Cell Science EERHARTICLE RESEARCH infcnl nrae ucinlEcdei n -ci in F-actin and 2426 E-cadherin junctional increased significantly RPTP of effector downstream recovery cortactin 7A). total (Fig. whereas comparable controls, was with compared as cells ntelvl fjntoa -ahrnadFatnidcdby induced F-actin and junctional reduction E-cadherin the the on junctional effect of of no RPTP levels had measure variant the 3YF in a the density or as the cortactin type and junctions tagged signal along fluorescently RPTP E-cadherin a F-actin of or of intensity – the 2009) monitored al., with RPTP et replaced variant were mouse (Ren phosphomimetic residues acid) tyrosine a three aspartic these or Y486, which phenylalanine) and in in (3YD, Y470 to – (3YF, Y421, variants mutated residues, variant cortactin were tyrosine of principal non-phosphorylatable number three a which a RPTP with cortactin, RPTP the together wild-type with by or how cells affected alone Caco-2 induced either examined cortactin transfected phenotype we of we Accordingly, mutants junctional possibility, phosphorylation the this of test expression To junctions. hsfnigsgetdta otci ih eafunctional a be might cortactin that suggested finding This a a kokonpeoye vrxrsino ihrwild- either of Overexpression phenotype. -knockdown elto.B otat xrsino otci 3YD cortactin of expression contrast, By depletion. a osrc (mRPTP construct a atvtdcSca cadherin-based at c-Src -activated a Cer) esubsequently We –Cherry). a knockdown. a siRNA, bevdwe mRPTP when observed RPTP euaini irbat,nuosadohrcl ye,b virtue by types, cell other and neurons fibroblasts, identified. in been RPTP regulation hardly phosphatase have tyrosine junctional junctions that The adherens factors mediate the at to However, c-Src 2007). control and al., activated et binding be (McLachlan to organization homophilic reported was E-cadherin signaling integrity, by epithelial c-Src v- disturb recently, to The more known crucial. and, been is long E-cadherin- has intercellular SFKs oncogene which by Src cellular in signaling coordinated phosphotyrosine processes collective require mediated communication, are and accordingly adhesion organization that and functions integrity Epithelial adherens epithelial of junctions organization the for important RPTP DISCUSSION htti euto srsosbefrteprubdjunctional perturbed the for suggest RPTP responsible with and is associated RPTP cortactin, organization reduction of that this phosphorylation that indicate tyrosine the results reduces These 7B–D). (Fig. a a kokonclst ee htwscmaal othat to comparable was that level a to cells -knockdown ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal otosacSccratnsgaigptwyta is that pathway signaling c-Src–cortactin a controls fwhich of el pedn nEa–c(ntepeec fteRGD mean the the of show presence Data the peptide). (in Ecad–Fc single on of spreading lamellipodia cells cadherin-based intensity the signal at pY419-Src measured the 10 of bar: Quantification Scale (E) upper-right image. the each in of shown corner are boxes) the cadherin-driven (white of of lamellipodia presence magnifications (in Higher minutes peptide). 90 RGD for allowed Ecad–Fc were on that spread cells to in (purple) E-cadherin and (pY419-Src, green) c-Src activated for immunofluorescence the with as contact expressed mean in is area substrate surface Ecad–Fc-coated cell the the 10 of bars: Quantification Scale (C) in substrate. area the surface with the contact delimits line dashed A RPTP cells. and spreading control of images contrast each 10 of bar: corner upper-right Scale the image. in shown are boxes) lamellipodia (white cadherin-driven of magnifications Higher needn xeiet,frec fwhich of each for experiments, independent o 0mnts Single minutes. 90 for eeaaye) ** analyzed). were hN cr hN)o poly- on shRNA) (ctrl shRNA human against shRNAs RPTP expressing cells Caco-2 of activation. Src and spreading cell RPTP 6. Fig. Ea–c-otdsrae.()Imnfursec for RPTP Immunofluorescence (A) surfaces. (Ecad–Fc)-coated 6 a a ...(he needn xeiet,freach for experiments, independent (three s.e.m. (RPTP nclsta eealwdt pedo Ecad–Fc on spread to allowed were that cells in § a 0clswr nlzd.()Co- (D) analyzed). were cells 60 a a sdt ecetephenotype the rescue to used was a srqie o E-cadherin-dependent for required is hN1adsRA)o non-targeting a or shRNA2) and shRNA1 P , a m .1 s non-significant. ns, 0.01; a z .()Rpeettv phase- Representative (B) m. a enipiae nSFK in implicated been has ofclpcue r shown. are pictures confocal depletion. D lsn PL rE-cadherin or (PDL) -lysine 6 ...(three s.e.m. elsraigassay spreading Cell m a m. -knockdown a m m. knockdown § cells 9

Journal of Cell Science nlsso -ahrn(-ah,RPTP (E-cadh), E-cadherin of analysis ahrno -ci.Rpeettv ofcliae hwtedsrbto ftefursettg(ht) -ahrn(re)o -ci prl)a (purple) F-actin or (green) E-cadherin (white), tag mean fluorescent the the show Data of 10 controls. bar: distribution Scale untreated the conditions. show various the images under confocal junctions RPTP Representative mouse or F-actin. RPTP cortactin, or against human siRNA cadherin cort3YD–GFP) and with (cort3YF–GFP transfected mutated cells or Caco-2 GFP) cortactin. of mutants phosphomimetic EERHARTICLE RESEARCH acnms(nwihSK r omnyfudt be to found RPTP commonly of are relevance these SFKs potential in the which residue of (in tyrosine spite on carcinomas C-terminal phosphate In inhibitory regulatory an kinases. remove crucial specifically to the ability its of corta after recovered cortactin –WCL total (Cort). to anti-cortactin relative or pY-cortactin (pY) of anti-phosphotyrosine quantification with densitometric immunoblotting Right, by mean control. analyzed no-antibody and –Ab, (input), control; lysates lysate cell from (IP) immunoprecipitated RPTP 7. Fig. ao2clsepesn hNsaanthmnRPTP human against shRNAs expressing cells Caco-2 6 ...(ieidpneteprmns.()Rsu fRPTP of Rescue (B) experiments). independent (five s.e.m. a euae ahrnbsdjntosb fetn yoiepopoyaino cortactin. of phosphorylation tyrosine affecting by junctions cadherin-based regulates a 6 n otci ne h aiu odtos h niGPHbo ssona odn control. loading a as shown is blot anti-GAPDH The conditions. various the under cortactin and ...(he needn experiments; independent (three s.e.m. m .()Qatttv nlsso ucinlEcdei uprgah rFatnitniy(oe rp) untr., graph). (lower intensity F-actin or graph) (upper E-cadherin junctional of analysis Quantitative (C) m. a (RPTP a hN1adsRA)o o-agtn hN cr hN) et otci was cortactin Left, shRNA). (ctrl shRNA non-targeting a or shRNA2) and shRNA1 a kokonidcdjntoa eet yoeepeso fnnpopoyaal versus non-phosphorylatable of overexpression by defects junctional -knockdown-induced a in n deesjntos hr ti ncoemlclrpoiiyto proximity molecular close in is it where junctions, adherens ciae) h pteilcl ilg fRPTP of biology cell epithelial unexplored. the activated), 5 1.* 21). a ee eosreta RPTP that observe we Here, ue oCer (mRPTP Cherry to fused a P oehrwt lsisepesn F-agdwl-ye(cortWT– wild-type GFP-tagged expressing plasmids with together , ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal , .5 ** 0.05; P , .1 *** 0.01; A nlsso otci yoiepopoyainin phosphorylation tyrosine cortactin of Analysis (A) a P Cer)wr ie n muotie o E- for immunostained and fixed were –Cherry) , .0;n,nnsgiiat D etr blot Western (D) non-significant. ns, 0.001; a serce tcadherin-based at enriched is tnI ssona the as shown is IP ctin owhole-cell no , a intercellular t remained 2427

Journal of Cell Science aha,gvnteipiaino h omri epithelial in former the of implication the given pathway, eedn grgtsadt ananteitgiyof integrity junctions. cell–cell of the linearity of the calcium- and knockdown maintain ring is form actin monolayers, to to effect cellular to cells RPTP In This structures, and epithelial junctions. of locale. 3D intercellular aggregates ability cellular well-ordered the dependent this in of at control organize by c-Src accompanied regulation epithelial and activation the in of participates it that and E-cadherin, ARTICLE RESEARCH 2428 might of RPTP distribution which c-Src, RPTP punctuated regulating upon of and observed discontinuous dysfunction E-cadherin the to recycling, contribute and/or adherens the 2011). endocytosis Yap, to and recruited (McLachlan is signaling and SFK 2010), through al., junction intercellular et at organization (Smutny E-cadherin contacts for required is which IIB, RPTP that indicate observations Our RPTP of Functions RPTP between link molecular RPTP the of a loss normalizes reveals with cortactin adequate This associated of distribution form that E-cadherin mutant show in aberrant 3YD we the and RPTP of 2009) Here, on expression depends al., shown). cortactin et of not phosphorylation (Ren tyrosine (data cells MCF7 cells in Caco-2 depletion cortactin RPTP of of effects junctional The RPTP of effector daetcls nti es,a diinlrlvn agtof target relevant additional between an tension sense, junctional this RPTP imbalanced In cells. is adjacent there RPTP of that 2003; consequence al., a suggests linearity et as junctional monolayers reduced Wichert of epithelial von observation in Our 2010; 2003). al., al., et et Zeng (Krndija RPTP activation require and SFK these stiffness sites; substrate adhesion on depend focal effects integrin-based 2011). at al., formation et fiber stress (Gomez junctions the adhesions, RPTP along E-cadherin-based tension to is of generating contributes strengthening by which and and binding extension contractility, homophilic the E-cadherin actomyosin by cortical promoted Junctional requires junctions. exploration. merits integrity junctions adherens RPTP epithelial of at at targets signaling additional Src E-cadherin for potential of the However, organization lead would aberrant This actin function. to peri-junctional cortactin deregulating the by by cytoskeleton, strengthening adhesion intercellular 2013). adhesion al., intercellular et thus Thomas for 2002; studies process al., et crucial our (Lambert RPTP a pool, cytoskeleton, showing cortactin actin homeostasis junctional By RPTP and the implicate regulator filaments. control positive of in actin a regulation is involved branched cortactin complex, 2009), of Arp2/3 al., the et Ren of 2004; al., et RPTP or cortactin the either of that effect observation the depletion. the rescue not by did cortactin supported mutant acid 3YF is aspartic it (c-Src-mediated) of between phosphotyrosine, caution, differences and behavior structural mimicking requires significant the interpretation the as given interpret this to Although mutant phosphorylation. possible 3YD is the It integrity. junction ee eietf h -r usrt otci sadownstream a as cortactin substrate c-Src the identify we Here, ufc raiaino -ahrnas eiso dynamic on relies also E-cadherin of organization Surface satre fEcdei-ciae F inln (Helwani signaling SFK E-cadherin-activated of target a As a a a a led enlne octseea otatlt and contractility cytoskeletal to linked been already has teihla ucin ih etennucemyosin nonmuscle the be might junctions epithelial at fet -ahrnognzto,tepr-ucinlF- peri-junctional the organization, E-cadherin affects a a ncodnto ewe -ahrnadthe and E-cadherin between coordination in atvtdcSca ahrnbsdjunctions. cadherin-based at c-Src -activated a tteeihla junctions epithelial the at a ol oto vnsi h endocytic the in events control could a ncdw r iia othose to similar are knockdown a ncdw.Ptnily by Potentially, knockdown. a elto ih reduce might depletion a n epithelial and a knockdown a a a a -mediated -mediated -mediated n the and , a a . dein ..terlvneo RPTP of rule relevance not the do RPTP e.g. data Takeichi, of adhesion, our functions and Finally, epithelial recycling. (Kametani out endocytic E-cadherin to flow to during and contribution basal-to-apical 2007), 1995) its e.g. as al., well dynamics, et as Yonemura intermediates maturation, junctional junctional junctions; transient of adherens fusion (spot-like RPTP and of will formation contribution It the the contacts. detail to in cell–cell RPTP characterize to to of informative recruitment intervention be early early its an with suggest consistent Ecad–Fc of on spreading RPTP question which the at RPTP maturation raises of ill-understood, stage the though maturation, receptor and neurotransmitter 2011). al., neuronal et and (Ohnishi in neurons Swaminathan in 2010; and trafficking al., 2012) et Cartwright, (Canel endocytosis E-cadherin yt ih ugs oei h aneac fapicobasal of maintenance the in role a polarity. suggest might cysts novmn fPP ncl–elahso.Rslsfrom the Results of adhesion. that suggested understanding cell–cell had inhibition expand in PTP global PTPs and involving experiments of confirm involvement findings junctions cadherin-based of Our integrity the and PTPs neato ih ewa n/rtaset uhinteraction Such transient. RPTP and/or co- the by weak of mediated be level be might their low might that suggests the interaction proteins although two the interaction, between FRET immunoprecipitation direct of for occurrence potential The them. RPTP integrin, with between or be associating NCAM as physically would such by molecules, adhesion adhesion cell from E-cadherin signals RPTP from to be to signals transduced remains It how engagement. E-cadherin elucidated upon signaling c-Src of RPTP that suggest results to Our E-cadherin–RPTP found of be aspects Mechanistic will contacts. cell–cell PTPs is, at additional activation It c-Src that adhesions. to contribute unlikely cadherin-based RPTP not at identifies however, signaling c-Src al., study et controlling Fuchs our 2010; at al., act knowledge, et also Espejo PTPs Mu 2006; 1996; al., other et Several (Anders junctions. RPTP junctions by implicate adherens controlled directly of negatively findings integrity our are contrast, that indirect By kinases reflect PTPs. merely tyrosine might of effects activation such However, 2011). and (McLachlan Yap, integrity junctional cadherin-based support PTPs oiiain sc ssrn n yoiepopoyainand phosphorylation tyrosine and serine known as (such are RPTP modifications mechanisms RPTP control of regulatory activation to multiple local Indeed, the junctions. of study. mechanism further for and topic rewarding a be RPTP likely will of partner dynamics The analogous RPTP 2005). for interactions, suggested RPTP intracellular that RPTP to involve altered the might to to able association in case, be signal this might conformational resulting in itself a 1999); E-cadherin al., transmit RPTP of et of engagement (Zeng association homophilic contactin the molecule for adhesion claimed been h ope n utse aueo ucinlorganization junctional of nature multistep and complex The eodiserqiigfrhrsuycnen h possibility the concerns study further requiring issue second A a kokonidcddfcsi elageainand aggregation cell in defects -knockdown-induced ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal le ta. 99 ht ta. 07.T h eto our of best the To 2007). al., et Sheth 1999; al., et ¨ller a n -ahrnidctscoepoiiyadthe and proximity close indicates E-cadherin and a nldn ieiain post-translational dimerization, including , a RPTP . a NA rstl Bdio tal., et (Bodrikov crosstalk –NCAM a a a a srsosbefrlclactivation local for responsible is a a enrpre otransduce to reported been has cooan iia owa has what to similar ectodomain, a ucin lentvl,the Alternatively, function. erimn sa E-cadherin an as recruitment eodE-cadherin-mediated beyond a a o Dognzto into organization 3D for cini otcrucial. most is action a a a interactions stefrtPTP first the as ihteneural the with a a tepithelial at ectodomain, a nthe in a a ,

Journal of Cell Science ytmwsue opouevrs o rdcino iu,5 virus, of production For virus. produce to used was system 3 rnfrvco a otasetdtgte ih10 with together co-transfected was vector transfer 5 was control non-targeting the of ,Adee129 n 5 and 12259) Addgene G, CCCTTCTGGAGCATTATCT-3 GGCGAAGAGAATACAGACTAT-3 between tandem sequence coding GFP–mCherry GFP the The cloning by Australia). the obtained Bioscience, was (Institute construct Stow Molecular Jenny al., from et gift Smutny for generous (Clontech; a was pEGFP-N1 mCherry–VAMP3 into cloning 2011). cDNA by E-cadherin obtained vector of murine was pmCherry-N1 the E-cadherin–GFP into ratio murine cDNA Similarly, corresponding 3:1 (Clontech). the cloning a by (Invitrogen) obtained (with siRNA). were nM RNAiMAX 50 instructions of transfections concentration final or manufacturer’s a siRNA and reagent:DNA, the 2000 Lipofectamine and to Lipofectamine DNA according Transient using (SIC001) Sigma. performed RNA from control scrambled were and (SASI_Hs01_00169093) duplex a loe osldf yicbto t37 plated at matrix incubation were Matrigel by solution solidify The and to Dickinson). Matrigel allowed 3440-100-01) (Becton this was slides (Trevigen, in chamber collagen embedded eight-well into Cells I HEPES. type M mg/ml 0.02 1 354230), (BD, o uuersac.Teasessol lob instructive be function. junctions appropriate also and for molecules PTPs should adhesion on of rely answers that types other The the question research. interesting concerning an is future processes these for junctions, of adherens several at Shalloway, or conditions one and affects or the Zheng factors and 1994; particular binding, of homophilic al., presence E-cadherin al., et how Su et and 1999; Whether (Groen 2001). al., changes et Jiang conformational 2008; and oxidation) cysteine ARTICLE RESEARCH DAi ak’Blne atSlto HS;4 glKl 0mg/l 60 KCl, mg/l mM 40 4 (HBSS; in Solution sequestration Salt calcium Balanced Hanks’ by in isolated EDTA were cells confluent Nearly assay aggregation Cell viral concentrated containing and 80,000 post-transfection, Supernatant at hours ultracentrifugation 48 transfection. by cells at 100-fold 293T harvested before HEK was particles hours into 24 coprecipitation phosphate seeded calcium using 12260) human for sequences targeting derivative. sense pLenti The a RPTP (Sigma). into 10 knockdown with introduced selected To were and cells A431 Venus Infected protein RPTP fluorescent human encoding infection and cDNA particles lentiviral of Generation and mutagenesis RPTP PCR-based vector. two-step pEGFP-N1 by into generated introduced were cortactin (3YF) mutants Y421F/Y470/Y486F and (3YD) transfection Y421D/Y470D/Y486D cell siRNA and Plasmids penicillin FBS, 10% with (100 supplemented streptomycin DMEM and units/ml) in (100 units/ cultured (100 penicillin were (100 (Gibco), streptomycin acids and bovine amino ml) fetal non-essential 10% containing (FBS), medium RPMI serum in cultured were cells Caco-2 culture Cell METHODS AND MATERIALS 0dy,te anandi 2 in maintained then days, 10 diino eim eimwsrnwdeey34dy pt 4days 14 to up days formation. 3–4 cyst every follow renewed to was Medium medium. of addition W2T oo Bcmn.Tevrlple a eupne nPSand PBS in resuspended was pellet at viral The stored (Beckman). rotor Ti SW32 el eetysnzdt banasnl-elsseso.Asseso of suspension A suspension. single-cell a 5.8 obtain to trypsinized were cells 9 Human 6 SgaTC. ln H02.Ascn eeainpackaging generation second A SHC002). clone TRC1.5 (Sigma Nhe 10 4 and I el/lwsmxdwt ouincnann 0 Matrigel 40% containing solution a with mixed was cells/ml 2 80 b ctnnmhryadmrn RPTP murine and -catenin–mCherry Xho ˚ .Ifce el eeslce n4 in selected were cells Infected C. ie fpmCherry-N1. of sites I a eue h LO1pr etvrlvector lentiviral pLKO.1-puro the used we , m m /l.MF7 41adHK23 cells 293T HEK and A431 MCF-7, g/ml). fpPx ggpl a-e;Addgene tat-rev; (gag-pol, psPax2 of g m a /lpuromycin. g/ml 9 9 -CAACAAGATGAAGAGCACCAA- (RPTP a ue tisCtriu othe to C-terminus its at fused was 9 m (RPTP /l.Fr3 rwh Caco-2 growth, 3D For g/ml). a g m ˚ o 0mntspirt the to prior minutes 30 for C hN2,adtesequence the and shRNA2), o or t4 at hours 2 for /lbatcdnfr1 days. 10 for blasticidin g/ml a a Nioligonucleotide RNAi hN1 n 5 and shRNA1) m m fpDG(VSV- pMD2G of g /lprmcnfor puromycin g/ml a PTPRA mhrywere –mCherry ˚ sn the using C ee5 were 9 m -AG- gof 9 - KH tipn ufr(25m rsHlp .,2 D,10mM 100 SDS, 2% 6.8, in pH 60 at enhanced incubated Tris-HCl hour 1 mM were by for (62.5 mercaptoethanol) membranes revealed buffer required, stripping and horseradish When with ImmunoResearch) coupled chemiluminescence. antibody (Jackson secondary with peroxidase hour 1 for incubated al .%N-0 0m C,2m CaCl mM 2 KCl, mM 50 mM 150 NP-40, 7.5, pH HEPES 0.5% mM 20 NaCl, containing buffer lysis using RPTP performed of Co-immunoprecipitation buffer. Laemmli hshts inhibitors. phosphatase rti xrcswr rprdi rtnlssbfe 5 MHEPES mM (50 buffer lysis Triton in prepared were extracts Protein analysis immunoblot and Extracts anti- polyclonal rabbit monoclonal 2007), mouse al., Technology), anti- Signaling et (Cell Sandilands family Tyr(P)-419-Src 2–17, anti-c- mouse monoclonal (antibody supernatant), mouse Src Sigma-Aldrich), (SHE78-7, culture (327, anti-v-Src antibody hybridoma mouse monoclonal NE), Millipore), (4G10, (4F11, blocking Omaha, anti-cortactin anti-phosphotyrosine Wheelock; monoclonal anti-E-cadherin mouse Peggy Invitrogen), from monoclonal gift mouse (a anti-E-cadherin the ectodomain monoclonal against HECD-1 E-cadherin mouse monoclonal mouse were Laboratories), Transduction used (BD antibodies primary The Antibodies bTet(bd)egtwl irsie eecae ihEa–c(50 poly- Ecad–Fc or with coated ml) were microslides eight-well (Ibidi) IbiTreat assay spreading Cell Protein 4 chromatography. at transiently dialyzed Protein-A-affinity was from were (Ecad–Fc) purified by was samples Fc protein medium secreted IgG The the cells. conditioned 293T to HEK into fused transfected region E-cadherin extracellular human the containing of backbone) (pEE14 vector purification Expression and production protein Recombinant 4 at overnight lysate of anti- mg 1.5 to with coupled incubated were Upstate-16266) antibody slurry, cortactin (50% beads Protein-G–agarose Immunoprecipitation MgCl mM 1.5 NaCl, mM 150 7.5, pH eetysnzdfr5mntsa 37 at minutes 5 RPTP for and Control trypsinized 2002a). were al., et (Kovacs previously Na mM 1 containing X-100), Triton 1% otiig01 Tween-20. 0.1% containing 0m/lBA MCaCl mM 5 BSA, mg/ml 10 upnin(2 suspension utr ihsta a enpeiul otdwt %bvn serum bovine 2% onto with CaCl plated coated mM EDTA, previously 10 mM been (BSA), 4 had albumin of that presence dishes the culture in or medium supplemented sltdclswr eupne t2 at resuspended were cells Isolated niio okal(oh)ad1m MF n lae t14,000 at cleared 20 and PMSF, minutes. mM 15 1 and (Roche) Cocktail Inhibitor t37 at 5 MTi-C H74 5 MNC,2m CaCl mM 2 NaCl, mM 150 7.4, pH Tris-HCl mM (50 hkna 5rmfr4husa 37 at hours 4 for rpm 75 at shaken ie n2 aaomleye(F)adpaecnrs mgswere microscope. inverted images DMR phase-contrast Leica and Upright an (PFA) using paraformaldehyde taken 2% in fixed narttn he.Tebaswr ahdsxtmswt rtnlysis Triton with times six Na washed mM were 1 beads containing The buffer wheel. rotating a on rat-abtIGcnuae oC3(ako ImmunoResearch). anti-mouse-IgG (Jackson donkey Cy3 and to (Invitrogen) conjugated 647 or anti-rabbit-IgG secondary 594 or conjugated 488, The anti-mouse-IgG Fluor or Alexa (Invitrogen). anti-rabbit-IgG to goat anti-ZO-1 were monoclonal used antibodies mouse and 1999) r5 S nTSfr1hu tro eprtr n incubated and temperature room at hour 1 for Immobilon 4 TBS to at in transferred overnight BSA milk and skimmed 5% 5% with gels or blocked were polyacrylamide Membranes (Millipore). 8% membrane on resolved 2 PO a ˚ tbln(im-lrc) abtplcoa anti-RPTP polyclonal rabbit (Sigma-Aldrich), -tubulin ne osataiain hr plcbe uigtelast the during applicable, Where agitation. constant under C 4 ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal / al 0m/ Na mg/l 90 NaCl, g/l 8 , D 6 lsn 01m/l vrih t4 at overnight mg/ml) (0.1 -lysine ˚ 10 ihpiayatbd.Mmrnswr washed, were Membranes antibody. primary with C m fpoenwsrsseddi aml buffer, Laemmli in resuspended was protein of g 5 el/li eu-otiigmdu)fr1hour 1 for medium) serum-containing in cells/ml 3 VO ˚ noclimcnann trg buffer storage calcium-containing into C 2 2 nHS o 0mnts sdescribed as minutes, 30 for HBSS in nHS vrih t4 at overnight HBSS in 4 ˚ n 0m a,adrsseddin resuspended and NaF, mM 10 and ne gtto n ahdi TBS in washed and agitation under C 2 ˚ oalwageain el were Cells aggregation. allow to C HPO 6 2 ˚ MET,1%gyeo and glycerol 10% EDTA, mM 1 , 10 n anandi single-cell in maintained and C 3 VO 4 5 N 7H el/li 0m calcium- mM 10 in cells/ml 4 0m a,4 Protease 4% NaF, mM 10 , 2 ,10mg/l 100 O, ˚ ,te lce with blocked then C, a 2 n -ahrnwas E-cadherin and lspoes and protease plus a kokoncells -knockdown 2 ). ˚ ,adfurther and C, a D S tal., et (Su -glucose). 2429 g m for ˚ b g/ C -

Journal of Cell Science eupne nLemibfe n oldbfr D-AEwsperformed. was SDS-PAGE before boiled and buffer Laemmli in resuspended nest ln h ie ooaiainaayi a efre on performed rounds. was randomization 100 of with analysis signal method perimeter Costes’ pY419-Src Colocalization using entire average line. contact the cell–cell the the individual covering measuring wide) along and pixels intensity cells (15 spread line performed individual a was assay drawing spreading the by in c-Src lamellipodia wide) of cadherin-driven pixels status the activation (10 the at line of analysis same The the junctions. individual along covering intensity by signal analyzed mean was monolayers the on measuring al., intensity signal et intercellular c-Src (Otani total images. between ZO-1 and previously pY419-Src from distance described junctions picked as linear randomly length, individual analyzing the junction 2006), intensity real of the the ratio to as the vertices signal, as the index calculated linearity junctional of was The distribution it. within E-cadherin) the E- (for deviation (for and standard intensity F-actin) signal individual total or covering average (ROIs) to cadherin the interest orthogonal measuring of and line contacts regions pixel contactscell–cell drawing 50 cell–cell by at a analyzed intensity along was Fluorescence plots the contacts. intensity from cell–cell measured signal was individual was Metamorph the signal of of E-cadherin function the spread Scan of spreading Line Briefly, the used. contacts, where cell–cell microscope, at software. ImageJ density Olympus confocal or TCS Metamorph IX81 Scanner Leica with a analyzed an resonant were with Images camera, or Tandem indicated. camera Leica ER AOBS Orca-1 DFC SP5 Hamamatsu a a and and microscope microscope (Sigma). inverted Mowiol with in stained mounted were were Nuclei Coverslips Sigma). temperature. ng/ml, room at (100 hour DAPI 1 for performed was 94 eeitoue.Tecl upninwste eddot coated 37 onto seeded at then incubated was H- and suspension (Bachem plates cell The (cRGD) introduced. were GPenGRGDSPGA or 3964) peptide SHE78-7 antibody integrin-blocking E-cadherin-blocking the the suspension, in minutes 30 ARTICLE RESEARCH 2430 NaCl, performed mM was 150 antibodies gelatin, 4 primary 0.2% other at with BSA, all overnight Incubation 0.5% X-100. For for with Triton PBS saponin. in blocked 0.1% for X-100 0.025% and Triton saponin FBS, 0.3% minutes 5– with 5 0.025% 10% permeabilized for in were in cells fixed blocked antibodies, permeabilized anti-E- and and were the minutes using confluence 30 cells immunofluorescence was until antibody, For PFA. immunofluorescence coverslips cadherin cells 1–4% Otherwise, with and glass minutes 2008). to on minutes, 15 al., 30 prior et grown (Jaffe temperature for were previously 50 room described PFA in at as digested 4% performed minutes was 15 in collagen for fixation cultures, III 3D collagenase in units/ml penetration antibody For analysis image and Immunofluorescence Human vitro In irsoeadwsaaye yuigMtmrho ImageJ. or on Metamorph determined using was by inverted cells analyzed DMR Leica was adherent Upright and of an microscope using The pixels) by Violet. obtained (in images Cresyl area phase-contrast 0.5% surface with cell staining total or immunofluorescence for PFA MPS,frhricbtda 4 at and incubated (Roche) Cocktail further Inhibitor Protease PMSF, 4% NaF, mM 1 mM 10 acid, i iodoacetic prepared were extracts Cell medium. o 0mntsa 4 at minutes 90 c then proteins for and fusion acid GST iodoacetic with unreacted incubated inactivate to (BioChemika) dithiothreitol 0mntsb dig1m Na mM 1 adding by minutes 20 iutnosymttdt eie iligteCSmtn DA The cDNA. 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Vfamn atrbutn the blunting (after fragment RV aayi iecsen eius42ad72were 732 and 442 residues cysteine site catalytic Sma ˚ .Baswr ahdfu ie ihlssbuffer, lysis with times four washed were Beads C. ieo GXX S-uinpoen were proteins GST-fusion pGEX3X. of site I ˚ o 0mnts el eefxdwt 1% with fixed were Cells minutes. 90 for C z ai iw ihteIae lgnJacoP, plugin ImageJ the with views -axis 3 VO uldt lttin–ehrs beads glutathione–Sepharose to oupled rtnlssbfe otiig5mM 5 containing buffer lysis Triton n 4 n MH mM 1 and ˚ o 5mntswt 0mM 10 with minutes 15 for C 2 80 PTPRA Xma ˚ C. 2 O I vragwith overhang III 2 noteculture the into a subcloned was a sd ttsia aaaesona olw:n,ntsignificant not ns, follows: as shown are data test ( Statistical comparisons multiple used. Tukey’s by was followed ANOVA one-way where Student’s two-tailed using values EF-1 mhryN n F–Cer admcntut rco- or construct, and with E-cad–GFP tandem transfected with GFP–mCherry transfected transiently and and pmCherry-N1 China) pEGFP-N1, Co., Biotechnology (Shengyou betv Pa pcrmt14N,Zis iharslto f0.14 of resolution a with Zeiss) NA, 1.4 Apochromat (Plan objective P1149;adKd acrPoeto h nooyRsac Foundation. Research Oncology the of Project APP1044041, Cancer 631683, Kids Medical numbers and & [grant APP1010489]; Health Australia le National of contre Council; Council Ligue Research Research La Australian (Denmark); the Bekæmpelse (France); Kræftens Cancer by supported was Work Funding manuscript. the A.S.Y., wrote M.T., the J.S. of experiments. and much performed V.D. supervised and G.A.G., V.D. conceived initial and study. the work, the made experimental conceived N.V. J.S. experiments. and performed A.S.Y. G.A.G. observations. and S.P.H. F.N., X.L., M.T., contributions Author interests. competing no declare authors The interests Competing suggestions. and comments the constructive thank We highly Australia). for Brisbane, NE, reviewers Bioscience, Omaha, Molecular from Wheelock, for gift (Institute generous Peggy a Stow from was Jenny mCherry–VAMP3 gift Takeichi. a Masatoshi (University of was Serrels permission HECD-1 Bryan with and and Frame UK), anti-Src-2-17 Margaret Edinburgh, from The of gift discussion. generous for a France) was and Paris, antibody experiments, Curie, pilot (Institut for Dufour Australia) Sylvie Brisbane, Bioscience, McLachlan Molecular Robert and for Denmark) (Institute Nun Copenhagen, Barbara Centre, imaging, Innovation Monod, regarding and Jacques training Research and (Institut advice facility for ImagoSeine France) the Paris, for assays, France) 3D Paris, Monod, with RPTP Jacques assistance the (Institut for Delacour Japan) Delphine University, construct, lentiviral (Kyoto Sakurai Takeshi thank We Acknowledgements the calculating by assessed was data the of significance Statistical analyses Statistical el eeicbtdi BSsplmne ih1 MHPSp 7.4 pH CaCl HEPES mM mM 10 5 with supplemented and HBSS in incubated were cells rmhryVM3 ieclswr mgda 8huspost-transfection hours 37 48 at at imaged were cells Live mCherry–VAMP3. or ta. 00 sn utmmd aLbruie(ahehe l,2012). al., et (Ratheesh (Grashoff routine MatLab previously custom-made a described using as 2010) al., performed et were calculations cFRET RTefcec a acltdfreeyiaeuigtefollowing the using construct. image 2012); tandem every al., for GFP–mCherry et FRET (Ferrari calculated the equation when was cytoplasm for efficiency cell determined FRET the was or identified signal, efficiency as cells, fluorescence of E-cad–GFP adherens zonula by the to corresponding ROIs selected sn h olwn qain(rsofe l,21;Fraie l,2012); al., et basis Ferrari pixel-by-pixel 2010; a al., et on (Grashoff obtained equation was following channel the using FRET the from images acquisition. line sequential by acceptor acquired the were in Images emission the regions. the using collecting emission and by excitation collected region for was line emission laser channel 546-nm donor nm), acceptor the 560 the pass addition, (long in In region emission respectively. emission acceptor and the nm) 488- collecting 500–530 a pass (band and using scanning line by laser recorded were nm channels FRET and Donor pixel. oo n cetrbedtruhfatosepesda ucin of functions as expressed fractions bleed-through ( donor acceptor and donor hr RTi h RTcanliae and image, channel FRET the is FRET where P . o RTaayi,Cc- el eeclue ngasbtoe dishes glass-bottomed on cultured were cells Caco-2 analysis, FRET For h RTefcec a acltdfrtoepxl oae nthe in located pixels those for calculated was efficiency FRET The o RTmaueet,tecretdFE cRT inlfrom signal (cFRET) FRET corrected the measurements, FRET For .5;* 0.05); ˚ na S50Mt es ofclmcocp.Drn imaging, During microscope. confocal Zeiss Meta LSM510 an on C I D ora fCl cec 21)17 4023 doi:10.1242/jcs.134379 2420–2432 127, (2014) Science Cell of Journal n cetr( acceptor and ) P , cFRET .5 ** 0.05; 2 mgswr curdwt 63 a with acquired were Images . ~ P FRET I , A E intensities. ) .1 *** 0.01; ~ 1 { { b I dbt b t I ctnnmhryo RPTP or -catenin–mCherry tss xetfrFE measurements FRET for except -tests, D ðÞ z P D , cFRET I dbt D | .0;**** 0.001; I ( D I D - b I abt and ) dbt ðÞ ( I A D abt and ) P | , ( 6 I I A 0.0001. A , ucin and functions ) abt oil-immersion a -venus e (Biotech ˜ez a ( I –mCherry A r the are ) m m/ P -

Journal of Cell Science oas .M,Ai .G,MCrak .J n a,A S. A. Yap, and J. A. McCormack, G., R. Ali, M., E. Kovacs, M. Takeichi, and P. Y. M. Kametani, Sheetz, and M. Tanase, Y., Cai, H., A. Huang, G., Jiang, oas .M,Gown . l,R . aesn .D n a,A S. A. Yap, and D. A. Paterson, G., R. Ali, M., Goodwin, M., E. Kovacs, T. Hunter, and J. Sap, J., Noel, J., Su, J., Hertog, den G., Jiang, af,A . ai . ugn .adHl,A. Hall, and J. Durgan, N., and Kaji, D. B., Shalloway, S., A. B. Jaffe, White, M., Wang, H., Wu, R., Xu, L., Yao, J., Huang, U. Tepass, and C. J. T. Harris, nes . etn,P,Lmih . uga . atan . atg P., Saftig, D., Hartmann, M., Murgia, S., Lammich, P., Mertins, L., J. Anders, S. Smith, and J. W. Nelson, L., C. Adams, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134379/-/DC1 online available material Supplementary material Supplementary ARTICLE RESEARCH re,A,Oevod,J,vndrWj,T n e etg J. Hertog, den and T. Wijk, der van J., Overvoorde, A., Groen, Paolo and N. 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