oii .Brkp,Mce .HdiansadJu and Hadjihannas V. Michel Bernkopf, B. signalling Dominic upstream to by insensitivity pathway involves Wnt conductin/axin2 the of regulation Negative-feedback REPORT SHORT ß eevd3Jl 04 cetd2 coe 2014 October 22 Accepted 2014; July 3 Received ([email protected]) correspondence for *Author Friedrich-Alexander II, Medicine Universita Experimental of Chair Nikolaus-Fiebiger-Center, proteins, Wnt of absence the In 2012). factors transcription Nusse, TCF and with conjunction (Clevers in genes target Wnt of allowing pathway, signalling of Stabilisation INTRODUCTION Wnt feedback, Negative Dvl, Conductin, Axin, WORDS: KEY related structurally are axin2) as Wnt/ of known inhibitors (also conductin and Axin ABSTRACT h oeo odci sangtv-edakrgltro Wnt of regulator negative-feedback a as of conductin feature important signalling. of an is role Dvl Dvl. to the insensitivity and that component propose axin, We to Dvl2. pathway compared functional Dvl2 Wnt of their to degradation in binding upstream reduced differ shows conductin the Conductin and with axin regulation. that negative-feedback interaction Wnt show a in we is implicated Here, conductin gene expressed, target constitutively Wnt is axin Whereas catenin. ihai ucini ifrn as rzldascae Dvl Frizzled-associated ways. causes which different complexes, receptor frizzled–LRP5/6 in to interfere axin can function recruits Dvl axin. axin to binds it with which through domain DIX a has mediating ineffectiveat head-to-tail axin render the and abolish oligomerisation axin that prevent domain interaction DIX the of mutations Specific for puncta Kishida cytoplasmic sites 2011; as interaction high-avidity seen provide al., to often thought are overexpression, et complexes, upon These (Fiedler 1999). domains al., DIX et their of interaction 2009; al., et Wu 2008). 1999; al., al., al., et of et et Smalley Zeng Cliffe inhibition 2008; al., 2007; the et al., Cselenyi in et 2003; resulting (Bilic degradation ultimately and (Dvl) phosphorylation 3 and 1, axin or dishevelled frizzled of phosphoprotein 2 recruitment receptors frizzled-associated membrane the their Weis, to by leads to and proteins (LRP5/6) ligands (Stamos LRP6 or axin2) Wnt LRP5 as of known Binding as (also known 2013). destruction (also kinases conductin proteasomal axin the proteins or a scaffolding APC, axin1) the and suppressor and by CK1, tumour and ubiquitylation the GSK3 mediated of consisting for phosphorylation complex by earmarked degradation is catenin 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,3–9doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. xnmlclsfr yai lgmr yhead-to-tail by oligomers dynamic form molecules Axin ¨ Erlangen-Nu t b ctnnadohrdsrcincmlxcomponents. complex destruction other and -catenin b ctnndgaain(ide ta. 01.Dlalso Dvl 2011). al., et (Fiedler degradation -catenin b b ctnnb odci sol orybokdby blocked poorly only is conductin by -catenin b ctnnsgaln htpooedgaainof degradation promote that signalling -catenin ctnni e tpi h Wnt/ the in step key a is -catenin ¨ neg Glu rnberg, ¨ kt.6 -15 ragn Germany. Erlangen, D-91054 6, ckstr. b ctnnt tmlt transcription stimulate to -catenin b b -catenin -catenin gnBehrens* rgen ¨ b b - - inligbcueo uain fAC(L1 W8)or SW480) (DLD1, APC exhibit of which mutations Wnt lines, conductin of of activation because cell and to signalling due carcinoma negative- expression axin conductin colorectal for high compared constitutively three factor also in strong decisive We exclude levels a largely regulation. as results feedback conductin These of chart). resting bar accumulation under 1A, of (Fig. downregulation levels after and axin lower conductin axin and of substantially upregulation than axin remained Wnt3a-induced surprisingly, lower of that and, showed were comparison conditions This 7–10). allowed levels and 3–6 blots conductin lanes 1A, same (Fig. levels the conductin GFP–axin and on GFP–conductin blot of al., western present dilutions et the serial Lustig of h, using Normalisation 1,2; by 6 1999). signals lanes for al., 1A, et treatment (Fig. Yamamoto the decreased Wnt3a 2002; axin reports, after of previous that Wnt3a- increased whereas with from conductin line extracts of In of amount cells. blotting MDA-MB-231 western treated signalling by Wnt during determined conductin and were axin of amounts relative The DISCUSSION AND RESULTS n inlig oee,i sucerhwcnutnescapes conductin how unclear is is it of the it Therefore, regulator of negative-feedback 2002). However, a al., activation signalling. as et Wnt after acts Lustig conductin upregulated 2002; that al., assumed gene et target (Jho 1999). pathway Wnt is al., conductin expressed, direct et constitutively Fagotto is a which 1998; axin, to al., contrast et In (Behrens elements sequence 2007). al., thereby et Schwarz-Romond and 1999; al., homopolymers et axin al., and et with of Dvl interfere Zeng self-assembly of 2009; interactions disturb al., domain et DIX–DIX axin the Wu addition, 2008; In al., 2008). et (Cselenyi receptors. LRP5/6 phosphorylated by mediated GSK3 axin-bound of inhibition ifrn oe fai n odci scntttv versus constitutive as conductin signalling. and Wnt axin of inhibitors of inducible Dvl2. the by roles determine differences than mediated differing expression inhibition Dvl2 than to with rather latter resistant functional efficiently Thus, largely less the levels, it interacts axin making only to conductin axin favours compared to that protein upstream leads (2) conductin that stimulation and in to Wnt increase that evidence modest susceptible (1) a showing, provide less by mechanism be We might available inhibition. thus degrading conductin saturate might continue addition, and to to able Wnt-receptor–Dvl-complexes high be at sufficiently sites levels binding to remain to accumulate signalling Wnt in of active activation upon inhibition upstream eesecee hs fcnutn(i.1) ofrigthat signalling. confirming Wnt by 1B), upregulated (Fig. moderately conductin only is axin of lines, conductin cell those these all exceeded In 2002). levels al., et (Lustig (HCT116) catenin xnadcnutnaerltdpoen htsaekey share that proteins related are conductin and Axin b ctnndgaain npicpe odci might conductin principle, In degradation. -catenin b ctnndgaain(ide ta. 01 Kishida 2011; al., et (Fiedler degradation -catenin b ctnn lentvl,o in or Alternatively, -catenin. 33 b -

Journal of Cell Science HR REPORT SHORT 34 Flag– of measurements densitometric show blot Flag the below Numbers vector). plasmids. (empty indicated GFP the with transfected cells B in 7–9 * and 4–6 lanes lan and (B, A, DLD1 and in SW480 7–10 HCT116, and cells 3–6 cancer (lanes colorectal GFP–axin of or lysates GFP–conductin in human 1,2), with lanes transfected (A, cells (–) HEK293T untreated of left lysates b or diluted h serially 6 in for and (+) 3), Wnt3a with treated cells and MDA-MB-231 levels Expression 1. Fig. degrading eeso vrxrse rtisbsdo h niGPatbd lt eut r mean are Results of blot. Quantification antibody (D) anti-GFP the on of based staining proteins overexpressed of levels o ufcett ul lc xnpoen.Wtatreatment Wnt3a proteins. of knockdown axin whereas BIO, block inhibitor apparently fully are might they to increased but that extent, sufficient 2004) certain al., a not to et pathway (Bafico the Wnts stimulate endogenous express cells degrading both that in suggesting active or 2A), are (Fig. factors axin ligands Wnt exogenous of small in of increase absence Depletion an using nuclear to staining. led cells monitored conductin immunofluorescence and MDA-MB-231 by knocked (siRNAs) levels we in signalling, RNAs expression Wnt interfering of their inhibition the down to conductin 1F). and (Fig. conductin of coexpression by lower less but its explaining expressed possibly degrading distributed, in activity diffusely conductin whereas puncta, more levels in present expression was was different axin of Notably, range 1E). a (Fig. over 1C,D) (Fig. conductin endogenous of reduction stronger P atnwsue salaigcnrl eaielvl fai bak n odci ge)wr eemndb estmti esrmnsadnormali and measurements densitometric by determined were (grey) conductin and (black) axin of levels Relative control. loading a as used was -actin , enx oprdteptnyo xnadcnutnin conductin and axin of potency the compared next We ocmaeteidvda otiuino noeosaxin endogenous of contribution individual the compare To .0 (Student’s 0.001 b b b b ctnn(e)i W8 el rnfce ihGPcnutno F–xn(re) otdlnsmr rnfce el.Saebr 20 bar: Scale cells. transfected mark lines Dotted (green). GFP–axin or GFP–conductin with transfected cells SW480 in (red) -catenin ctnnitniy iia otetetwt h GSK3 the with treatment to similar intensity, -catenin ctnnwssrnl eue ycepeso faxin of coexpression by reduced strongly was -catenin ctnn rnfcino xni W8 el e oa to led cells SW480 in axin of Transfection -catenin. b t 2 ts) E ltigo F versus GFP of Blotting (E) -test). ctnnfursec neste ftreidpneteprmnsa nC t nrnfce.Rslsaemean are Results untransfected. Ut, C. in as experiments independent three of intensities fluorescence -catenin lgol vector. Flag-only , b ctnn(i.1) iial,transiently Similarly, 1C). (Fig. -catenin b ctnndgaaincpct fai n conductin. and axin of capacity degradation -catenin b b ctnn fnt,MDA-MB-231 note, Of -catenin. ctnnsann nest nthe in intensity staining -catenin b ctnnta rnfcinof transfection than -catenin b ctnnrdcdstaining. reduced -catenin b ctnnitniiso niiulclsfo .()Wsenbotn o lgadGPi yae fSW480 of lysates in GFP and Flag for blotting Western (F) C. from cells individual of intensities -catenin b -catenin motnl,kokono odci ute nrae the increased further conductin of Wnt3a-induced knockdown Importantly, ncdw fai i o Fg A.TeWnt3a-induced The 2A). (Fig. not did dephosphorylated of axin increase of knockdown ncdw fcnutnldt togrices fWnt3a- Moreover, of increase 2B–D). (Fig. stronger a rates to TCF/ led knockdown induced cell conductin different three of similar in knockdown axin, at not but lines, conductin, of knockdown by eetr yDli ieycniee h nta tptowards step initial the the considered widely of is inhibition Dvl by receptors strongly more under is activity activity activity axin regulatory inhibited. whereas and negative activation, level retain Wnt expression sustained to lower appears its despite conductin Thus, 2D). (Fig. n oxrsino v2CA nrae h rprinof proportion was the Axin increased 1999). Dvl2–CAAX of al., coexpression fractions, et cytoplasmic and and Smalley membrane by between 3A; determined distributed (Fig. equally was Dvl2, fractionation of tested cell form CAAX-tagged by membrane-associated therefore conductin with a and interaction We axin Dvl2, their of 2011). recruitment in this, differ Bienz, For might Dvl. conductin and and Metcalfe axin whether 2009; al., et erimn fai n odci rtist frizzled to proteins conductin and axin of Recruitment 6 AB etr ltigfrcnutn(d) xnadGPi yae of lysates in GFP and axin (Cdt), conductin for blotting Western (A,B) ...o ieidpneteprmns C Immunofluorescence (C) experiments. independent five of s.e.m. ora fCl cec 21)18 33 doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal b ctnnrpre ciiyta ncdw faxin of knockdown than activity reporter -catenin b b ctnnsann nest,whereas intensity, staining -catenin ctnndsrcincmlx(MacDonald complex destruction -catenin b ctnn(B)wsaugmented was (ABC) -catenin b ctnnnraie ocoexpressed to normalised -catenin 6 ...( s.e.m. n 5 20); m ainto sation m. s1– es ).

Journal of Cell Science 01.W rps htti eut nihbto fDvl2 diffuse a of had alone inhibition Conductin scheme). in visible see longer results 3C, no (Fig. complexes this puncta smaller as that to leading al., propose et polymerisation, tail (Fiedler We Dvl2 the with leaves interaction 2011). for but intact surface DIX homopolymerisation, axin interaction its the of The preventing surface but, 3C). interaction domain 2011) head (Fig. the al., puncta impairs et Dvl2 mutation M3 (Fiedler of puncta formation domain abolished form DIX The surprisingly, not 3C). does (Fig. axinM3 puncta such mutant in Dvl2 with colocalises lower 3B, the (Fig. with Dvl2–CAAX associated of become panels). coexpression not did upon conductin) membrane of domain DIX HR REPORT SHORT ae –2.Ipraty elcmn fteDXdmi of Cdt domain generating DIX axin, the of of that with replacement and 3A, 2011) conductin Importantly, (Fig. fraction Dvl2–CAAX axin, cytoplasmic 9–12). by al., membrane to the lanes the contrast to in recruited In et poorly present 5–8). only mainly lanes Fiedler was 3A, DIX–DIX-mediated (Fig. conductin specific Dvl2 axinM3; with the interaction domain on (construct DIX dependence its demonstrating Membrane when mutated 1–4). reduced lanes strongly was 3A, was (Fig. axin fraction of membrane recruitment the in axin ucaepteno F–xni h yols a less was GFP–axin 3B, contrast, cytoplasm (Fig. in membrane In plasma resulting panels). the the upper at Dvl2–CAAX, proteins in The both of of conductin. GFP–axin colocalisation coexpression and conductin upon axin of this of predominant that that domains pattern and DIX show axin, the punctate than results by Dvl2 determined with These is interaction weaker 13–16). a Dvl2–CAAX exhibits lanes by protein 3A, this (Fig. of recruitment membrane increased ieai,Dl oaie nctpamcpnt,adaxin and puncta, cytoplasmic in localises Dvl2 axin, Like CdtDIX ai otiigthe containing (axin AxinDIX eutdin resulted , hra odci a esefcet eutn nresidual in resulting degraded efficient, efficiently less was Axin conductin 4A). (Fig. whereas cells readout, a endogenous SW480 As of Dvl2. staining by immunofluorescence used inhibited we differently is these conductin and the disrupt axin of to Dvl2. strength with the fails interaction in conductin differences therefore and support axin and data 3C). (Fig. These axinM3 Dvl2 polymers. in extent than lower puncta. pattern a in Dvl2 to polymers of but, integrates its formation conductinM3 Dvl2 that the by staining propose abolish of We not indicated presence did axinM3, the as to diffuse in contrast axin, diffuse than remained partially ConductinM3 efficiency puncta, in lower Dvl2 remaining a with colocalised with and albeit 1C), (Fig. pattern staining ae oehr hs eut hwta h ifrn aaiiyof capability different the that show results these together, Taken eitdb Cdt by mediated u o fteDXdmi uatDlM–AX reduced Dvl2M2–CAAX, mutant domain axin-mediated Dvl2–CAAX, DIX of of level the the Coexpression of not 4A,a,f). (Fig. but staining catenin otiigteDXdmi fai (GFP–Cdt axin of conductin whereas domain 4A,d,e), DIX (Fig. the Dvl2–CAAX containing by inhibition to eut r uniidi i.4,.Smlry erdto of degradation Similarly, 4B,C. Fig. in expressed These transiently quantified 4A,h,i). (Fig. are Dvl2–CAAX by results inhibition to sensitive more eitdb odci Fg Ag.Ai otiigteDIX the containing Axin degradation (GFP–axin 4A,g). conductin affected (Fig. of domain conductin weakly by only mediated Dvl2–CAAX Importantly, oeefcetyihbtdb v2CA hndegradation than Dvl2–CAAX Axin Conversely, by conductin. inhibited by efficiently more enx nlsdwhether analysed next We CdtDIX a nfetdb v2CA,weesdegradation whereas Dvl2–CAAX, by unaffected was ora fCl cec 21)18 33 doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal AxinDIX b a lce yDl–AX(i.4D,E). (Fig. Dvl2–CAAX by blocked was ctnnuo oxrsino xnwas axin of coexpression upon -catenin b b ctnndgaain(i.4A,b,c). (Fig. degradation -catenin ctnndgaainmdae by mediated degradation -catenin b ctnndgaainmdae by mediated degradation -catenin * needn xeiet ( experiments independent eut r mean are Results activity. reporter TCF/ catenin-dependent of D changes in fold chart shows bar The control. loading h. 6 for Wnt3a without or with incubated and indicated siRNAs, the with transfected (D) cells HEK293T and X- lysates) Triton 100 panels, two lower hypotonic lysates; panels, three upper (C; U2OS (B), lysates MDA-MB-231 in from conductin and axin (ABC), (active) for dephosphorylated blotting Western (B–D) (ANOVA). ( experiments mean are Results cells. in individual quantified was nuclei DAPI-stained or (+Wnt) h untreated. 6 left for Wnt3a with were treated siRNAs indicated the with transfected cells MDA-MB-231 signalling. (A) Wnt axin on of conductin depletion and of Impact 2. Fig. CdtDIX P , .0 (Student’s 0.001 6 eaelreyresistant largely became ) ...o he independent three of s.e.m. n b . b ctnnsann in staining -catenin atnwsue sa as used was -actin 20;* 1200); 6 AxinDIX ...o three of s.e.m. t -test). b P ctnnin -catenin , b b n became ) -catenin -catenin, 5 0.05 b - 6); 35 b -

Journal of Cell Science HR REPORT SHORT nlsshspeitdta hr r e mn cd htare that acids amino bioinformatical ten 36 are previous there that A predicted interactions. has analysis domain respective the on depends DIX–DIX conductin and axin with interfere to Dvl2 ewe xnadcnutnad hrfr,mgtmediate might therefore, differ and, acids amino conductin these and of interactions domain Four axin DIX 2009). between of Arias, specificity and for required (Ehebauer be to likely ora fCl cec 21)18 33 doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal eetv I ed wn othe mutation. to M3 owing heads indicates DIX ‘X’ defective (green). Dvl2 conductin and and (red) axin of of domains models DIX interaction show right the F–xno GFP–axin or GFP–axin with co-transfected cells HEK293T of staining Immunofluorescence (B) band. unspecific *, of fractions. purity show blots pan- cadherin and Tubulin plasmids. indicated with transfected cells HEK293T of and fractions (M) (C) membrane cytoplasmic in HA and GFP interactions. domain DIX–DIX are by Dvl mediated with conductin and of axin association Differential 3. Fig. F–v2(re) cl a:20 bar: Scale (green). coexpressing CFP–Dvl2 cells U2OS in constructs (red) Flag indicated of staining ( experiments independent three of mean are Results were quantified. Dvl2-CAAX of in presence construct the GFP the of recruitment bar: Scale 20 right. the on are magnified regions boxed The (red). vector HA–Dvl2–CAAX vector or HA-only (+HA) a either with together xeiet ( experiments F–v2lclsto.Rslsare Results localisation. diffuse CFP–Dvl2 with cells of Quantification mean n 5 m 0) C Immunofluorescence (C) 600). .Clswt nfr membrane uniform with Cells m. 6 ...o he independent three of s.e.m. n A etr ltigfor blotting Western (A) 5 0) cee on Schemes 400). CdtDIX 6 (green) s.e.m. m m.

Journal of Cell Science HR REPORT SHORT i.4. Fig. e etpg o legend. for page next See ora fCl cec 21)18 33 doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal 37

Journal of Cell Science xeiet ( experiments etro ADl.Rslsaemean are HA-only Results with HA–Dvl2. together or siRNAs vector indicated with transfected cells TCF/ HEK293T of changes Fold (G) ftreidpneteprmns( experiments independent three of show blots SW480 vector). Flag from Flag– below lysates of in Numbers measurements HA plasmids. densitometric and indicated GFP with Flag, transfected for cells blotting Western (D,E) versus A. intensities from GFP of Blotting (C) rd nS40clstasetdwt F–xn(–) GFP–axin (a–c), GFP–axin with transfected DIX– cells by SW480 mediated in conductin (red) and interactions. axin domain of DIX inhibition Differential 4. Fig. REPORT SHORT 38 37 at penicillin-streptomycin 1% and serum with bovine (DMEM) medium fetal Eagle’s modified 10% Dulbecco’s in grown were treatments Cells and transfections culture, Cell temporal METHODS for AND activity. allow MATERIALS pathway to Wnt axin that of by tuning found block fine abrupt spatial we and an conductin clear, by over signalling favoured of not is suppression signalling). gradual are a hedgehog that this conceivable in in active for less was patched reasons conductin (e.g. the modes Although constitutive both inducible in act in and regulators modes negative feedback where with pathways signalling, contrasts other and Wnt remarkable of is regulators respectively, negative inducible and constitutive 2005). viable conductin Costantini, neutralise and to and axin can leads between mechanisms cDNA differences compensatory functional conductin/axin2 that of suggesting knock-in Replacement mice, upstream signalling. by Wnt axin of towards qualitative of regulation specific has the the here for responsiveness whether revealed consequences mode determined based feedback be quantitative reduced is than to regulator rather remains of its negative-feedback It a basis signalling. on as the act as mainly to capacity This the conductin conductin that Dvl2. idea of the of to favours and overproduction insensitive regulation pathway mere relatively negative is out of conductin that rules that than lower and remained cells axin amount Wnt-stimulated the in that conductin showing by of signalling Wnt in regulation TCF/ feedback the of 4G). activity (Fig. reporter Dvl2-stimulated of dependent not catenin increased versus but axin conductin further of of Axin inhibition knockdown axin in Dvl2, by differences by mediated the mediated with degradation conductin line not In 4F). but (Fig. axin by inhibited mediated Dvl3 material and (supplementary Dvl1 Dvl2, Dvl Like S1). with Fig. interaction their in differences odtoe eim(ilr ta. 03 t4 fe siRNA after h 48 at Wnt3a- 2003) with al., treated et were (Willert Cells using using cells). Lipofectamine medium transfected transfected or (SW480 conditioned were cells) were (Invitrogen) U2OS 2006) 2000 plasmids (HEK293T, al., (Sigma) and polyethylenimine et (Invitrogen), (Hadjihannas Oligofectamine conductin and 0 CO 10% xrsigidctdpamd.– Aol etr eut r mean are Results vector. HA-only –, plasmids. indicated expressing of Quantification (F) 20 lines bar: Dotted Scale HA–Dvl2M2–CAAX. cells. or transfected HA–Dvl2–CAAX mark (–), vector HA-only a F–odci Ct fg rGFP–Cdt or (f,g) (Cdt) GFP–conductin loecneitniisfo .Rslsaemean ( are experiments Results independent A. from intensities fluorescence u eut hdlgto h oeua ai fnegative- of basis molecular the on light shed results Our h eaaino ak ewe xnadcnutnas conductin and axin between tasks of separation The 2 2 Aol etr ,0.4 +, vector; HA-only , iNsaantai Tneegre l,2011), al., et (Tanneberger axin against siRNAs . n 5 ) * 6); b P ctnnfursec neste fS40cells SW480 of intensities fluorescence -catenin , .1( 0.01 b n ctnndpnetrpre ciiyin activity reporter -catenin-dependent A muoloecnesann of staining Immunofluorescence (A) . 5;* 65); t -test). b m ctnndgaainta xn tis It axin. than degradation -catenin n P ;+,0.8 ++, g; . , m 6 b b AxinDIX 5;** 65); .1 ** 0.01, .()Qatfcto of Quantification (B) m. ctnnnraie oGP(empty GFP to normalised -catenin ctnnitniiso niiulcells individual of intensities -catenin ...o he independent three of s.e.m. hi gen oehrwt either with together (green) (h,i) P m , P HA–Dvl2-CAAX. g , .0 (Student’s 0.001 .0 (Student’s 0.001 6 b ...o three of s.e.m. ctnndegradation -catenin nvivo in b b -catenin CdtDIX -catenin t -test). b ˚ t under C -catenin -test). 6 CdtDIX (Chia s.e.m. (d,e), b - ta. 07 a eemndi E23 cells. HEK293T in determined was 2007) al., et TCF/ Sigma. from n ebaefatoswr banduigteProteoJET the using Cytoplasmic obtained 2D). 2002) were Fig. al., fractions in membrane et (experiments and Lustig (Promega) panels, Buffer or upper Lysis Passive 2C, buffer or Fig. lysis in X-100-based (experiments Triton buffer in hypotonic prepared were blotting extracts western Whole-cell and fractionation lysis, Cell GFP–axin generate To biology Molecular aio . i,G,Gli,L,Hri,V n aosn .A. S. Aaronson, and V. Harris, L., Goldin, G., Liu, A., Bafico, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.159145/-/DC1 online available material Supplementary material Supplementary J.B.]. to Friedrich- KFO257 the number of [grant funding Forschungsgemeinschaft Initiative Fields Emerging Universita by Alexander supported was study This Funding manuscript. the wrote J.B. and J.B. D.B.B. and M.V.H., and D.B.B., data, experiments, analysed performed and designed M.V.H. and D.B.B. contributions Author interests. competing no declare authors The M. interests and Competing reagents, for Sussman D. and Dale, T. Bienz, M. Bru Kikuchi, after A. thank tested We was 2A). (Fig. Acknowledgements significance method Bonferroni or the on 4B,F,G) based Fig. testing and post-hoc by 2D ANOVA Fig. 1D; (Fig. P determined. were 4) Statistics 1, (Figs cells nuclear whole the of of or intensities 2) (Fig. background-free using regions and microscope at times, Axioplan2 acquired exposure were an images constant on measurements, intensity acquired For were (Zeiss). (Hadjihannas Metamorph Images previously 2006). described al., as et stained were cells Methanol-fixed microscopy Immunofluorescence rnfcin (2 transfection. lgai rt yA iuh OaaUiest,Jpn,GFP–axin by Japan), HA–Dvl2-CAAX UK). and University, University, UK) (Cardiff Cambridge, (Osaka Dale (MRC, T. Kikuchi Bienz M. site-directed A. by Dvl3 MD), (human) by Baltimore, and Quikchange 2011, Maryland, (rat) and of Dvl1 al., (University the Flag–axin for were axin Sussman D. et plasmids by of conductin using provided Expression Fiedler were mutants (Stratagene). mouse and generated M3 kit of 2007 mutagenesis and were 700–840 al., Dvl2 et respectively) acids of (Schwarz-Romond mutant amino conductin M2 and The axin exchanged. rat of os niGP(oh)adrtanti- rat and anti-active- (Roche) mouse anti-GFP 2002), al., mouse et Lustig G7; (C/ anti-conductin mouse Signaling), (Cell anti-axin rabbit as Sigma), from (all performed were: was antibodies Primary anti- blotting 2011). mouse al., Western et (Tanneberger (Fermentas). previously described Kit Extraction Protein i.1,,sga aisotie ihat-xnadanti-conductin and anti-axin in with antibodies. amounts anti-GFP with obtained conductin obtained those Densitometry to and ratios normalised were axin antibodies 2D signal of AIDA 1A,B, comparison Fig. with For performed (raytest). software was analysis Densitometric ern,J,Jrhw .A,Wu A., B. Jerchow, J., Behrens, vle eedtrie sn nard w-aldStudent’s two-tailed unpaired, using determined were -values Ku cancer human in activation pathway Wnt cells. constitutive for mechanism autocrine cnrfrtcnclassistance. technical for ¨ckner l . elc,D n icmir W. Birchmeier, and D. Wedlich, M., hl, ¨ acrCell Cancer b atn abtat-lg abtat-A abtanti-PanCadherin rabbit anti-HA, rabbit anti-Flag, rabbit -actin, ora fCl cec 21)18 33 doi:10.1242/jcs.159145 33–39 128, (2015) Science Cell of Journal 9 Z,3 tErlangen-Nu ¨t 6 b 9 497-506. , E)-6-Bromoindirubin-3 ctnndpnetpA eotratvt (Major activity reporter pBAR -catenin-dependent CdtDIX n GFP–Cdt and tl,M,Gim . srn,C,Wrz R., Wirtz, C., Asbrand, J., Grimm, M., rtele, ¨ rbr;adteDeutsche the and ¨rnberg; 19) ucinlitrcino an of interaction Functional (1998). a tbln(eoe)antibodies. (Serotec) -tubulin AxinDIX 9 oie(I)wsobtained was (BIO) -oxime b ctnn(B;Millipore), (ABC; -catenin mn cd 719–832 acids amino , TM Membrane 20) An (2004). t -tests

Journal of Cell Science HR REPORT SHORT aot,F,Jo . eg . ut,T,Jo,T,Kumn,C n Costantini, and C. Kaufmann, T., Joos, M. T., Kurth, L., A. Zeng, E., Jho, Arias, F., Fagotto, and T. M. Ehebauer, Lee, and A. L. Lee, A., C. Thorne, E., Tahinci, K., K. Jernigan, S., C. Cselenyi, M. Bienz, and F. Hamada, A., Cliffe, R. Nusse, and H. Clevers, F. Costantini, and V. I. Chia, M. Bienz, M., C. Cruciat, T., Zimmermann, G., Davidson, L., Y. Huang, J., Bilic, utg . eco,B,Scs . elr . itc,T,Krtn . a de van U., Karsten, T., Pietsch, S., Weiler, A. M., Sachs, Kikuchi, B., and Jerchow, M. B., Lustig, Kishida, S., Ikeda, S., Hino, F. H., Costantini, and Yamamoto, N. S., J. Kishida, Freund, K., C. Joo, C., Domon, T., Zhang, H., E. Jho, Bru V., M. Hadjihannas, Bienz, and J. Mieszczanek, J., T. Rutherford, C., Mendoza-Topaz, M., Fiedler, niiin n nrclua localization. intracellular and inhibition, F. domains. DIX Dishevelled and Axin the of determinants beta-catenin. of USA phosphorylation Sci. GSK3’s Acad. inhibiting by degradation E. signaling. wingless during membrane plasma 966. the to Axin 1192-1205. vivo. in equivalent functionally phosphorylation. LRP6 C. dishevelled-dependent Niehrs, and GSK3beta. and APC, beta-catenin, 596-599. with conductin, homolog, axin eeig . lvr,H,Sha,P . icmir .e al. et W. Birchmeier, M., P. Schlag, H., Clevers, M., and interactions Wetering, protein stability. for beta-catenin necessary are regulate axin to and ability Dvl their a of domains Axin2, DIX (1999). of pathway. transcription signaling the the of induces regulator negative signaling Wnt/beta-catenin/Tcf (2002). cancer. colon in instability J. 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