ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 000, 000–000 doi:10.1242/jcs.165704

CORRECTION In vivo analysis of formation and endocytosis of the Wnt/b-Catenin signaling complex in zebrafish embryos

Anja I. H. Hagemann, Jennifer Kurz, Silke Kauffeld, Qing Chen, Patrick M. Reeves, Sabrina Weber, Simone Schindler, Gary Davidson, Tomas Kirchhausen and Steffen Scholpp

There was an error published in J. Cell Sci. 127, 3970-3982.

The first line of the Abstract should read as follows: After activation by Wnt/b-Catenin ligands, a multi-protein complex assembles at the plasma membrane as membrane-bound receptors and intracellular signal transducers are clustered into the so-called Lrp6-signalosome.

We apologise to the readers for any confusion that this error might have caused.

1

Journal of Cell Science JCS165704.3d 10/11/14 12:36:43 The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 9.0.225/W (Oct 13 2006) tmlto,Wtrcpo rzld(z n h low-density the ligand and Wnt (Fz) Upon Frizzled receptor degradation. Wnt proteasomal stimulation, subsequent (b and protein repeat-containing beta-transducing ligase ubiquitin phosphorylates colo eiie o nee,909C,USA. CA, Keck 90089 California, Angeles, Southern Los of Medicine, University of Center, School at CIRM USA. Medicine Broad MA, Molecular address: 02115 and *Present Boston, Cellular Hospital, in Children’s Program Boston and School Medical Harvard IG,701Krrh,Germany. Karsruhe, 76021 (ITG), inln ope nzbaihembryos zebrafish in complex signaling naI .Hagemann H. I. Anja vivo In ARTICLE RESEARCH 3970 2014 July 7 Accepted 2013; December 31 attributed. Received properly is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` 1 complex destruction Clevers Kinase multi-protein Casein 1 (APC), a 2009; coli state, polyposis adenomatous by Axin1, Moon, Wnt-off containing degradation the and for In targeted (Angers 2012). human Nusse, cancer and and biology including cell stem disease, development, embryonic in pathway Wnt/ INTRODUCTION Zebrafish Endocytosis, Signalosome, Development, signaling, Wnt WORDS: KEY of reduction Wnt/ consequently, maintain and, Ap2 that membrane conclude We plasma signaling. the at formation Blockage endocytosis. during Ap2m stability of its maintain to endocytic Dvl2 with this interacts also but The that internalization signaling. ligand-receptor show for for essential We only not aggregates. is complex, process intracellular transducer the forms to bound Dvl2 still when However, Rab-positive path. a endocytic follows complex Wnt–Fz–Lrp6 The routes. separate complex take transducer the and after complex that, ligand-receptor find the We internalization, endocytosis. by disassembled partially activated the and to receptors complex transducer the assembled of continuously recruitment is Dvl2-mediated It by structure. dynamic the highly that a and show is embryos zebrafish signalosome is live of dissolution studies receptors imaging and Our clear. Lrp6-signalosome. formation not yet signalosome so-called membrane-bound of the mechanism the clustering into However, transducers the signal intracellular at Wnt/ assembles by activation After ABSTRACT ioeSchindler Simone uhrfrcrepnec ([email protected]) correspondence for Author alrh nttt fTcnlg KT,Isiueo oiooyadGenetics and Toxicology of Institute (KIT), Technology of Institute Karlsruhe a 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,37–92doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. (CK1 b Ctnnsgaigi nesnil vltoaiyconserved evolutionarily essential, an is signaling -Catenin ucinlast v2dgaain niio fsignalosome of inhibiton degradation, Dvl2 to leads function 2 a n lcgnSnhs iae3 Kinase Synthase Glycogen and ) b ctnnsgaigi vertebrates. in signaling -catenin nlsso omto n noyoi fteWnt/ the of endocytosis and formation of analysis b Ctnnpiigi o bqiiainb h E3 the by ubiquitination for it priming -Catenin m -uui fteedctcCahi dpo Ap2 adaptor Clathrin endocytic the of 2-subunit 1, b ,Gr Davidson Gary *, Ctnnlgns ut-rti complex multi-protein a ligands, -Catenin 1 enfrKurz Jennifer , 2 eatet fCl ilg n Pediatrics, and Biology Cell of Departments m -eitdedctssi motn to important is endocytosis 2-mediated b Ctnni constantly is -Catenin 1 1 b oa Kirchhausen Tomas , ik Kauffeld Silke , (GSK3b .GSK3 ). -TrCP) b 1 igChen Qing , eoe cieol uiglt atuain(lihe l,2003). al., et (Ulrich gastrulation Wnt/ late during stage, only this active becomes At signaling Wnt/PCP whereas organism. detectable, already intact is signaling Catenin an in pathways signaling 01 aaooe l,20)weesi a lopooe by proposed also for important has is Wnt/ it (CME) whereas endocytosis Clathrin-mediated 2006) that al.,others al., et Ohkawara et 2011; al., Yamamoto et Glinka 2011; 2013; al., endocytosis et Demir 2007; of al., (Bilic et activity type Lrp6 for the required is endocytosis about Caveolin-dependent Furthermore, discussion Wnt/ 2010). for on-going al., required an et Taelman is 2012; there al., et signaling(Dobrowolski maintain to late (MVBs) bodies in multi-vesicular all or sequestered endosomes al., that become suggested Wnt, et was including it components, (Li model signaling alternative for complex an found in ligand-receptor contrast, in indication In activated study the 2012). no an this was of In throughout there inhibition. endocytosis however, and non-disintegrating culture, activation cell signal that human is proposed of was cycle complex it entire Recently, destruction 2006). Bellen, and the Seto 2010; Metcalfe al., 2013b; signal al., et et for (Kim discussed internalization controversially ligand-receptor is maintenance of necessity the However, hs rvd noptimal as stage an a blastula provide in at embryos signaling these zebrafish chose Wnt we non-canonical tissue, and functional canonical Ap2 Wnt/ of in for requirement endocytosis endocytosis the of dissect Ap2 necessity To partner the signaling. clarify Catenin binding to postulated us its allow would of analysis functional Wnt/ during that and signaling Wnt/PCP of Wnt/PCP protein for hub essential is binding which Xenopus cargo CME, the the for fact, Fz4 to In Clathrin pits. Ap2 subunit coated links and forming selection into cargo It is for recruitment subunits responsible adaptor 2009). is and four Clathrin membrane, Traub, cytoplasmic This the 2012; (Ap2). with 2 (Kirchhausen, protein heterotetramer adaptor complexes a linker the on relies as but receptors such cargo to or 2007). membrane al., the et Yu non- (Chen 2008; regulate al., signaling et to Kim (Wnt/PCP) found 2003; polarity al., been et also cell has Wnt/planar CME canonical addition, In 2009). emdLp-inlsm Blce l,20) hc niisthe inhibits which GSK3 2007), of al., activity congruously et kinase complex (Bilic multi-protein Lrp6-signalosome to membranous termed bind a Lrp5/6 and form Dvl, complex to binds GSK3 Fz Lrp6; the and 2004). to Axin1 Axin1 al., and and et Dvl (Cong (Lrp5 as membrane such ligand-receptor 6 proteins This cytoplasmic together. recruits and cluster complex Lrp5/6) as 5 to referred proteins hereafter receptor-related lipoprotein 2 uigteedctcpoes ltrnde o iddrcl to directly bind not does Clathrin process, endocytic the During n tfe Scholpp Steffen and b Ctnnsgaig(lte n us,20;Kkcie al., et Kikuchi 2006; Nusse, and (Blitzer signaling -Catenin atuain(ue l,20) ic v2i h common the is Dvl2 Since 2007). al., et (Yu gastrulation 1 arc .Reeves M. Patrick , m a hw obn oDl,wih–i un–recruits – turn in – which Dvl2, to bind to shown was 2 b b h iadrcpo ope n h transducer the and complex ligand-receptor The . Ctnnsgaig thsbe ugse that suggested been has It signaling. -Catenin b ycmeiieybnigi oLrp5/6. to it binding competitively by nvivo in 1, ` b Ctnnsgaig ereasoned we signaling, -Catenin 2 arn Weber Sabrina , ytmt isc ohWnt both dissect to system a b 1, -Catenin b 1, m m 2-mediated 2and 1 , s m b b 2 1 - -

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Journal of Cell Science EERHARTICLE RESEARCH oreadfudte nrae .-odatracidification after 1.7-fold Wnt8 increased the to them next appeared found the cells quantified in rather and punctae We punctae source Dvl2 D). endocytosed 4C, supplementary Dvl2 (Fig. of Lrp6 aggregates number and whereas Wnt8 and observed 4A,B to S5B), (Fig. juxtaposed we Wnt8 Fig. embryos A1, of control material bafilomycin to accumulation compared with intracellular treatment overlapping 3; Movie After every material cell supplementary receiving and cells). 4I,J per (Fig. event minutes together endocytic 33 Wnt8 one of revealed Dvl2 events with internalization material by signalosomes of supplementary of eight Quantification induction and Wnt8. paracrine the of 4A,C at suggesting Dvl2 (Fig. S5A), for Fz1-positive Fig. out or membrane Lrp6 and Wnt-producing and plasma Wnt8 one a for Dvl2- the to positive adjacent clusters in a found cells we In and clone cell 4J). Wnt8 to (Fig. Lrp6-positive ligand-secreting embryo of aimed a zebrafish small in we overexpression blastomers generated be 2012), by of to we internalization al., clones order paracrine Therefore, et in and Wnt8. 2010) (Gross autocrine between Basler, exosomes distinguish and in on endocytosed (Port are secreted fashion ligands Wnt autocrine that suggested an been had function a it Ap2 in Since on Lrp6 depends and that Dvl2 fashion with paracrine together internalized is Wnt8 n 530 n onst nesnilrl fedctssdrn Wnt/ during endocytosis of role essential an signaling. Catenin to function remaining Ap2 knockdown points requires internalization partial The and Dvl2 the and Wnt8 of 4F,H). that (Fig. suggest result a reduced detectable Ap2 be of embryos might again still Wnt8 was intracellular control-treated but Ap2 accumulation treatment to in Wnt8 threefold compared and 4B,D,H) aggregates bafilomycin-treated (Fig. sixfold Wnt8 of In volume increased 4A,C,E,H). intracellular (Fig. the however, control embryos, to compared eaeams neetbei Ap2 Wnt8 in internalized of undetectable volume almost total The became 4E-G). of (Fig. knockdown treatment after internalized much as Ap2 half conditions. only was Dvl2 control reduced that was under formation Dvl2 Ap2 signalosome the in detection that of found fraction we our a Consistently, that escapes suggesting population also 4G), (Fig. blockage oivsiaeteitaellrlclzto ftetransducer stable of the use made of we complex, localization ligand-receptor the intracellular and complex the investigate endosomes late To Rab7-positive with colocalizes Wnt8 m ucini eevn el needn ftebafilomycin the of independent cells receiving in function 2 m m ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal i.2 inlsm omto ttectpamcmembrane Ap2m cytoplasmic on the dependent at is formation Signalosome 2. Fig. fWt RAo Ap2 expression or by induced mRNA be Wnt8 can of signalosome Fz1 the membrane. (I) of plasma (green). Formation the CFPGPI (J–L) to with Dvl2 Dvl2 of recruits Control co-expression supplementary (H) in S4. control constructs Fig. and expression material S3 indicated Fig. the material of supplementary images in shown are B-G CK1 Lrp6, for GSK3 the positive to signalosomes lead code of mRNA color Wnt8-GFP formation indicated of Expression the constructs. to injected related represent for channels I split and with CFP-GPI B-G areas in marker boxed images membrane Magnified the A,B,D,E–G,L,M). colors of in indicated mRNA (blue the ng in 1,2 shown with are together and indicated as expressed single represent images Confocal xetAi1adDl ue t2n)adAp2 and mRNA, ng) ng 2 at 1 (used at Dvl2 used and at were Axin1 constructs constructs except indicated all the stage; express epiboly that 30–50% embryos zebrafish live of opoio(O lgmr agtn Ap2 targeting oligomers (MO) morpholino s+..o e needn ape ah * given each. are samples bars independent ten Error of software. +s.e. Imaris as at with signalosomes membrane of plasma measurment the puncta. and Dvl2 quantification intracellular Manual arrow, (N,O) red puncta; arrow, Wnt8 green the intracellular signalosome; at arrow, signalosomes Yellow Dvl2-positive membrane. and plasma Wnt8- of reduction strong ucinuigtemrhln prah hs results These approach. morpholino the using function 2 opatebys(i.4) diinly efound we Additionally, 4E). (Fig. embryos morphant 2 b xn n v2(–,JLM.Snl hnesfrimages for channels Single J,L,M). (A–D, Dvl2 and Axin1 , m opatebysatrbafilomycin after embryos morphant 2 m function. 2 RA() LM neto f05mM 0.5 of Injection (L,M) (K). mRNA 2 z scin.Cntut were Constructs -sections. ofclmcocp analysis microscopy Confocal m opatembryos morphant 2 m aadAp2 and 2a P , m .5 **** 0.05, ue t16ng). 1,6 at (used 2 c , b -Catenin, m blasto leads 2b P , 0.001. 3973 b -

Journal of Cell Science nooe,icuigmlivsclrbde,fs ogigantic Although to 2004). fuse al., bodies, et fusion multi-vesicular (Cerny is homotypic including lysosomes aggregates a endosomes, small and to Dvl2 the endosomes leading with of of provide Vacuolin-1, embryos treated To formation inhibitor we molecule endosomes. the endosomes, late of that independent and evidence early further with colocalize Wnt8 EERHARTICLE RESEARCH 3974 (Rab5: treatment bafilomycin after vesicles Rab5 0.063 and aggregates overlap Wnt8 between positive strong correlation higher a and towards (0.02460.008) (0.172 Rab7 Rab5 with with colocalization endosomes. late and cases early from both 0.023 aggregates positive in (Rab5: observed bafilomycin 0.017 was with treatment coefficient after 0.008 colocalization the of of shown coefficient signals Rab7 Manders (mean or Rab5 a either by with measuring 1 Dvl2 by of of colocalization Wnt8 (Coefficient 0 or coefficient colocalization, Dvl2 colocalization of colocalization Manders either the the with quantified We markers 2011). al., Rab5 endosomal et tagged (Clark fluorescently Rab7 expressing or lines zebrafish transgenic h oa olo h nlzdWt hwdsm u little but some showed Wnt8 analyzed the of pool total The .0,Rb:0.104 Rab7: 60.005, Dvl2- of separation a suggesting 5B,D,I), (Fig. 60.005 ..) epciey(i.5,,) iia increase minimal A 5A,C,I). (Fig. respectively 6s.e.m), admlclzto) efudams no almost found We localization). 5random .4;Fg EGI.Ti itiuinshifted distribution This 5E,G,I). Fig. 60.041; .4;Fg FHI,sgetn that suggesting 5F,H,I), Fig. 60.046; .0 r0.003 or 60.001 .0;Rab7: 60.006; 60.002 5full iadrcpo ope cusa eyerytm on after point time the early from gastrulation. very zebrafish complex a during at endocytosis transducer occurs intracellular complex ligand-receptor the of dissociation otdt a7pstv aeedsmsfrdgaainor Rab5-positive degradation and for Dvl2- of form endosomes colocalization vesicles is late little to complex The Rab7-positive recycling. complex ligand-receptor to the ligand-receptor routed while Wnt8-positive aggregates, Lrp6-signalosome dissociates intracellular complex, the entire transducer the from the Dvl-positive of part the intracellular that signalosome, the results Subsequently, These observation internalized. shown). becomes not our data and cells 5J,K receiving support (Fig. the treatment in visible detectable the the not after of was any Wnt8 with overlap whereas not not vacuoles, is do aggregates aggregates Dvl2 Dvl2 of and appearance altered the cells, gastrula in vesicles ta. 99.Hwvr ti tl nla htteoii n the aggregates and these origin that the reasoned We what are. unclear punctae still Smalley these is 2007b; of it function al., However, an et 1999). Schwarz-Romond al., – et 2007a; al., punctae et cytoplasmic Romond form polymerize in dynamically to and Wnt/ to during domain reported process DIX essential been has its Dvl through past, the In Ap2 m -eitdedctssi eurdfrDl stability Dvl2 for required is endocytosis 2-mediated rspiamelanogaster Drosophila ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal hnesrltdt h niae oo oefrijce constructs. injected for code color split to indicated the with going to areas is related boxed that channels represent Dvl2 images and Magnified Wnt8 internalized. of be cluster show membranous scanning a before or indicates (F,G) hour Lrp6 1 with Wnt8 for of treated A1 colocalization Embryos bafilomycin plasma (F–I) nM the (B,C). 150 in Wnt8 with unless by Lrp6, induction does with after Dvl2 nor membrane (B–E) Wnt8 signalosome. with a colocalize (green) from neither Wnt8 (red) of Dvl2 (from endocytosis with analysis of together time-lapse 2) a Movie of material markers pictures supplementary membrane Still with (A) together (blue). colors CFP-GPI indicated the in shown CFP- ng), (1 Wnt8 Ap2 single except and ng, ng) 2 (1,2 at epiboly GPI used 30–50% at were constructs constructs indicated all the stage; of that mRNA embryos ng zebrafish 2 live express signalosome. of the analysis of microscopy endocytosis Confocal autonomous Cell 3. Fig. 2 vni aioyi-rae embryos bafilomycin-treated in even z scin.Cntut eeepesda niae n are and indicated as expressed were Constructs -sections. m 16n) ofcliae represent images Confocal ng). (1,6 2 b Ctnnsgaigi elculture cell in signaling -Catenin Aerde l,19;Schwarz- 1998; al., et (Axelrod b Ctnn(,) ro nA in Arrow (H,I). -Catenin 2 ugssthat suggests

Journal of Cell Science hs eut hwta v2saiiyad hrfr,Wnt/ therefore, and, Ap2 was of on stability zebrafish. depend Dvl2 stability activation signaling that protein Catenin mutual show results Dvl2 Ap2 the these However, Notably, after to 6H). unaltered 6G,K). led (Fig. (Fig. and expression and Dynamin 6L-N) I,J) (Fig. Ap2 ectopically and of dominant-negative overexpression was Ap2 6E,F either (Fig. with genes or co-injected embryos embryos target in Dvl2-injected reduced in of endocytosis. immunoblot impaired induced expression to by due protein Furthermore, protein Dvl2 of Dvl2 stability decreased endogenous Ap2 of dominant-negative impaired less analysis Consistently, the 6C). detected embryos Fig. of we Dynamin; to (DN overexpression mutant similar by to Dynamin2-K44A compared 6A,B), endocytosis (Fig. aggregates for Dvl2 embryos cytosolic of control amount decreased a EERHARTICLE RESEARCH hrfr,w etdwehredctssi eurdfrDvl2 for endosomes. required linked Wnt/Lrp-positive is Ap2 In still endocytosis the formation. whether complex from aggregate tested transducer release we the Therefore, after of Dvl2 assemblies to be might m ncdw mro Fg D,pitn oa to pointing 6D), (Fig. embryos knockdown 2 m vrxrsin(i.6) Together, 6D). (Fig. overexpression 2 m nrae v2cutrsz threefold size cluster Dvl2 increased 2 m opatebys eobserved we embryos, morphant 2 m eitdedctssin endocytosis mediated 2 m morpholinos 2 axin2 b - xn nctpamcpnte(i.7) etw addressed we We Next localization. Axin1 7M). impairs (Fig. endocytosis of punctae with blockage colocalized whether cytoplasmic is Dvl2 Dvl2 (DIX-domain) wild-type in the that Axin1 Axin1 found with in we interacts However, Notably, that intact. 7I-L). domain (Fig. the mutants membrane Axin1-GFP cytoplasmic at of aggregates reduction the Axin1-GFP-formed a remaining to the led and co- mutants fluorescence that Dvl2 Dvl2 found the We the of behavior. with subcellular expression its Axin1 analyzed and co-expressed mutants we mutants Therefore, Dvl2 Wnt/ of endocytosis. inability activate the whether to asked we Next, signaling. a eue hnDl igemtnsadtedul mutant of double interaction Ap2 that and the suggests Dvl2 This and 7A-E,F). single-mutants (Fig. co-expressed Dvl2 were of when expression that reduced found We was 2010). Ap2 al., for et Yu sites 2007; binding described were essential domains be both to because C-terminus, the at (AHEA) K4M n nte ntetrsn-ae Yxx tyrosine-based domain the DEP in the another in one and Dvl2, transducer (K340M) into the mutations point of two Ap2 introduced internalization requires and signaling complex whether test To Ap2 of Binding ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal m sncsayfrteatvto fWnt/ of activation the for necessary is 2 m oDl srqie o Wnt/ for required is Dvl2 to 2 b Ctnnsgaigi ikdt impaired to linked is signaling -Catenin m idn oDl nzbaih we zebrafish, in Dvl2 to binding 2 CF.Errbr r ie s+..o ten P of * +s.e. each. as samples given independent from are clone bars Wnt8 Error to (C–F). next cells in vesicles Dvl2-Cherry intracellular of Manual quantification (G) constructs. indicated injected for the code to color related channels split areas with boxed scanning. represent before images hour Magnified 1 for A1 with bafilomycin treated were Embryos (B,D,F) stage. cell Ap2 of injection represent single images by Confocal (indicated lines). CFP dashed nuclear by marked were only. clones cell Wnt8-producing one convenience, into For and H2B Wnt8 CFP-Histone ng ng eight- 0.25 0.1 at with later injected and stage stage Ap2 cell ng one-cell 1,6 at CFP-GPI, Fz1) of or ng constructs 1,2 indicated Dvl2, of ng mRNAs (2 of set injected a embryos with zebrafish live of analysis signalosome of internalization. analysis Clonal 4. Fig. lnsdslydi AFad(I). and (A–F in displayed generate clones to procedure Experimental (J) 3). Movie material signalosome (supplementary a from with (red) together Dvl2 (green) Wnt8 of of analysis endocytosis time-lapse a of pictures ouei oto el (1 cells control in to normalized volume cell per volume embryos. in different Represented three of 15 cells in responding volume total Wnt8-GFP of intracellular software) Imaris (using surface application after quantification Automatic (H) , .0;ns,ntsgiiat( significant not n.s., 0.005; z scin.(,)Cnrl.()Co- (E) Controls. (A,C) -sections. m ncl utr Y tal., et (Yu culture cell in 2 m opoio Ms tone- at (MOs) morpholinos 2 ofclmicroscopy Confocal b Ctnnactivity -Catenin 5 W 25,6 otn motive sorting axin2 P P , m § .5 *** 0.05; m b 0.05). 3 -Catenin and .()Still (I) ). 3975 lef1 m 2

Journal of Cell Science h niie noyoi fAi1atrc-xrsinwith co-expression after Axin1 of Dvl2 endocytosis the inhibited Ap2 whether the interested of also co-expression were of independently al., clusters et in Ap2 proteins (Schwarz-Romond two Dvl2 the of of colocalization showed domain Dvl2 with co-expression indeed, DIX and, 2007a) the interaction to and directly dynamics their Dvl2 investigate with to embryos zebrafish EERHARTICLE RESEARCH 3976 the tagged of fluorescently GSK3 members Axin1, Therefore, other of together. holding versions Wnt/ complex by their gain transducer activity aggregates signaling Dvl2 these Catenin whether asked we Next intracellularly cluster Dvl2 complex with transducer the of Members Wnt/ for prerequisite a is the which Dvl2 with complex, of interaction observation the Ap2 that our with suggest findings to plasma These mutants. similarly the Dvl2 at Axin1-GFP 7G,H), predominantly (Fig. reduced found membrane Dynamin was Axin1 DN and of fluorescence co-expression that found oaie rdmnnl otepam ebaewe expressed when membrane plasma the to predominantly localized m Fg AD upeetr aeilFg 3-) We S3A-C). Fig. material supplementary 8A-D; (Fig. 2 K340M/AHEA m nvivo in sncsayt lo noyoi ftetransducer the of endocytosis allow to necessary is 2 h cfodpoenAi1wsdsrbdt bind to described was Axin1 protein scaffold The . obemtn Fg L.Hwvr Axin1 However, 7L). (Fig. double-mutant b and b Ctnnwr mgdi live in imaged were -Catenin m sal ocmest for compensate to able is 2 b Ctnnsignaling. -Catenin b - v2adAp2 and Dvl2 membrane cytoplasmic with colocalization lead obvious the not to did Dvl2 to of Expression localize S4G). Fig. material did (supplementary – junctions adherens Wnt/ canonical of effector main GSK3 of clustering cytoplasmic oehrwt Ap2 with together hneo idtp v2t Dvl2 to Dvl2 wild-type GSK3 of and change Dvl2 of clusters larger Ap2 times of GSK3 co-expression and recruited 8E), Dvl2 (Fig. overexpression However, S4E). material (supplementary Fig. embryos in expressed when cytoplasm the for Ser/Thr-kinase main opee nisiatv omta lospoogdmaintenance prolonged the allows that of Wnt/ form of inactive pre-stage its in a transducer complexes the resembles that hypothesize We complex endocytosed. is that and GSK3 Axin1, catenin includes that complex transducer intracellular of clustering oprdt v2cutr ln Fg JL;tec-xrsinof co-expression the times 8J,L); four (Fig. size alone Ap2 in clusters increased Dvl2 which to clusters, compared same the to recruited hs eut ugs htDl-oiiecutr ersn an represent clusters Dvl2-positive that suggest results These m n Dvl2 and 2 b ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal Ctnnsgaigfloigsgaooeendocytosis. signalosome following signaling -Catenin b Ctnn(i.8K). (Fig. -Catenin m eeco-expressed, were 2 K340M/AHEA m n Dvl2 and 2 b Ctnn perdeel itiue in distributed evenly appeared -Catenin, n03 MOfr15husbefore hours 1.5 scanning. for DMSO Vacuolin-1 0.3% with in treated Embryos 0.3% (K) with treated DMSO, embryos Control samples (J) independent each. five of +s.e. represent bars was Error Wnt8 automatically. or calculated Dvl2 either Manders for and Coefficient manually set were function. Thresholds module software Coloc the Imaris using performed Wnt8 was Analysis and Rab7. Rab5 with with Dvl2 of of colocalization Quantification (I) CFP-GPI. transiently expressing line transgenic zebrafish (C,D) Rab7-GFP or in (A,B) His-CFP Rab5-GFP marker nuclear Wnt8 with of together expression Clonal (E–H) (blue). CFP- GPI marker membrane indicated with the together in colors shown are and as indicated expressed were Constructs single sections. represent tissue images host Confocal and (right). (left) Wnt8- clone between cell border positive the indicate E–H lines in Dashed line. transgenic Rab7-GFP zebrafish or (C,D) (A,B) Rab5-GFP in Dvl2 of Expression (A–D) stages. at epiboly constructs 30–50% indicated of mRNA that express embryos zebrafish Rab7- transgenic or GFP Rab5-GFP of analysis microscopy Dvl2 markers. and endosomal Wnt8 with of Colocalization 5. Fig. b i o edt n cytoplasmic any to lead not did K340M/AHEA Ctnn(i.8) oee,when However, 8I). (Fig. -Catenin b b Ctnnsgaigada atof part as and signaling -Catenin K340M/AHEA Fg 8G). (Fig. b b Ctnnwsefficiently was -Catenin Fg FH hra the whereas 8F,H) (Fig. Fg C.GSK3 8C). (Fig. b b Ctnndestruction -Catenin b i o edt any to lead not did nopnteupon punctae into Ctnn–a the as – -Catenin m Confocal omdfour formed 2 b and b ,the z b - -

Journal of Cell Science EERHARTICLE RESEARCH lyarl nvrosohrsgaigptwy,sc steFfand Fgf the as such Tgf- pathways, signaling the other various in role a play Wnt/ during crucial to and Lrp6-signalosomes Ap2 of overexpressing formation the Wnt/ induce enhance studies to able previous We Lrp6-signalosomes. structures were these with termed 2007), accordance al., et in (Bilic and, of aggregates cells membranous Wnt-responding Wnt/ the found of transducers we and far, receptors stage, ligands, blastula So zebrafish In at 2013a). organism. al., et embryos Kim vertebrate 2007; al., et living (Bilic cells culture in tissue only a reported been have signaling in Wnt canonical of signalosomes but Wnt/ level to subcellular of process regard the with investigate Lrp6-signalosome this to of endocytosis was subsequent the study and our formation Lrp6-signalosome of aim The zebrafish. Wnt/ of mechanism biological b cell the investigated we study, this In Ap2 DISCUSSION ftasuto acdsadtepoeso endocytosis. of process the and cascades transduction regulationof the processes: biological cell important two connects that Ap2 that hypothesize we Thus, 2012). al., ctnnsgaigb sn ihrslto ofcliaigin imaging confocal high-resolution using by signaling –catenin m ucindrn r6sgasm formation signalsome Lrp6 during function 2 b inln ahasi erfs mro Uaakret (Umasankar embryos zebrafish in pathways signaling b Ctnnsgaigidpnetyo h iadby ligand the of independently signaling -Catenin b m Ctnnsgaig p a ensgetdto suggested been had Ap2 signaling. -Catenin ,wihipista p n noyoi are endocytosis and Ap2 that implies which 2, b m Ctnnsgaigo a on signaling -Catenin sacnrlhbprotein hub central a is 2 b Ctnnptwyin pathway -Catenin lsuasae.Hwvr ubro noyi vnsmight events endocytic of detection. number our escaped a 15–20 have completely of in However, diameter have stages. a microscopy (with blastula which cells confocal large embryos, and these translucent zebrafish high-resolution monitor with to using able combination were by ligand-bound We three clathrin- 2012). to had processes al., that one et that only proposed (Cocucci border visible, endocytosis been to receptors able of became had are vesicles event It Lrp6 coated analysis. transient with previous and escaped fast colocalizing large a Wnt A1, suggesting bafilomycin the of with in vesicles aggregates endocytic blocking Wnt8 of by However, tagged maturation assay. very imaging the fluorescently clonal detected al., our of of had et cells we Yamamoto receiving signal Initially, 2005; intracellular 2008a). al., al., little et al., et Piddini et Yamamoto 2012; Glinka 2006; al., 2006; be et Nusse, to Jiang and 2011; (Blitzer endocytosis signaling. of for type necessary the Wnt/ for concerning required conclusions their in a Wnt/ to Drosophila leads of which Ap2 endocytosis, decrease and of formation knockdown Lrp6-signalosome that found We signaling and Endocytosis ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal n nmueadhmncl ie r controversial are lines cell human and mouse in and b opoio(O lgmr against and oligomers constructs (MO) indicated morpholino the express of that mRNA embryos synthetic zebrafish live of on analysis depends activity and Ap2 stability Dvl2 6. Fig. CAwsue slaigcnrl (E–K) control. with loading hybridization as used was embryos. PCNA double-morphant in of Dvl2 reduction endogenous a Ap2m2- shows and embryos blot embryos mRNA-expressing Western morphant (D) Ap2m2 control. of to analysis compared dominant-negative (C) express Dyn that (DN) embryos in and (B) are Ap2 Dvl2 in of detectable clusters hardly Intracellular (A–C) stage. epiboly r ie s+..o ieidpnetsamples independent five **** of each. bars +s.e. Error as M. and given L are in aggregate. shown Dvl2 data of of Quantification size (N) Ap2 increased of to is expression leads side Ectopic mRNA dorsal (L,M) (the right). shown the is on view stained lateral 30–40 the of embryos; representative are blastula-stage. shown at Embryos constructs indicated with injected Ctnnsgaigadwehredctssis endocytosis whether and signaling -Catenin b Ctnnsgaig rvossuisin studies Previous signaling. -Catenin m function. 2 P , 0.001. ACLM ofclmicroscopy Confocal (A–C,L,M) axin2 m ed oardcinof reduction a to leads 2 m and obemrhn embryos double-morphant 2 lef1 rbdi embryos in probed ap2 m m 2a/b )during m) nsitu In m t40% at 2 3977

Journal of Cell Science EERHARTICLE RESEARCH v2ageae eed nteitrcinwt Ap2 a 3978 from with of stem concentrations interaction high all that Taken aggregates the event. Dvl2 internalization on previous cytoplasmic of that depends function Wnt/ suggesting and during formation aggregates that function show Dvl2 and data stability Our signaling. Dvl2 Catenin of on interaction effects shown dramatic previously the on focused Ap2 we study, this In The (A) right). the on is side dorsal (the shown Ap2 is on of view induction dependent lateral the is by activity reflected pathway and signaling internalization Dvl2 7. Fig. 45-ipopae ncnrs oordt,itraiainof not was internalization aggregate activation signal data, this Wnt observed. our to phosphatidylinositol for response to of in essential Lrp6-signalosomes contrast presence the In is on (4,5)-bisphosphate. function depends suggest but Lrp6 Ap2 study of formation site this the and of at accumulates authors assembly clathrin The activation, cell Wnt 2013a). after mammalian al., that, Ap2 using et of (Kim study interaction receptor recent describes a which by lines, confirmed This reduced. was same Lrp6-signalosomes of the result formation At the comparable Wnt8. found Ap2 we any of time, accumulation find of extracellular not or did function intracellular we Dynamin2, the dominant-negative down knocking I oto.Ai1ageae ntectpam JL oepeso fDl ige JK n obemtns()tgte ihAi1 M ooaiaino Axin1 stage. AP2 of one-cell Colocalization and (M) at Dvl2 Axin1. each) between with single ng together interaction represent (L) (2 when images double-mutants mRNA reduced and Confocal synthetic stability (J,K) punctae. as single- its cytoplasmic Dvl2 injected and of in membrane Co-expression Dvl2 (J–L) cytoplasmic wild-type cytoplasm. the the with at in aggregates blocked Axin1 is Control. Axin1 (I) (I–M) stage. one-cell at yra-iePRwt rmr gis xn bu)adLf rd nebynccN netdwt h niae osrcs ro asaegvna +s.e. as given cytoplasm. are the bars in Error signal constructs. Axin1-GFP indicated the the reduces with endocytosis injected Dynamin-dependent cDNA of embryonic Blockage on (G,H) (red) duplicates. 12 Lef1 show in images and scanned Confocal (blue) experiments Axin2 independent against two primers of with PCR real-time by hnw lce noyoi ttepam ebaeby membrane plasma the at endocytosis blocked we When m m ihDl Y ta. 07 ue l,21) hc has which 2010), al., et Yu 2007; al., et (Yu Dvl2 with 2 uui srqie opoetDl rmdegradation from Dvl2 protect to required is subunit 2 m m z - tcso ieebysa 0 pbl tg.Idctdcntut eeijce t1n ssnhtcmN,ecp xn 2ng), (2 Axin1 except mRNA, synthetic as ng 1 at injected were constructs Indicated stage. epiboly 40% at embryos live of stacks axin2 m m rb expressing by or 2 ihteLp co- Lrp6 the with 2 xrsini lsuasaeebysa 0 pbl.Ebyswr netdwt niae osrcs the constructs; indicated with injected were Embryos epiboly. 30% at embryos blastula-stage in expression n 5 2 B 33;(C) 33/39; (B) 72; m -idn sites. 2-binding m b 2, - n 5 z 72;(D) 27/29; scin flv mro t4%eioysae niae osrcswere constructs Indicated stage. epiboly 40% at embryos live of -sections osiuiepoeltcdsrcin(lfee l,2003). the of al., activation that et its suggested (Cliffe report prevents lysosomal recent formation destruction and a the proteasomal Furthermore, polymer proteolytic Indeed by Dvl–Axin membrane. controlled constitutive and is from the Dvl2 Dvl degradation from protect of removal to stability important after The be 2010). degradation al., might et complex (Metcalfe before transducer culture cell human in reported opnnso h inactive the of cytoplasmic combination of a Ap2 endocytosis, complexes, components al., transducer to After the et form 6). binds aggregates (Fig. Dvl2 Dvl2 (Gao protein membrane endocytosis partners Dvl2 plasma does stabilizes binding the why stabilizing quickly At is So, 2010). needs Dvl2 that 2007). and suggsted al., been degraded has et It stability? (Bryja Dvl2 maintain promote pool to important Dvl is hyperosmolaric endocytosis the with that culture suggested in have is the SN4741 sucrose Dvl2 using line cell that experiments Ap2 neuronal and previous mouse and complex, Indeed, transducer degraded. Dvl2 signal subsequently the between of binding endocytosis with interference Wnt/ activate can Dvl2 v2wt te ebr ftetasue ope uhas such complex transducer of the colocalization of showing GSK3 members data Axin, our other by with supported Dvl2 is This Dvl2. (A–E) nsitu In n ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal 5 32;(E) 23/29; b yrdzto hw erae ciiyo h Wnt/ the of activity decreased shows hybridization and n 5 b Ctnni h yols,wihhsbeen has which cytoplasm, the in -Catenin 33.()Rltv agtgn xrsinanalyzed expression gene target Relative (F) 33/35. b Ctnntre ee,w rsm that presume we genes, target -Catenin b Ctnndsrcincmlxand complex destruction -Catenin m simpaired. is 2 m m blocks 2 ,which 2, b -catenin

Journal of Cell Science oddvsce u eprrl typlmrzdi the Clathrin in the subunit polymerized of its dismantling with intracellular stay the Ap2 including that temporarily endocytosis, coat speculate ligand-receptor but We after in the cytoplasm. shape vesicles from that their dissociate loaded al., alter suggests components et not (Cerny signalosome do this Vacuolin-1 chemical and small 2004), Rab7 the or not with do combination Rab5 aggregates Dvl2 our early route: with in However, different a found overlap endosomes. takes be Dvl late that can show in and data endocytosis subsequently by and up endosomes taken together Wnt8 is that Lrp6 demonstrate we with Indeed, 2014). al., et Vinyoles EERHARTICLE RESEARCH ihAi1 GSK3 Dvl of Axin1, colocalization signalosome showing Lrp6 by with lysosomes entire or the MVBs that to suggested localizes internalization cells after HeLa Dvl2 in and Wnt/ Studies Wnt8 of promote routing Endocytic thus, transducer and, the zebrafish. Dvl2 of in signaling formation stabilize subsequent complex the therefore, and We, 2013). signalosomes al., Ap2 et that (Jung in HEK293T hypothesize de-ubiquitylation line by cell Dvl human of the stabilization to leads pathway Wnt b m n yorce Temne l,2010; al., et (Taelman Lysotracker and -eitdrcuteto v2t Lrp6- to Dvl2 of recruitment 2-mediated m ih elne othe to linked be might 2 b -Catenin erdto.Bnigo h n iadt eetr z and Fz1 receptors to ligand Wnt the of Binding degradation. sebyo h etuto ope n the and complex plasma the on destruction the is balance the and the 9A) cytoplasm of (Fig. state the assembly Wnt-off between the In the Dvl2 membrane. of and members complex between al., exists et balance (Kim Axin1 PP1-mediated by protein state scaffold inactive 2013b). the which an in of into state dephosphorylation degradation active the an for in from routed assembly converted complex after destruction be this maintained Furthermore, might 2012). is al., and et (Li is structure cytoplasm complex stable destruction the very that In a proposed Dvl2 1999). recently al., was so-called et it Smalley the addition, 2007b; – al., et aggregates (Schwarz-Romond biological Dvl2 punctae the a intracellular recognizing as of studies previous exists relevance hypothesis with This Dvl2 accordance complex. that in transducer is to indicate intracellular back the data translocated of Our are member membrane. (Kirchhausen, Clathrin and plasma Hsc70 Ap2 the protein process this cognate In heatshock 2002). the the by vesicle. of dismantled the are activity vesicles from Clathrin-coated signalosome endocytosis, Dvl2-positive After the of dissociation nsmay epooeamdl(i.9,i hc naffinity an which in 9), (Fig. model a propose we summary, In ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal Dvl oueatrsraeapiainuigIai software. Imaris as using GSK3 (A,B) application in surface size after punctae volume of quantification Automatic (D) 2n ah , go Ap2 of ng 1,6 mRNA each, synthetic ng as (2 injected were constructs Indicated single cluster complex Dvl2. transducer with the intracellularly of Members 8. Fig. ie s+..o e needn ape each. samples independent ten ****P are of bars +s.e. Error (E,F). as in given size punctae of Quantification (L) yc-xrsino Ap2 of improved co-expression be by can colocalization but aggregates, positive in size punctae (I) of (E,F). Quantification (H) mRNA. mutant aeilFg 3)atrc-xrsino Ap2 of co-expression after S3G) supplementary Fig. with material (compare unaltered is distribution ooaiaincnb mrvdb oepeso of co-expression by Ap2 improved be can colocalization uca htclclz ihDl A vni the in even Ap2 (A) of Dvl2 presence with intracellular colocalize as that distributed punctae is Axin1 (A,B) expression. GSK3 and (A–D), (D–H) Axin1 colors. of indicated distribution with Subcellular displayed and stage cell npam ebaecutr fe oepeso of co-expression after Ap2 clusters membrane plasma in cmaewt upeetr aeilFg 3)after Ap2 S3E) of Fig. co-expression material supplementary with (compare K340M/AHEA m m , RA G GSK3 (G) mRNA. 2 n Dvl and 2 b z .0;ns,ntsgiiat( significant not n.s., 0.001; scin flv mro t4%eioystage. epiboly 40% at embryos live of -sections DH lsesi v2pstv grgtsand aggregates Dvl2-positive in clusters (D–H) b Ctnncutr r esaprn nDvl2- in apparent less are clusters -Catenin b ctnn(–)i h otx fDl co- Dvl2 of context the in (I–L) -catenin obemtn mRNA. mutant double K340M/AHEA m B.()Ai1i on predominantly found is Axin1 (C) (B). 2 m n Dvl and 2 m b RA() (G) (J). mRNA 2 m obemtn mRNA. mutant double ofclIae represent Images Confocal ,12n F-P)a one- at CFP-GPI) ng 1,2 2, itiuini unaltered is distribution b Ctnndestruction -Catenin K340M/AHEA P § 0.05). b b Ctnnis -Catenin double -Catenin b b m -Catenin and 2 3979

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(Bilic and al., signalosome activated et an Davidson of formation the to EERHARTICLE RESEARCH 3980 Technology were of and Institute ( Protection Karlsruhe zebrafish Breeding Animal the (KIT). and on (Regierungspra¨sidium Az.35-9185.64) law Committee Karlsruhe, Animal-Protection German Local by the in approved performed with were procedures experimental accordance and husbandry zebrafish All manipulation and embryos Zebrafish METHODS AND MATERIALS destruction Axin1-positive the of GSK3 turn including in recruitment – which complex Lrp6, the of Ap2 phosphorylation to initiates and binds and is polymerization Dvl2 receptor the enough, the high is to receptors When recruited Fz1 9B). accumulated (Fig. of complex number receptor the the of clustering initiates Lrp6 im,S oi,M)atr2 p.Tedt epeeti hsstudy this in present we pigment data prevent The hpf. To (PTU, 20 2002). 1-phenyl-2-thiourea after al., mM MO) 0.2 et Louis, in St (Brand raised Sigma, cycle were dark embryos h formation, light/10 h 14 m oehriiit noyoi ffatoso the of fractions of endocytosis initiate together 2 ai rerio Danio b otercpo ope,leading complex, receptor the to – eemitie t28 at maintained were ) m .Ti a induce may This 2. ˚ Cona niae osrcswr netda n-elsaeo,frclone for All or, stage injection. mRNA, capped one-cell as before cells at 8–16 stage of injected one into one-cell were formation, constructs at indicated (Sigma-Aldrich) Pronase from gift kind a were Rab5- lines AB2O2. Link. transgenic zebrafish Brian expressing wild-type Rab7-eGFP KIT and of eGFP analysis from acquired were zAp2 morpholinos and Constructs ihtemesg ahn i (Ambion). Kit Machine mMessage the with GTCCA;Ap2 TGGTCCTCAAA; eekn it rmJmSih(aeane l,2009). al., et (Hagemann Smith Jim from xRab5-GFP gifts (HistoneH2B-CFP), kind His-CFP were CFP-GPI, Brand 2005). Michael by al., provided et kindly (Rhinn was hAxin1- zWnt8-GFP Davidson. Addgene. Gary from from are #17048 2001); hCK1c al., GFP, et (Lekven xGSK3 ORF1 17086, zWnt8 x # #29680, 2004); 2010); al., al., et et (Waxman zDvl2 LTD. 1994). GeneTools from obtained were ATG the spanning AAC) h -adCtriio Dl pamdfo hroSinii)( Scientific) Thermo Fidelity from (plasmid to High zDvl2 introduced of C-termini were Phusion and sites N- using Restriction the Biolabs). directions England (New manufacturer polymerase per performed ihpie (5 A primer with GyetCTP[idgf rmUeStra Uwe Ap2 from Ap2 for for 2012)] gift pCS2+ into [kind introduced was pGlyserteC-TFP product resulting the and GAGTATGACCGATGGGGAAA-3 mro eehretdadezmtclydcointdwith dechorionated enzymatically and harvested were Embryos Ap2 al., et (Rupp pCS2+ in subcloned were constructs expression All o Dl-hryadzv2mtns l C ecin were reactions PCR all mutants, zDvl2 and zDvl2-Cherry For m abmrhln lgmr (Ap2 oligomers morpholino 2a/b m awsapiidfo 4hfzbaiheby DAlibrary cDNA embryo zebrafish hpf 24 from amplified was 2a ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal GP Lp-F n Lp-hrywr idyprovided kindly were hLrp6-Cherry and hLrp6-GFP -GFP, m 2a-TFP. 9 -CCATGGAATGTGAGGTTTGA-3 b CtnnGP(ilradMo,19) #16839, 1997); Moon, and (Miller -Catenin-GFP m b 5 2b: 9 -CCTCCAATCATGGTGGCGGTTA- 9 eitdb h neato fDvl2 Ap2 of with interaction the by mediated is internalization Subsequent (C) membrane. cytoplasmic the at and formation Fz signalosome to Dvl2 of recruitment Ap2-dependent co-receptor, inducing Lrp6 thereby and receptor bound Fz is to ligand situation, Wnt-on the eusrto fAi,CK1 Axin, of sequestration cieaddegrades and active the state, b Wnt-off the In (A) signaling. 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Journal of Cell Science ba)wr sdi :00dilution. 1:1000 a in used were from from (ab137727 abcam) AP2M1 blot (GTX129968 human western against Dvl2 antibody polyclonal for zebrafish and and GeneTex) against assay antibody reporter luciferase Polyclonal Wnt analysis. for buffer lysis Triton normalized assayed subsequently and to was stage activity shield luciferase at The harvested activity. luciferase were for each embryos ten indicated. of as pools mRNA synthetic Four plus Gradl) Dietmar from (gift DNA plasmid mg rcsigwspromdb sn nIai .. software 6.3.1 Imaris an using by performed AG). 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Beretta, N., Kirsch, K., Demir, aisn . u . hn . ii,J,Fne,U,Sank . lna A. Glinka, P., Stannek, U., Fenger, J., Bilic, J., Shen, W., Wu, G., Davidson, H. Varmus, and L. Schweizer, F., Cong, ouc,E,Aut . oln,S n ichue,T. Kirchhausen, and S. Boulant, F., Aguet, E., Cocucci, M. Bienz, and F. Hamada, A., Cliffe, orwlk,R n eRbri,E M. E. Robertis, De and R. Dobrowolski, a,C n hn Y.-G. Chen, and C. Gao, lvr,H n us,R. Nusse, and H. Clevers, lr,B . itr . oe,A .adLn,B A. B. Link, and R. A. Cohen, M., Winter, S., B. Clark, hn . e eg,D,Bon . h,S,H,L . ilr .E,Caron, E., W. Miller, A., L. Hu, S., Ahn, J., Brown, D., Berge, ten W., Chen, en,J,Fn,Y,Y,A,Myk,K,Brooo . lmemn J., Klumperman, B., Borgonovo, K., Miyake, A., Yu, Y., Feng, J., Cerny, lna . od,C,Krc,N,Hag .L,Kznky,O,Igligr D., Ingelfinger, O., Kazanskaya, Y.-L., Huang, N., Kirsch, C., Dolde, A., Glinka, rn,M,Gaao .adN¨sli-ohr,C. Nu¨ sslein-Volhard, and M. Granato, M., Brand, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.148767/-/DC1 online available material Supplementary material Supplementary GM- numbers release. [grant immediate Health P.R. for of 1036. PMC Institutes FOR in National DFG Deposited the group Noether 075252]. by research Emmy supported Wnt DFG are DFG the T.K. the by and and supported the were and S.S. 847/2 and fellowship S.W. S.K., J.K., A.I.H.H., Funding performed constructs. Schindler zDvl2 S. the generated and P.M.R. S.W. analysis. and blot Q.C. Western assays. the the reporter performed tested TOPFlash G.D. and the generated performed S.K. J.K. constructs. analysis. Wnt8-tagged data and experiments performed A.I.H.H. contributions Author interests. competing no declare authors The interests Competing lines. fish transgenic Dosch Roland for and Germany) WI) School, Milwaukee, Medical Wisconsin, of University (Go¨ttingen constructs College cDNA (Medical for Link Brian Germany) and Technology, and UK) of London, Institute (MRC, (Karlsruhe Stra¨hle Smith Jim Uwe Gradl Germany), Dietmar Technology, the Germany), of on Dresden, Institute (TU comments (Karlsruhe Brand valuable Michael for thank also Heidelberg) We (DKFZ, manuscript. Gross Julia thank We Acknowledgements rj,V,Caja V., Bryja, R. Nusse, and T. J. Blitzer, M. Bienz, C.-M., Cruciat, T., Zimmermann, G., Davidson, Y.-L., Huang, J., Bilic, T. R. Moon, and S. Angers, eindtersac,AIHH,TK,S cop rt h manuscript. the wrote Scholpp S. T.K., A.I.H.H., research, the designed xlo,J . ilr .R,Sumn .M,Mo,R .adPrio,N. Perrimon, and T. R. Moon, M., J. Shulman, R., J. Miller, D., J. 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Journal of Cell Science tmlto,Wtrcpo rzld(z n h low-density the ligand and Wnt (Fz) Upon Frizzled receptor degradation. Wnt proteasomal stimulation, subsequent (b and protein repeat-containing beta-transducing ligase ubiquitin phosphorylates colo eiie o nee,909C,USA. CA, Keck 90089 California, Angeles, Southern Los of Medicine, University of Center, School at CIRM USA. Medicine Broad MA, Molecular address: 02115 and *Present Boston, Cellular Hospital, in Children’s Program Boston and School Medical Harvard IG,701Krrh,Germany. Karsruhe, 76021 (ITG), inln ope nzbaihembryos zebrafish in complex signaling naI .Hagemann H. I. Anja vivo In ARTICLE RESEARCH 3970 2014 July 7 Accepted 2013; December 31 attributed. Received properly is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` 1 complex destruction Clevers Kinase multi-protein Casein 1 (APC), a 2009; coli state, polyposis adenomatous by Axin1, Moon, Wnt-off containing degradation the and for In targeted (Angers 2012). human Nusse, cancer and and biology including cell stem disease, development, embryonic in pathway Wnt/ INTRODUCTION Zebrafish Endocytosis, Signalosome, Development, signaling, Wnt WORDS: KEY of reduction Wnt/ consequently, maintain and, Ap2 that membrane conclude We plasma signaling. the at formation Blockage endocytosis. during Ap2m stability of its maintain to endocytic Dvl2 with this interacts also but The that internalization signaling. ligand-receptor show for for essential We only not aggregates. is complex, process intracellular transducer the forms to bound Dvl2 still when However, Rab-positive path. a endocytic follows complex Wnt–Fz–Lrp6 The routes. separate complex take transducer the and after complex that, ligand-receptor find the We internalization, endocytosis. by disassembled partially activated the and to receptors complex transducer the assembled of continuously recruitment is Dvl2-mediated It by structure. dynamic the highly that a and show is embryos zebrafish signalosome is live of dissolution studies receptors imaging and Our clear. Lrp6-signalosome. formation not yet signalosome so-called membrane-bound of the mechanism the clustering into However, transducers the signal intracellular at Wnt/ assembles by activation After ABSTRACT ioeSchindler Simone uhrfrcrepnec ([email protected]) correspondence for Author alrh nttt fTcnlg KT,Isiueo oiooyadGenetics and Toxicology of Institute (KIT), Technology of Institute Karlsruhe a 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,37–92doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. (CK1 b Ctnnsgaigi nesnil vltoaiyconserved evolutionarily essential, an is signaling -Catenin ucinlast v2dgaain niio fsignalosome of inhibiton degradation, Dvl2 to leads function 2 a n lcgnSnhs iae3 Kinase Synthase Glycogen and ) b ctnnsgaigi vertebrates. in signaling -catenin nlsso omto n noyoi fteWnt/ the of endocytosis and formation of analysis b Ctnnpiigi o bqiiainb h E3 the by ubiquitination for it priming -Catenin m -uui fteedctcCahi dpo Ap2 adaptor Clathrin endocytic the of 2-subunit 1, b ,Gr Davidson Gary *, Ctnnlgns ut-rti complex multi-protein a ligands, -Catenin 1 enfrKurz Jennifer , 2 eatet fCl ilg n Pediatrics, and Biology Cell of Departments m -eitdedctssi motn to important is endocytosis 2-mediated b Ctnni constantly is -Catenin 1 1 b oa Kirchhausen Tomas , ik Kauffeld Silke , (GSK3 b .GSK3 ). -TrCP) b 1 igChen Qing , eoe cieol uiglt atuain(lihe l,2003). al., et (Ulrich gastrulation Wnt/ late during stage, only this active becomes At signaling Wnt/PCP whereas organism. detectable, already intact is signaling Catenin an in pathways signaling 01 aaooe l,20)weesi a lopooe by proposed also for important has is Wnt/ it (CME) whereas endocytosis Clathrin-mediated 2006) that al.,others al., et Ohkawara et 2011; al., Yamamoto et Glinka 2011; 2013; al., endocytosis et Demir 2007; of al., (Bilic et activity type Lrp6 for the required is endocytosis about Caveolin-dependent Furthermore, discussion Wnt/ 2010). for on-going al., required an et Taelman is 2012; there al., et signaling(Dobrowolski maintain to late (MVBs) bodies in multi-vesicular all or sequestered endosomes al., that become suggested Wnt, et was including it components, (Li model signaling alternative for complex an found in ligand-receptor contrast, in indication In activated study the 2012). no an this was of In throughout there inhibition. endocytosis however, and non-disintegrating culture, activation cell signal that human is proposed of was cycle complex it entire Recently, destruction 2006). Bellen, and the Seto 2010; Metcalfe al., 2013b; signal al., et et for (Kim discussed internalization controversially ligand-receptor is maintenance of necessity the However, hs rvd noptimal as stage an a blastula provide in at embryos signaling these zebrafish chose Wnt we non-canonical tissue, and functional canonical Ap2 Wnt/ of in for requirement endocytosis endocytosis the of dissect Ap2 necessity To partner the signaling. clarify Catenin binding to postulated us its allow would of analysis functional Wnt/ during that and signaling Wnt/PCP of Wnt/PCP protein for hub essential is binding which Xenopus cargo CME, the the for fact, Fz4 to In Clathrin pits. 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Journal of Cell Science EERHARTICLE RESEARCH oreadfudte nrae .-odatracidification after 1.7-fold Wnt8 increased the to them next appeared found the cells quantified in rather and punctae We punctae source Dvl2 D). endocytosed 4C, supplementary Dvl2 (Fig. of Lrp6 aggregates number and whereas Wnt8 and observed 4A,B to S5B), (Fig. juxtaposed we Wnt8 Fig. embryos A1, of control material bafilomycin to accumulation compared with intracellular treatment overlapping 3; Movie After every material cell supplementary receiving and cells). 4I,J per (Fig. event minutes together endocytic 33 Wnt8 one of revealed Dvl2 events with internalization material by signalosomes of supplementary of eight Quantification induction and Wnt8. paracrine the of 4A,C at suggesting Dvl2 (Fig. S5A), for Fz1-positive Fig. out or membrane Lrp6 and Wnt-producing and plasma Wnt8 one a for Dvl2- the to positive adjacent clusters in a found cells we In and clone cell 4J). Wnt8 to (Fig. Lrp6-positive ligand-secreting embryo of aimed a zebrafish small in we overexpression blastomers generated be 2012), by of to we internalization al., clones order paracrine Therefore, et in and Wnt8. 2010) (Gross autocrine between Basler, exosomes distinguish and in on endocytosed (Port are secreted fashion ligands Wnt autocrine that suggested an been had function a it Ap2 in Since on Lrp6 depends and that Dvl2 fashion with paracrine together internalized is Wnt8 n 530 n onst nesnilrl fedctssdrn Wnt/ during endocytosis of role essential an signaling. Catenin to function remaining Ap2 knockdown points requires internalization partial The and Dvl2 the and Wnt8 of 4F,H). that (Fig. suggest result a reduced detectable Ap2 be of embryos might again still Wnt8 was intracellular control-treated but Ap2 accumulation treatment to in Wnt8 threefold compared and 4B,D,H) aggregates bafilomycin-treated (Fig. sixfold Wnt8 of In volume increased 4A,C,E,H). intracellular (Fig. the however, control embryos, to compared eaeams neetbei Ap2 Wnt8 in internalized of undetectable volume almost total The became 4E-G). of (Fig. knockdown treatment after internalized much as Ap2 half conditions. only was Dvl2 control reduced that was under formation Dvl2 Ap2 signalosome the in detection that of found fraction we our a Consistently, that escapes suggesting population also 4G), (Fig. blockage oivsiaeteitaellrlclzto ftetransducer stable of the use made of we complex, localization ligand-receptor the intracellular and complex the investigate endosomes late To Rab7-positive with colocalizes Wnt8 m ucini eevn el needn ftebafilomycin the of independent cells receiving in function 2 m m ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal i.2 inlsm omto ttectpamcmembrane Ap2m cytoplasmic on the dependent at is formation Signalosome 2. Fig. fWt RAo Ap2 expression or by induced mRNA be Wnt8 can of signalosome Fz1 the membrane. (I) of plasma (green). Formation the CFPGPI (J–L) to with Dvl2 Dvl2 of recruits Control co-expression supplementary (H) in S4. control constructs Fig. and expression material S3 indicated Fig. the material of supplementary images in shown are B-G CK1 Lrp6, for GSK3 the positive to signalosomes lead code of mRNA color Wnt8-GFP formation indicated of Expression the constructs. to injected related represent for channels I split and with CFP-GPI B-G areas in marker boxed images membrane Magnified the A,B,D,E–G,L,M). colors of in indicated mRNA (blue the ng in 1,2 shown with are together and indicated as expressed single represent images Confocal xetAi1adDl ue t2n)adAp2 and mRNA, ng) ng 2 at 1 (used at Dvl2 used and at were Axin1 constructs constructs except indicated all the stage; express epiboly that 30–50% embryos zebrafish live of opoio(O lgmr agtn Ap2 targeting oligomers (MO) morpholino s+..o e needn ape ah * given each. are samples bars independent ten Error of software. +s.e. Imaris as at with signalosomes membrane of plasma measurment the puncta. and Dvl2 quantification intracellular Manual arrow, (N,O) red puncta; arrow, Wnt8 green the intracellular signalosome; at arrow, signalosomes Yellow Dvl2-positive membrane. and plasma Wnt8- of reduction strong ucinuigtemrhln prah hs results These approach. morpholino the using function 2 opatebys(i.4) diinly efound we Additionally, 4E). (Fig. embryos morphant 2 b xn n v2(–,JLM.Snl hnesfrimages for channels Single J,L,M). (A–D, Dvl2 and Axin1 , m opatebysatrbafilomycin after embryos morphant 2 m function. 2 RA() LM neto f05mM 0.5 of Injection (L,M) (K). mRNA 2 z scin.Cntut were Constructs -sections. ofclmcocp analysis microscopy Confocal m opatembryos morphant 2 m aadAp2 and 2a P , m .5 **** 0.05, ue t16ng). 1,6 at (used 2 c , b -Catenin, m blasto leads 2b P , 0.001. 3973 b -

Journal of Cell Science nooe,icuigmlivsclrbde,fs ogigantic Although to 2004). fuse al., bodies, et fusion multi-vesicular (Cerny is homotypic including lysosomes aggregates a endosomes, small and to Dvl2 the endosomes leading with of of provide Vacuolin-1, embryos treated To formation inhibitor we molecule endosomes. the endosomes, late of that independent and evidence early further with colocalize Wnt8 EERHARTICLE RESEARCH 3974 (Rab5: treatment bafilomycin after vesicles Rab5 0.063 and aggregates Wnt8 between positive correlation higher a towards (0.024 (0.172 Rab7 Rab5 with with colocalization endosomes. late and cases early from both 0.023 aggregates positive in (Rab5: observed bafilomycin 0.017 was with treatment coefficient after 0.008 colocalization the of of shown coefficient signals Rab7 Manders (mean or Rab5 a either by with measuring 1 Dvl2 by of of colocalization Wnt8 (Coefficient 0 or coefficient colocalization, Dvl2 colocalization of colocalization Manders either the the with quantified We markers 2011). al., Rab5 endosomal et tagged (Clark fluorescently Rab7 expressing or lines zebrafish transgenic h oa olo h nlzdWt hwdsm u little but some showed Wnt8 analyzed the of pool total The .0,Rb:0.104 Rab7: 60.005, Dvl2- of separation a suggesting 5B,D,I), (Fig. 60.005 ..) epciey(i.5,,) iia increase minimal A 5A,C,I). (Fig. respectively 6s.e.m), admlclzto) efudams no almost found We localization). 5random .4;Fg EGI.Ti itiuinshifted distribution This 5E,G,I). Fig. 60.041; .4;Fg FHI,sgetn that suggesting 5F,H,I), Fig. 60.046; .0)adsrn overlap strong and 60.008) .0 r0.003 or 60.001 .0;Rab7: 60.006; 60.002 5full iadrcpo ope cusa eyerytm on after point time the early from gastrulation. very zebrafish complex a during at endocytosis transducer occurs intracellular complex ligand-receptor the of dissociation otdt a7pstv aeedsmsfrdgaainor Rab5-positive degradation and for Dvl2- of form endosomes colocalization vesicles is late little to complex The Rab7-positive recycling. complex ligand-receptor to the ligand-receptor routed while Wnt8-positive aggregates, Lrp6-signalosome dissociates intracellular complex, the entire transducer the from the Dvl-positive of part the intracellular that signalosome, the results Subsequently, These observation internalized. shown). becomes not our data and cells 5J,K receiving support (Fig. the treatment in visible detectable the the not after of was any Wnt8 with overlap whereas not not vacuoles, is do aggregates aggregates Dvl2 Dvl2 of and appearance altered the cells, gastrula in vesicles ta. 99.Hwvr ti tl nla htteoii n the aggregates and these origin that the reasoned We what are. unclear punctae still Smalley these is 2007b; of it function al., However, an et 1999). Schwarz-Romond al., – et 2007a; al., punctae et cytoplasmic Romond form polymerize in dynamically to and Wnt/ to during domain reported process DIX essential been has its Dvl through past, the In Ap2 m -eitdedctssi eurdfrDl stability Dvl2 for required is endocytosis 2-mediated rspiamelanogaster Drosophila ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal hnesrltdt h niae oo oefrijce constructs. injected for code color split to indicated the with going to areas is related boxed that channels represent Dvl2 images and Magnified Wnt8 internalized. of be cluster show membranous scanning a before or indicates (F,G) hour Lrp6 1 with Wnt8 for of treated A1 colocalization Embryos bafilomycin plasma (F–I) nM the (B,C). 150 in Wnt8 with unless by Lrp6, induction does with after Dvl2 nor membrane (B–E) Wnt8 signalosome. with a colocalize (green) from neither Wnt8 (red) of Dvl2 (from endocytosis with analysis of together time-lapse 2) a Movie of material markers pictures supplementary membrane Still with (A) together (blue). colors CFP-GPI indicated the in shown CFP- ng), (1 Wnt8 Ap2 single except and ng, ng) 2 (1,2 at epiboly GPI used 30–50% at were constructs constructs indicated all the stage; of that mRNA embryos ng zebrafish 2 live express signalosome. of the analysis of microscopy endocytosis Confocal autonomous Cell 3. Fig. 2 vni aioyi-rae embryos bafilomycin-treated in even z scin.Cntut eeepesda niae n are and indicated as expressed were Constructs -sections. m 16n) ofcliae represent images Confocal ng). (1,6 2 b Ctnnsgaigi elculture cell in signaling -Catenin Aerde l,19;Schwarz- 1998; al., et (Axelrod b Ctnn(,) ro nA in Arrow (H,I). -Catenin 2 ugssthat suggests

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Journal of Cell Science h niie noyoi fAi1atrc-xrsinwith co-expression after Axin1 of Dvl2 endocytosis the inhibited Ap2 whether the interested of also co-expression were of independently al., clusters et in Ap2 proteins (Schwarz-Romond two Dvl2 the of of colocalization showed domain Dvl2 with co-expression indeed, DIX and, 2007a) the interaction to and directly dynamics their Dvl2 investigate with to embryos zebrafish EERHARTICLE RESEARCH 3976 the tagged of fluorescently GSK3 members Axin1, Therefore, other of together. holding versions Wnt/ complex by their gain transducer activity aggregates signaling Dvl2 these Catenin whether asked we Next intracellularly cluster Dvl2 complex with transducer the of Members Wnt/ for prerequisite a is the which Dvl2 with complex, of interaction observation the Ap2 that our with suggest findings to plasma These mutants. similarly the Dvl2 at Axin1-GFP 7G,H), predominantly (Fig. reduced found membrane Dynamin was Axin1 DN and of fluorescence co-expression that found oaie rdmnnl otepam ebaewe expressed when membrane plasma the to predominantly localized m Fg AD upeetr aeilFg 3-) We S3A-C). Fig. material supplementary 8A-D; (Fig. 2 K340M/AHEA m nvivo in sncsayt lo noyoi ftetransducer the of endocytosis allow to necessary is 2 h cfodpoenAi1wsdsrbdt bind to described was Axin1 protein scaffold The . obemtn Fg L.Hwvr Axin1 However, 7L). (Fig. double-mutant b and b Ctnnwr mgdi live in imaged were -Catenin m sal ocmest for compensate to able is 2 b Ctnnsignaling. -Catenin b - v2adAp2 and Dvl2 membrane cytoplasmic with colocalization lead obvious the not to did Dvl2 to of Expression localize S4G). Fig. material did (supplementary – junctions adherens Wnt/ canonical of effector main GSK3 of clustering cytoplasmic oehrwt Ap2 with together hneo idtp v2t Dvl2 to Dvl2 wild-type GSK3 of and change Dvl2 of clusters larger Ap2 times of GSK3 co-expression and recruited 8E), Dvl2 (Fig. overexpression However, S4E). material (supplementary Fig. embryos in expressed when cytoplasm the for Ser/Thr-kinase main opee nisiatv omta lospoogdmaintenance prolonged the allows that of Wnt/ form of inactive pre-stage its in a transducer complexes the resembles that hypothesize We complex endocytosed. is that and GSK3 Axin1, catenin includes that complex transducer intracellular of clustering oprdt v2cutr ln Fg JL;tec-xrsinof co-expression the times 8J,L); four (Fig. size alone Ap2 in clusters increased Dvl2 which to clusters, compared same the to recruited hs eut ugs htDl-oiiecutr ersn an represent clusters Dvl2-positive that suggest results These m n Dvl2 and 2 b ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal Ctnnsgaigfloigsgaooeendocytosis. signalosome following signaling -Catenin b Ctnn(i.8K). (Fig. -Catenin m eeco-expressed, were 2 K340M/AHEA m n Dvl2 and 2 b Ctnn perdeel itiue in distributed evenly appeared -Catenin, n03 MOfr15husbefore hours 1.5 scanning. for DMSO Vacuolin-1 0.3% with in treated Embryos 0.3% (K) with treated DMSO, embryos Control samples (J) independent each. five of +s.e. represent bars was Error Wnt8 automatically. or calculated Dvl2 either Manders for and Coefficient manually set were function. Thresholds module software Coloc the Imaris using performed Wnt8 was Analysis and Rab7. Rab5 with with Dvl2 of of colocalization Quantification (I) CFP-GPI. transiently expressing line transgenic zebrafish (C,D) Rab7-GFP or in (A,B) His-CFP Rab5-GFP marker nuclear Wnt8 with of together expression Clonal (E–H) (blue). CFP- GPI marker membrane indicated with the together in colors shown are and as indicated expressed were Constructs single sections. represent tissue images host Confocal and (right). (left) Wnt8- clone between cell border positive the indicate E–H lines in Dashed line. transgenic Rab7-GFP zebrafish or (C,D) (A,B) Rab5-GFP in Dvl2 of Expression (A–D) stages. at epiboly constructs 30–50% indicated of mRNA that express embryos zebrafish Rab7- transgenic or GFP Rab5-GFP of analysis microscopy Dvl2 markers. and endosomal Wnt8 with of Colocalization 5. Fig. b i o edt n cytoplasmic any to lead not did K340M/AHEA Ctnn(i.8) oee,when However, 8I). (Fig. -Catenin b b Ctnnsgaigada atof part as and signaling -Catenin K340M/AHEA Fg 8G). (Fig. b b Ctnnwsefficiently was -Catenin Fg FH hra the whereas 8F,H) (Fig. Fg C.GSK3 8C). (Fig. b b Ctnndestruction -Catenin b i o edt any to lead not did nopnteupon punctae into Ctnn–a the as – -Catenin m Confocal omdfour formed 2 b and b ,the z b - -

Journal of Cell Science EERHARTICLE RESEARCH lyarl nvrosohrsgaigptwy,sc steFfand Fgf the as such Tgf- pathways, signaling the other various in role a play Wnt/ during crucial to and Lrp6-signalosomes Ap2 of overexpressing formation the Wnt/ induce enhance studies to able previous We Lrp6-signalosomes. structures were these with termed 2007), accordance al., et in (Bilic and, of aggregates cells membranous Wnt-responding Wnt/ the found of transducers we and far, receptors stage, ligands, blastula So zebrafish In at 2013a). organism. al., et embryos Kim vertebrate 2007; al., et living (Bilic cells culture in tissue only a reported been have signaling in Wnt canonical of signalosomes but Wnt/ level to subcellular of process regard the with investigate Lrp6-signalosome this to of endocytosis was subsequent the study and our formation Lrp6-signalosome of aim The zebrafish. Wnt/ of mechanism biological b cell the investigated we study, this In Ap2 DISCUSSION ftasuto acdsadtepoeso endocytosis. of process the and cascades transduction regulationof the processes: biological cell important two connects that Ap2 that hypothesize we Thus, 2012). al., ctnnsgaigb sn ihrslto ofcliaigin imaging confocal high-resolution using by signaling –catenin m ucindrn r6sgasm formation signalsome Lrp6 during function 2 b inln ahasi erfs mro Uaakret (Umasankar embryos zebrafish in pathways signaling b Ctnnsgaigidpnetyo h iadby ligand the of independently signaling -Catenin b m Ctnnsgaig p a ensgetdto suggested been had Ap2 signaling. -Catenin ,wihipista p n noyoi are endocytosis and Ap2 that implies which 2, b m Ctnnsgaigo a on signaling -Catenin sacnrlhbprotein hub central a is 2 b Ctnnptwyin pathway -Catenin lsuasae.Hwvr ubro noyi vnsmight events endocytic of detection. number our escaped a 15–20 have completely of in However, diameter have stages. a microscopy (with blastula which cells confocal large embryos, and these translucent zebrafish high-resolution monitor with to using able combination were by ligand-bound We three clathrin- 2012). to had processes al., that one et that only proposed (Cocucci border visible, endocytosis been to receptors able of became had are vesicles event It Lrp6 coated analysis. transient with previous and escaped fast colocalizing large a Wnt A1, suggesting bafilomycin the of with in vesicles aggregates endocytic blocking Wnt8 of by However, tagged maturation assay. very imaging the fluorescently clonal detected al., our of of had et cells we Yamamoto receiving signal Initially, 2005; intracellular 2008a). al., al., little et al., et Piddini et Yamamoto 2012; Glinka 2006; al., 2006; be et Nusse, to Jiang and 2011; (Blitzer endocytosis signaling. of for type necessary the Wnt/ for concerning required conclusions their in a Wnt/ to Drosophila leads of which Ap2 endocytosis, decrease and of formation knockdown Lrp6-signalosome that found We signaling and Endocytosis ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal n nmueadhmncl ie r controversial are lines cell human and mouse in and b opoio(O lgmr against and oligomers constructs (MO) indicated morpholino the express of that mRNA embryos synthetic zebrafish live of on analysis depends activity and Ap2 stability Dvl2 6. Fig. CAwsue slaigcnrl (E–K) control. with loading hybridization as used was embryos. PCNA double-morphant in of Dvl2 reduction endogenous a Ap2m2- shows and embryos blot embryos mRNA-expressing Western morphant (D) Ap2m2 control. of to analysis compared dominant-negative (C) express Dyn that (DN) embryos in and (B) are Ap2 Dvl2 in of detectable clusters hardly Intracellular (A–C) stage. epiboly r ie s+..o ieidpnetsamples independent five **** of each. bars +s.e. Error as M. and given L are in aggregate. shown Dvl2 data of of Quantification size (N) Ap2 increased of to is expression leads side Ectopic mRNA dorsal (L,M) (the right). shown the is on view stained lateral 30–40 the of embryos; representative are blastula-stage. shown at Embryos constructs indicated with injected Ctnnsgaigadwehredctssis endocytosis whether and signaling -Catenin b Ctnnsgaig rvossuisin studies Previous signaling. -Catenin m function. 2 P , 0.001. ACLM ofclmicroscopy Confocal (A–C,L,M) axin2 m ed oardcinof reduction a to leads 2 m and obemrhn embryos double-morphant 2 lef1 rbdi embryos in probed ap2 m m 2a/b )during m) nsitu In m t40% at 2 3977

Journal of Cell Science EERHARTICLE RESEARCH v2ageae eed nteitrcinwt Ap2 a 3978 from with of stem concentrations interaction high all that Taken aggregates the event. Dvl2 internalization on previous cytoplasmic of that depends function Wnt/ suggesting and during formation aggregates that function show Dvl2 and data stability Our signaling. Dvl2 Catenin of on interaction effects shown dramatic previously the on focused Ap2 we study, this In The (A) right). the on is side dorsal (the shown Ap2 is on of view induction dependent lateral the is by activity reflected pathway and signaling internalization Dvl2 7. Fig. 45-ipopae ncnrs oordt,itraiainof not was internalization aggregate activation signal data, this Wnt observed. our to phosphatidylinositol for response to of in essential Lrp6-signalosomes contrast presence the In is on (4,5)-bisphosphate. function depends suggest but Lrp6 Ap2 study of formation site this the and of at accumulates authors assembly clathrin The activation, cell Wnt 2013a). after mammalian al., that, Ap2 using et of (Kim study interaction receptor recent describes a which by lines, confirmed This reduced. was same Lrp6-signalosomes of the result formation At the comparable Wnt8. found Ap2 we any of time, accumulation find of extracellular not or did function intracellular we Dynamin2, the dominant-negative down knocking I oto.Ai1ageae ntectpam JL oepeso fDl ige JK n obemtns()tgte ihAi1 M ooaiaino Axin1 stage. AP2 of one-cell Colocalization and (M) at Dvl2 Axin1. each) between with single ng together interaction represent (L) (2 when images double-mutants mRNA reduced and Confocal synthetic stability (J,K) punctae. as single- its cytoplasmic Dvl2 injected and of in membrane Co-expression Dvl2 (J–L) cytoplasmic wild-type cytoplasm. the the with at in aggregates blocked Axin1 is Control. Axin1 (I) (I–M) stage. one-cell at yra-iePRwt rmr gis xn bu)adLf rd nebynccN netdwt h niae osrcs ro asaegvna +s.e. as given cytoplasm. are the bars in Error signal constructs. Axin1-GFP indicated the the reduces with endocytosis injected Dynamin-dependent cDNA of embryonic Blockage on (G,H) (red) duplicates. 12 Lef1 show in images and scanned Confocal (blue) experiments Axin2 independent against two primers of with PCR real-time by hnw lce noyoi ttepam ebaeby membrane plasma the at endocytosis blocked we When m m ihDl Y ta. 07 ue l,21) hc has which 2010), al., et Yu 2007; al., et (Yu Dvl2 with 2 uui srqie opoetDl rmdegradation from Dvl2 protect to required is subunit 2 m m z - tcso ieebysa 0 pbl tg.Idctdcntut eeijce t1n ssnhtcmN,ecp xn 2ng), (2 Axin1 except mRNA, synthetic as ng 1 at injected were constructs Indicated stage. epiboly 40% at embryos live of stacks axin2 m m rb expressing by or 2 ihteLp co- Lrp6 the with 2 xrsini lsuasaeebysa 0 pbl.Ebyswr netdwt niae osrcs the constructs; indicated with injected were Embryos epiboly. 30% at embryos blastula-stage in expression n 5 2 B 33;(C) 33/39; (B) 72; m -idn sites. 2-binding m b 2, - n 5 z 72;(D) 27/29; scin flv mro t4%eioysae niae osrcswere constructs Indicated stage. epiboly 40% at embryos live of -sections osiuiepoeltcdsrcin(lfee l,2003). the of al., activation that et its suggested (Cliffe report prevents lysosomal recent formation destruction and a the proteasomal Furthermore, polymer proteolytic Indeed by Dvl–Axin membrane. controlled constitutive and is from the Dvl2 Dvl degradation from protect of removal to stability important after The be 2010). degradation al., might et complex (Metcalfe before transducer culture cell human in reported opnnso h inactive the of cytoplasmic combination of a Ap2 endocytosis, complexes, components al., transducer to After the et form 6). binds aggregates (Fig. Dvl2 Dvl2 (Gao protein membrane endocytosis partners Dvl2 plasma does stabilizes binding the why stabilizing quickly At is So, 2010). needs Dvl2 that 2007). and suggsted al., been degraded has et It stability? (Bryja Dvl2 maintain promote pool to important Dvl is hyperosmolaric endocytosis the with that culture suggested in have is the SN4741 sucrose Dvl2 using line cell that experiments Ap2 neuronal and previous mouse and complex, Indeed, transducer degraded. Dvl2 signal subsequently the between of binding endocytosis with interference Wnt/ activate can Dvl2 v2wt te ebr ftetasue ope uhas such complex transducer of the colocalization of showing GSK3 members data Axin, our other by with supported Dvl2 is This Dvl2. (A–E) nsitu In n ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal 5 32;(E) 23/29; b yrdzto hw erae ciiyo h Wnt/ the of activity decreased shows hybridization and n 5 b Ctnni h yols,wihhsbeen has which cytoplasm, the in -Catenin 33.()Rltv agtgn xrsinanalyzed expression gene target Relative (F) 33/35. b Ctnntre ee,w rsm that presume we genes, target -Catenin b Ctnndsrcincmlxand complex destruction -Catenin m simpaired. is 2 m m blocks 2 ,which 2, b -catenin

Journal of Cell Science oddvsce u eprrl typlmrzdi the Clathrin in the subunit polymerized of its dismantling with intracellular stay the Ap2 including that temporarily endocytosis, coat speculate ligand-receptor but We after in the cytoplasm. shape vesicles from that their dissociate loaded al., alter suggests components et not (Cerny signalosome do this Vacuolin-1 chemical and small 2004), Rab7 the or not with do combination Rab5 aggregates Dvl2 our early route: with in However, different a found overlap endosomes. takes be Dvl late that can show in and data endocytosis subsequently by and up endosomes taken together Wnt8 is that Lrp6 demonstrate we with Indeed, 2014). al., et Vinyoles EERHARTICLE RESEARCH ihAi1 GSK3 Dvl of Axin1, colocalization signalosome showing Lrp6 by with lysosomes entire or the MVBs that to suggested localizes internalization cells after HeLa Dvl2 in and Wnt/ Studies Wnt8 of promote routing Endocytic thus, transducer and, the zebrafish. Dvl2 of in signaling formation stabilize subsequent complex the therefore, and We, 2013). signalosomes al., Ap2 et that (Jung in HEK293T hypothesize de-ubiquitylation line by cell Dvl human of the stabilization to leads pathway Wnt b m n yorce Temne l,2010; al., et (Taelman Lysotracker and -eitdrcuteto v2t Lrp6- to Dvl2 of recruitment 2-mediated m ih elne othe to linked be might 2 b -Catenin erdto.Bnigo h n iadt eetr z and Fz1 receptors to ligand Wnt the of Binding degradation. sebyo h etuto ope n the and complex plasma the on destruction the is balance the and the 9A) cytoplasm of (Fig. state the assembly Wnt-off between the In the Dvl2 membrane. of and members complex between al., exists et balance (Kim Axin1 PP1-mediated by protein state scaffold inactive 2013b). the which an in of into state dephosphorylation degradation active the an for in from routed assembly converted complex after destruction be this maintained Furthermore, might 2012). is al., and et (Li is structure cytoplasm complex stable destruction the very that In a proposed Dvl2 1999). recently al., was so-called et it Smalley the addition, 2007b; – al., et aggregates (Schwarz-Romond biological Dvl2 punctae the a intracellular recognizing as of studies previous exists relevance hypothesis with This Dvl2 accordance complex. that in transducer is to indicate intracellular back the data translocated of Our are member membrane. (Kirchhausen, Clathrin and plasma Hsc70 Ap2 the protein process this cognate In heatshock 2002). the the by vesicle. of dismantled the are activity vesicles from Clathrin-coated signalosome endocytosis, Dvl2-positive After the of dissociation nsmay epooeamdl(i.9,i hc naffinity an which in 9), (Fig. model a propose we summary, In ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal Dvl oueatrsraeapiainuigIai software. Imaris as using GSK3 (A,B) application in surface size after punctae volume of quantification Automatic (D) 2n ah , go Ap2 of ng 1,6 mRNA each, synthetic ng as (2 injected were constructs Indicated single cluster complex Dvl2. transducer with the intracellularly of Members 8. Fig. ie s+..o e needn ape each. samples independent ten ****P are of bars +s.e. Error (E,F). as in given size punctae of Quantification (L) yc-xrsino Ap2 of improved co-expression be by can colocalization but aggregates, positive in size punctae (I) of (E,F). Quantification (H) mRNA. mutant aeilFg 3)atrc-xrsino Ap2 of co-expression after S3G) supplementary Fig. with material (compare unaltered is distribution ooaiaincnb mrvdb oepeso of co-expression by Ap2 improved be can colocalization uca htclclz ihDl A vni the in even Ap2 (A) of Dvl2 presence with intracellular colocalize as that distributed punctae is Axin1 (A,B) expression. GSK3 and (A–D), (D–H) Axin1 colors. of indicated distribution with Subcellular displayed and stage cell npam ebaecutr fe oepeso of co-expression after Ap2 clusters membrane plasma in cmaewt upeetr aeilFg 3)after Ap2 S3E) of Fig. co-expression material supplementary with (compare K340M/AHEA m m , RA G GSK3 (G) mRNA. 2 n Dvl and 2 b z .0;ns,ntsgiiat( significant not n.s., 0.001; scin flv mro t4%eioystage. epiboly 40% at embryos live of -sections DH lsesi v2pstv grgtsand aggregates Dvl2-positive in clusters (D–H) b Ctnncutr r esaprn nDvl2- in apparent less are clusters -Catenin b ctnn(–)i h otx fDl co- Dvl2 of context the in (I–L) -catenin obemtn mRNA. mutant double K340M/AHEA m B.()Ai1i on predominantly found is Axin1 (C) (B). 2 m n Dvl and 2 m b RA() (G) (J). mRNA 2 m obemtn mRNA. mutant double ofclIae represent Images Confocal ,12n F-P)a one- at CFP-GPI) ng 1,2 2, itiuini unaltered is distribution b Ctnndestruction -Catenin K340M/AHEA P § 0.05). b b Ctnnis -Catenin double -Catenin b b m -Catenin and 2 3979

Journal of Cell Science nooe(i.9) hc ed ofrhrmitnneo the of maintenance further The to early 9E). leads state. (Fig. the Wnt-on which membrane from 9D), dissociates the (Fig. however, to endosome complex, recycled in transducer are degraded signal ligand. or either the become lysosome and subsequently, the Lrp6 receptors, Fz1, and including Dvl2-positive Ligand complex, complex) intracellular receptor destruction the the inactivated from (the split complex to transducer required signal process further recycling Dvl2 Clathrin be subsequently, following may The that, 9C). propose (Fig. 2007; signalosome We al., Ap2 et 2005). (Bilic and al., signalosome activated et an Davidson of formation the to EERHARTICLE RESEARCH 3980 Technology were of and Institute ( Protection Karlsruhe zebrafish Breeding Animal the (KIT). and on (Regierungspra¨sidium Az.35-9185.64) law Committee Karlsruhe, Animal-Protection German Local by the in approved performed with were procedures experimental accordance and husbandry zebrafish All manipulation and embryos Zebrafish METHODS AND MATERIALS destruction Axin1-positive the of GSK3 turn including in recruitment – which complex Lrp6, the of Ap2 phosphorylation to initiates and binds and is polymerization Dvl2 receptor the enough, the high is to receptors When recruited Fz1 9B). accumulated (Fig. of complex number receptor the the of clustering initiates Lrp6 im,S oi,M)atr2 p.Tedt epeeti hsstudy this in present we pigment data prevent The hpf. To (PTU, 20 2002). 1-phenyl-2-thiourea after al., mM MO) 0.2 et Louis, in St (Brand raised Sigma, cycle were dark embryos h formation, light/10 h 14 m oehriiit noyoi ffatoso the of fractions of endocytosis initiate together 2 ai rerio Danio b otercpo ope,leading complex, receptor the to – eemitie t28 at maintained were ) m .Ti a induce may This 2. ˚ Cona niae osrcswr netda n-elsaeo,frclone for All or, stage injection. mRNA, capped one-cell as before cells at 8–16 stage of injected one into one-cell were formation, constructs at indicated (Sigma-Aldrich) Pronase from gift kind a were Rab5- lines AB2O2. Link. transgenic zebrafish Brian expressing wild-type Rab7-eGFP KIT and of eGFP analysis from acquired were zAp2 morpholinos and Constructs ihtemesg ahn i (Ambion). Kit Machine mMessage the with GTCCA;Ap2 TGGTCCTCAAA; eekn it rmJmSih(aeane l,2009). al., et (Hagemann Smith Jim from xRab5-GFP gifts (HistoneH2B-CFP), kind His-CFP were CFP-GPI, Brand 2005). Michael by al., provided et kindly (Rhinn was hAxin1- zWnt8-GFP Davidson. Addgene. Gary from from are #17048 2001); hCK1 al., GFP, et (Lekven xGSK3 ORF1 17086, zWnt8 x # #29680, 2004); 2010); al., al., et et (Waxman zDvl2 LTD. 1994). GeneTools from obtained were ATG the spanning AAC) h -adCtriio Dl pamdfo hroSinii)( Scientific) Thermo Fidelity from (plasmid to High zDvl2 introduced of C-termini were Phusion and sites N- using Restriction the Biolabs). directions England (New manufacturer polymerase per performed ihpie (5 A primer with GyetCTP[idgf rmUeStra Uwe Ap2 from Ap2 for for 2012)] gift pCS2+ into [kind introduced was pGlyserteC-TFP product resulting the and GAGTATGACCGATGGGGAAA-3 mro eehretdadezmtclydcointdwith dechorionated enzymatically and harvested were Embryos Ap2 al., et (Rupp pCS2+ in subcloned were constructs expression All o Dl-hryadzv2mtns l C ecin were reactions PCR all mutants, zDvl2 and zDvl2-Cherry For m abmrhln lgmr (Ap2 oligomers morpholino 2a/b m awsapiidfo 4hfzbaiheby DAlibrary cDNA embryo zebrafish hpf 24 from amplified was 2a ora fCl cec 21)17 9038 doi:10.1242/jcs.148767 3970–3982 127, (2014) Science Cell of Journal c GP Lp-F n Lp-hrywr idyprovided kindly were hLrp6-Cherry and hLrp6-GFP -GFP, m 2a-TFP. 9 -CCATGGAATGTGAGGTTTGA-3 b CtnnGP(ilradMo,19) #16839, 1997); Moon, and (Miller -Catenin-GFP m b 5 2b: 9 -CCTCCAATCATGGTGGCGGTTA- 9 eitdb h neato fDvl2 Ap2 of with interaction the by mediated is internalization Subsequent (C) membrane. cytoplasmic the at and formation Fz signalosome to Dvl2 of recruitment Ap2-dependent co-receptor, inducing Lrp6 thereby and receptor bound Fz is to ligand situation, Wnt-on the eusrto fAi,CK1 Axin, of sequestration cieaddegrades and active the state, b Wnt-off the In (A) signaling. 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Journal of Cell Science ba)wr sdi :00dilution. 1:1000 a in used were from from (ab137727 abcam) AP2M1 blot (GTX129968 human western against Dvl2 antibody polyclonal for zebrafish and and GeneTex) against assay antibody reporter luciferase Polyclonal Wnt analysis. for buffer lysis Triton normalized assayed subsequently and to was stage activity shield luciferase at The harvested activity. luciferase were for each embryos ten indicated. of as pools mRNA synthetic Four plus Gradl) Dietmar from (gift DNA plasmid mg rcsigwspromdb sn nIai .. software 6.3.1 Imaris an using by performed AG). 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Promega with real-time from Zymo Primers a StepOnePlus). transcriptase from in reverse Kit analysed MMLV Prep using and prepared Mini was RNA cDNA Direct-zol 300 in using lysed prepared were each embryos Fifty PCR quantitative Real-time Whole-mount PBS. in paraformaldehyde 4% in mRNA fixed were Embryos situ In or scanning before hour Sigma-Aldrich). 1 from for (V7139, DMSO 1% 30 in with (SAFSB1793-10UG, International) A1 bafilomycin VWR nM from 150 with treated were Embryos treatment Inhibitor DLAHEA. zDVL TACGGATGTCCAC-3 DLK4Mby K340M zDVL AHEA. pZero-zDVL CCGCCAGCCCATGAGGCCTCTAACTACAGCTAC-3 (5 two- similar E a primers TGGGCTGGCGGAGGCTGGGTG-3 in using Y55A simultaneously manner, The pZero, introduced K340M. step into were pZero-zDVL introduced mutations yield purified, L554A to was and sequencing PCR by fusion and confirmed this A and primers of containing product reaction The second B. a to concentration molar equal (5 D and TCCCAATG-3 B (5 primer C with generated primer was and fragment GCCACATTCGATCT-3 flanking A left a primer First steps. using two mutation in K340M performed the introduce was to Mutagenesis into (Clontech). introduced C-1 pmCherry product resulting the and CACAAAAAACTCACTGg-3) n e1(owr:5 (forward: Lef1 and GAAAGATCC-3 5 6 9 -TGTGATGTGAGAACCAACC-3 h rpemtn a eeae ylbrtn rgetfo pZero- from fragment a liberating by generated was triple-mutant The b igrsslto.Iae eeotie yuigaLiaTSS5X SP5 TCS Leica a using by obtained were Images solution. 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Beretta, N., Kirsch, K., Demir, aisn . u . hn . ii,J,Fne,U,Sank . lna A. Glinka, P., Stannek, U., Fenger, J., Bilic, J., Shen, W., Wu, G., Davidson, H. Varmus, and L. Schweizer, F., Cong, ouc,E,Aut . oln,S n ichue,T. Kirchhausen, and S. Boulant, F., Aguet, E., Cocucci, M. Bienz, and F. Hamada, A., Cliffe, orwlk,R n eRbri,E M. E. Robertis, De and R. Dobrowolski, a,C n hn Y.-G. Chen, and C. Gao, lvr,H n us,R. Nusse, and H. Clevers, lr,B . itr . oe,A .adLn,B A. B. Link, and R. A. Cohen, M., Winter, S., B. Clark, hn . e eg,D,Bon . h,S,H,L . ilr .E,Caron, E., W. Miller, A., L. Hu, S., Ahn, J., Brown, D., Berge, ten W., Chen, en,J,Fn,Y,Y,A,Myk,K,Brooo . lmemn J., Klumperman, B., Borgonovo, K., Miyake, A., Yu, Y., Feng, J., Cerny, lna . od,C,Krc,N,Hag .L,Kznky,O,Igligr D., Ingelfinger, O., Kazanskaya, Y.-L., Huang, N., Kirsch, C., Dolde, A., Glinka, rn,M,Gaao .adN¨sli-ohr,C. Nu¨ sslein-Volhard, and M. Granato, M., Brand, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.148767/-/DC1 online available material Supplementary material Supplementary GM- numbers release. [grant immediate Health P.R. for of 1036. PMC Institutes FOR in National DFG Deposited the group Noether 075252]. by research Emmy supported Wnt DFG are DFG the T.K. the by and and supported the were and S.S. 847/2 and fellowship S.W. S.K., J.K., A.I.H.H., Funding performed constructs. Schindler zDvl2 S. the generated and P.M.R. S.W. analysis. and blot Q.C. Western assays. the the reporter performed tested TOPFlash G.D. and the generated performed S.K. J.K. constructs. analysis. Wnt8-tagged data and experiments performed A.I.H.H. contributions Author interests. competing no declare authors The interests Competing lines. fish transgenic Dosch Roland for and Germany) WI) School, Milwaukee, Medical Wisconsin, of University (Go¨ttingen constructs College cDNA (Medical for Link Brian Germany) and Technology, and UK) of London, Institute (MRC, (Karlsruhe Stra¨hle Smith Jim Uwe Gradl Germany), Dietmar Technology, the Germany), of on Dresden, Institute (TU comments (Karlsruhe Brand valuable Michael for thank also Heidelberg) We (DKFZ, manuscript. Gross Julia thank We Acknowledgements rj,V,Caja V., Bryja, R. Nusse, and T. J. Blitzer, M. Bienz, C.-M., Cruciat, T., Zimmermann, G., Davidson, Y.-L., Huang, J., Bilic, T. R. Moon, and S. Angers, eindtersac,AIHH,TK,S cop rt h manuscript. the wrote Scholpp S. T.K., A.I.H.H., research, the designed xlo,J . ilr .R,Sumn .M,Mo,R .adPrio,N. Perrimon, and T. R. Moon, M., J. Shulman, R., J. Miller, D., J. Axelrod, o ciiyadcvoa noyoi fLRP6. of endocytosis M. caveolar Boutros, and and activity C. for Niehrs, M., Carl, F., Argenton, octpamcsga transduction. signal cytoplasmic to C. Niehrs, and LRP. containing and oligomers Frizzled forming by receptors, pathway its beta-catenin the activate to membrane eod ntelf facahi-otdpit. clathrin-coated a of life the in seconds signaling. wingless during membrane plasma 966. the to Axin 1192-1205. atrsgaln:mlieiua oisa inligorganelles. signalling Biol. as Cell bodies multivesicular signalling: factor Signal. ae rngnclnsfri iosuiso nooebooyi zebrafish. in biology endosome of studies vivo Dyn. in for lines transgenic based .G,Brk .S,Nse .adLfoiz .J. 4. R. Frizzled Lefkowitz, of and endocytosis Wnt5A-stimulated R. mediate Nusse, Science to S., 2 beta-arrestin L. recruits Barak, G., M. auln1ihbt a2)dpnetlssmleoyoi u o cell not but exocytosis lysosomal Ca(2+)-dependent T. resealing. 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