eerh sk nvriy - aaak,Sia sk 6-81 Japan. 565-0871, Osaka Suita, Yamadaoka, 3-2 University, Japan. Osaka 565-0871, Research, Osaka Suita, Yamadaoka, 2-2 University, Osaka Medicine, ieiYamamoto Hideki is formation cells lumen epithelial apical polarized for in required Wnt5a of secretion Basolateral ARTICLE RESEARCH eevd2 etme 04 cetd1 eebr2014 December 17 Accepted 2014; September 21 Received Japan. 615-8245, ` International Kyoto Foods Nishikyo-ku, Pharma Goryo-Ohara, Project, 1-49 Medical Ltd., Bio Co., Dept. R&D address: *Present 2 developmental of in family role 1 large 2013). important a an is al., which play et family, that (Gon Wnt molecules the cells) secreted of (IEC6 member a cells is polarization epithelial Wnt5a single-cell soluble intestinal of a rat induction their of in example the of an promotes and orientation As Wnt5a factors, 2008). the factor, Mostov, growth support and (Bryant and to recognize polarity pathways morphogens also signaling be as Cells activate such can proteins. factors, which matrix, membrane soluble extracellular membrane the environment, and basement with cells their including of basolateral interaction synthesized sense apical-basolateral the establish by or must mediated newly To cells 1999). apical polarity of al., polarity, et cell to (Yeaman delivery epithelial domains proteins correct of endocytosed maintenance the and requires generation The INTRODUCTION Lumen, lumen Polarity, Wnt5a Glycosylation, apical cells, Epithelial for WORDS: KEY necessary the cyst. is epithelial that the Wnt5a and in mechanisms formation of different secretion by sorted destination basolateral are Wnt5a-depleted receptors correct a its their and in Wnt5a to that apoptosis cells suggest cyst, luminal results These wild-type eliminating cyst. a for in necessary formation was lumen not a was apical by apoptosis for not Although apically. required but secreted Wnt5a, is that wild-type and mutant by cyst, Wnt5a epithelial rescued the were of phenotypes Knockdown formation these the cells. lumen Wls. epithelial apical at of delayed require Wnt5a side efficiently of apical not the more than did stimulated side basolateral sorting was their signaling Wnt5a-induced but the to membranes, localized The also were basolateral cells. receptors and Wnt5a was clathrin (AP-1). epithelial 1 (Wls), protein Wnt5a Wntless adaptor kidney required that Wnt5a polarized of secretion found in basolateral in sorted We involved basolaterally are polarization. is receptors secreted signaling basolateral Wnt5a its and how and apical and Wnt5a basolaterally, or whether remains apically it determined but cells, be epithelial in to polarity cell planar regulates Wnt5a ABSTRACT ohfm Takao Toshifumi uhrfrcrepnec ([email protected]) correspondence for Author aoaoyo rti rfln n ucinlPoemc,IsiuefrProtein for Institute Proteomics, Functional and Profiling Protein of of School Laboratory Graduate Biochemistry, and Biology Molecular of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,15–03doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 2 1 n kr Kikuchi Akira and hhr Awada Chihiro , 2, ,Sij Matsumoto Shinji *, 1, ` 1 oouiKaneiwa Tomoyuki , n cino ns n eemnto fWtamodifications Wnt5a post- of Thus, determination and 2012). secretion Wnts, al., the of in et action roles recognition and important (Janda the have for receptors modifications Wnts translational of of activation modification suggesting Fz8, lipid and mouse of of necessity domain extracellular the trans- the of the have groove of from analyses the modification structural Wnt11 lipid Crystal the of that 2013). sorting revealed al., apical et the recycling Wls (Yamamoto its for 2014). Golgi and al., required the membranes et ER not basolateral Yu in the the is 2010; to and trafficked Basler, role membranes is and plasma itself crucial (Port the Golgi a between the cycles through plays and in ER which Wnts the palmitoleic-acid-modified from recognizes (Wls), exit the Wnt, their of in Wntless for trafficking occurs essential Wnt11 is ER. and and porcupine the Wnt3a Yamamoto by of 2006; catalyzed lipidation is al., ER, et The (Takada 2013). residue al., Ser et conserved a at acid of secretion apical the The for necessary Wnt11. 2013). is their glycosylation is region al., and N-terminal and the unique, et at and residues, Wnt11 to Wnt3a (Yamamoto attached Asn glycan 2002). complex-type differed multiple (Spiro, profiles at ER glycosylation glycosylated the from were exit Wnt11 endoplasmic or the in (ER) folding reticulum their for necessary basolateral is proteins of secretary sorting al., the et Fo Deborde for 2008; 2001; clathrin, Bonifacino, important and and be (Boehm (AP-1) proteins to membrane 1 known protein are adaptor which requires basolateral Wnt3a and The of 2013). kidney basolaterally al., traffic canine et secreted (Yamamoto the Madin-Darby cells are epithelial that polarized (MDCK) Wnt11 revealed in also and respectively, study apically, Wnt3a previous of Our 2003). majority 2006; al., al., et Takada et 2007; al., Willert et Kurayoshi 2012; 2007; Korswagen, al., and et (Harterink Komekado synthesis their during lipids and hs n5 n t eetr pert ott h basolateral the to sort to appear 2010). receptors its al., thereby and et (FAK), Wnt5a (Matsumoto Thus, kinase adhesion adhesion cell-to-substrate focal enhancing with with interacts complex cancer a signaling, In forms 2013). receptor, al., Wnt5a integrin a et (Fz2), (Gon Frizzled-2 phenotype membrane knockdown cells, this Matrigel; an to inhibited basement with side Wnt5a opposite autonomously, of the cell containing on polarized forming were cap was Matrigel cells F-actin cell IEC6 single thick a when proteins, that on showed Polakis, study seeded 2004; previous Nusse, and Our Logan 2011; 2007). al., disease et causes (Kikuchi life humans postnatal in in signaling Wnt defective processes; h omto faia-aoaea polarity. for apical-basolateral required be of might formation and the cells epithelial polarized of membranes nadto,WtaadWt1aemdfe ihpalmitoleic with modified are Wnt11 and Wnt3a addition, In n rtisaepost-translationa are proteins Wnt lc,20) ti eeal huh htgyoyainof glycosylation that thought generally is It 2005). ¨lsch, a ,adDl yolsi rti htmdae Wnt5a mediates that protein cytoplasmic a Dvl, and 2, 1 aauiSugimoto Takayuki , l oiidwt o with modified lly Xenopus n8isrsinto inserts Wnt8 1 ligosaccharides , 1051

Journal of Cell Science ta. 00 aaooe l,20) lhuhtercross- their although 2004; al., 2006), et (Lu al., reported been also et has Wnts Yamamoto other with and 2010; reactivity Nishita 2008; (Ror2) al., al., for et co-receptors (Kim et 2 respectively as Wnt11, and function receptor Wnt5a (Ryk) Wnt3a, orphan kinase tyrosine kinase-like receptor-like tyrosine of receptor understanding the for necessary signaling. is Wnt5a spectrometry mass using ARTICLE RESEARCH 1052 than lower was expression cells, Wnt3 I MDCK In trans-epithelial 2001). claudin- and al., higher lack et they have (Furuse because al., expression cells cells et 2 II (Dukes MDCK difference I than lines MDCK resistance The cell electric addition, parent I) II). of (MDCK In low passages I (MDCK on 2011). II) based type (MDCK II is cells MDCK high MDCK type and cells: of types MDCK basolateral MDCK two the 3D of and between and types and I) apical two (2D) (MDCK in are two-dimensional There used with cultures. commonly experiments are polarization cells MDCK Wnt5a of secretion Polarized RESULTS that apical demonstrate enhancing apoptosis. thereby we without cell-to- Rac, formation promotes and activates lumen Wnt5a and we cyst, secreted adhesion apically substrate epithelial Furthermore, lumen not apical but the receptors. in basolaterally secretion of its polarized Wnt5a formation of and of roles sorting the Wnt5a examine polarized trafficking basolateral of the the show of we mechanisms examined Here, secretion receptors. we its polarized and Wnt5a members, the the in family elucidate mechanisms To Wnt unique unknown. largely and specific is common to their destinations sorted as are specific location these they how their same efficiently, underlying the mechanism signals to the However, sorted transmit Wnts. be To to need 2003). receptors al., et Yoshikawa n5 n n eetr nMC Iclst xmn its examine to function. its cells examine to II cells in MDCK I Wnt5a the endogenous MDCK apical in down knocked the overexpressed receptors and in mechanisms, we in trafficking Wnt experiments, observed not and subsequent but In Wnt5a was membranes 1C). (Fig. basal Wnt5a lumen of II/Wnt5a permeabilization, vicinity MDCK In immediate 1C). with (Fig. membranes whereas cells basal was lumens, was Wnt5a the apical permeabilization, Wnt5a on the without detected stained permeabilized, in in were not were cells the but expressed cells when cytoplasm transiently the the in and was detected cells Wnt5a II When MDCK culture. 1B). (Fig. Matrigel II/Wnt5a; lines cell MDCK both and in basolaterally I I/Wnt5a Wnt5a secreted MDCK expressed was (MDCK Exogenously in S1C). cells Fig. expressed material supplementary II stably of was MDCK trafficking the Wnt5a and show clearly, cells To I more 1A). MDCK Wnt5a (Fig. in basolaterally Wnt5a apical a secreted these endogenous was Under of to (TfR, 1A). majority sorted (Fig. the receptor conditions, respectively clearly membranes, transferrin were basolateral and receptor) and membrane marker) (an basolateral podocalyxin a membrane 1995), on al., monolayer et apical a (Gottardi as culture) (2D cultured material support were in (supplementary filter cells cells MDCK detected II When MDCK was S1B). in Fig. Wnt5a not Western but endogenous S1A). cells, I that Fig. MDCK material revealed (supplementary analysis cells blot II MDCK in o-est iorti eetrrltdpoen6(LRP6), 6 protein receptor-related lipoprotein Low-density hs idnswr ofre sn DKI yt n3D in cysts II MDCK using confirmed were findings These Wnt7a , Wnt5b RA eeepesda ihrlvl than levels higher at expressed were mRNAs and Wnt11 Wnt4 RA,whereas mRNAs, and Wnt7a RAexpression mRNA Wnt5a Wnt4 , mRNA Wnt5a Wnt1 , C61,plioecai.Test flpdto a clearly was lipidation of site The acid acid. fatty monounsaturated palmitoleic the with (C16:1), peptide S2B), the Fig. as material identified (supplementary containing was 1455.9 peptide m/z The at observed S1). S244, Table (MALDI-MS/ material spectrometry (supplementary of mass MS) structure MALDI-tandem by folded identified for overall sufficient the but is maintaining N114 alone, alone, for at Wnt5a. N326 N114 glycosylation important that or at suggesting is thereby N312 glycosylation secretion, at Wnt5a that or glycosylation show secretion. not Wnt5aN114Q/N120Q/N312Q results or impaired contrast, These Wnt5aN326Q completely wild- By to Wnt5aN312Q, Wnt5aN114Q/N120Q/N326Q Wnt5a. levels mutated), secretion type similar is (supplementary showed Wnt5aN312Q/N326Q sites glycosylated N114 is glycosylation (N120 when Wnt5a Wnt5aN114Q/N120Q S2A). the Fig. mutations material to of each combinations added of various role secretion, were the in investigate Wnt5a To of 2007). glycan al., et Wnt5a (Kurayoshi of secreted N326 and N312 S2). Table N114, material supplementary to 2; attached (Fig. were glycans Further that confirmed S1). (LC/ESI-MS) spectrometry mass ionization Table electrospray chromatography/ liquid nano-flow material using peptides tryptic of (supplementary analyses glycosylated was N120 not but and respectively, hybrid high-mannose, oligosaccharides, with high-mannose-type glycosylated were N326 N114, covered; and was N312 sequence acid amino Wnt5a entire the of About 74.3% (MALDI-MS). matrix-associated spectrometry mass by desorption/ionization to laser followed subjected chromatography were (Kurayoshi Wnt5a liquid purified N326 of nano-flow peptides and Tryptic 2007). N312 al., N120, et N114, including of X-Ser/Thr, profiles glycan possible the four has determined Wnt5a Wnt5a. we trafficking, their are for proteins important secretory of modifications post-translational Because Wnt5a of modification Post-translational r motn o h otn fWtat h basolateral the to AP-1 Wnt5a and of clathrin sorting Wls, the that for off-target suggest important (si)RNA results are interfering These small the excluding effects. in S1D). affect 1G), (Fig. observed not cells phenotypes Fig. did 1F; the (Fig. AP-4) TfR rescued material and of podocalyxin subunit supplementary Wnt5a, severely of medium distribution was a polarized these of Wnt5a AP4M1; depletion Under of the as However, S1D,E). secretion 1E). (Fig. Fig. basolateral suppressed the material podocalyxin of conditions, supplementary that not respectively, but 1E; AP-1B, TfR (Fig. of and sorting AP-1A polarized of the disturbed subunits medium are or clathrin which of and Knockdown 2002). (Boehm al., and Fo et cells Simmen 2008; 2012; epithelial al., al., et et in Deborde proteins membranes 2001; cargo sort Bonifacino, basolateral AP-4 the and Fig. AP-1B material to supplementary AP-1A, 1D; and podocalyxin (Fig. Clathrin impaired of S1D). not distribution was II/ polarized TfR MDCK the and Wls polarized although the of in cells, al., from knockdown secretion Wnt5a et Wnt5a observations, clathrin Yamamoto suppressed these completely 2010; and with Basler, Consistent AP-1 and 2013). (Port by network membranes trans-Golgi basolateral the membrane. ii-oiidppiewsas bevdaogtepeptides the among observed also was peptide lipid-modified A never was Wnt5a mutated, were sites glycosylation all When l sesnilfrtesceino ns n stafce to trafficked is and Wnts, of secretion the for essential is Wls m B(lokona PM n PM,respectively), AP1M2, and AP1M1 as known (also 1B ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal N gyoyainsts aey Asn- namely, sites; -glycosylation m A and 1A- m lc,20;Gravotta 2005; ¨lsch, BH expression 1B–HA m m as known (also 4 1B-depeleted m 1A

Journal of Cell Science EERHARTICLE RESEARCH h oiiainwt amtli cdi seta o Wnt for essential is acid to palmitoleic failed that with confirming secretion. Wnt5aS244A S2D), modification addition, Fig. with 2006; material the In al., modified (supplementary et 2013). secreted (Takada is be al., Wnt11 Wnt5a and et Wnt3a that (supplementary Yamamoto are as indicating MALDI-MS/MS acid S2C), palmitoleic by Fig. S244 material as identified 1. Fig. e etpg o legend. for page next See ak lcslto iei h -emnlrgo,Lu8was Leu68 region, N-terminal Wnt5a the that in that Given site 2013). and apical glycosylation al., the secretion, a et for apical lacks (Yamamoto required its Wnt11 is of for lectin, secretion essential galactose-binding is a Wnt11 galectin-3, of attached glycan N40 complex-type the to that shown previously have We induces Wnt5a secretion of apical region its N-terminal the to glycans of Addition ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal 1053

Journal of Cell Science sda pcladbsltrlmmrn rti akr,respectively. means markers, as protein shown membrane are were basolateral Results and (TfR) and (Ap) receptor apical apical transferrin as between and used Wnts Podocalyxin of fractions. the ratio (Bl) and the basolateral Imaging as NIH expressed using were quantified results basolateral were the signals from Wnt5a Neutravidin–agarose was membranes. using Wnt5a precipitates Blue– membrane-associated the using and in precipitates media, observed the apical in the detected from was Sepharose apical-basolateral Wnt5a the Soluble to assay. subjected sorting were filter Wnt5a. polycarbonate of Transwell secretion Polarized 1. Fig. ARTICLE RESEARCH 1054 migration different showed cells Wnt5a II/Wnt5a Wild-type 3A). MDCK (Fig. N68-X-S70, from site sequence secreted glycosylation acid possible amino a the is generate which to Asn to mutated assay. sorting apical-basolateral the to subjected then were cells cells the II/Wnt5a and MDCK (G) assay. expressing sorting stably with apical-basolateral transfected the were to cells (E), subjected II/Wnt5a (Cla) MDCK clathrin (E,F) for 100%. siRNAs fraction, to basolateral set control was were the was which cells signal in the Wnt5a Neutravidin–agarose The and using assay. siRNA, sorting precipitated Wls apical-basolateral the or to (cont) subjected control then with transfected were 10 bar: cells Scale respectively. markers, basolateral and and by F-actin observed (DIC). or microscopy or F-actin, relief-contrast (+) visualize with to cysts phalloidin the Alexa-Fluor-633-conjugated and Matrigel, stably 3D ( or in without transiently cultured cells were the II Wnt5a to subjected MDCK expressing were (C) cells assay. II/Wnt5a sorting MDCK apical-basolateral and I/Wnt5a Neutravidin– MDCK using (B) precipitation agarose. N, Blue–Sepharose; using precipitation 2 emaiiainwr tie ihteidctdatbde or antibodies indicated the with stained were permeabilization ) m BH eetasetdwt iNsfor siRNAs with transfected were 1B–HA 6 m Aand 1A ...fo oridpneteprmns B, experiments. independent four from s.e.m. m B()or (E) 1B A DKIclssee na on seeded cells I MDCK (A) b ctnnwr sda apical as used were -catenin m F,adteclswere cells the and (F), 4 m .()MC II/Wnt5a MDCK (D) m. m Aand 1A m 1B, PGs ) hc lae -ikdgyas n pntreatment H upon endoglycosidase and with glycans, N-linked cleaves which F), (PNGase F N-glycosidase peptide: with treatment upon SDS-PAGE by patterns n h muto noH Endo of spectrometric glycan, amount of by the addition mass the mobility and to lower owing of Wnt5a exhibited wild-type than S1G) SDS-PAGE results glycosylated, Fig. cells the II/Wnt5aL68N heterogeneously material MDCK (supplementary with from is secreted Wnt5aL68N consistent analyses. Wnt5a is that which indicates 3B). This (Fig. glycan complex-type the not but glycans, hybrid-type and ta. 90 urane l,18) n- as nw as known (also Gnt-I 1984). al., et six of one Fuhrmann is which 1990; MGAT1), al., Golgi- et and I similar mannosidase a at cells 3C), active. NIH3T3 (Fig. is Wnt5aL68N medium in conditioned that Wnt5a suggesting Dvl2 control of the of medium that with to shift conditioned efficiency interact mobility the might a in Wnt5a induced 3B), Wnt5aL68N that Fig. suggesting membranes. (see in medium 3B), basement Fig. the ‘N’ in in (see present ‘B’ was membranes Wnt5a secreted with apically whereas associated secreted Basally was 3B). Wnt5a (Fig. cells in II/Wnt5aL68N apically In secreted MDCK Wnt5aL68N. was polarized to Wnt5aL68N Wnt5a, attached wild-type was to type contrast complex the that indicating n5L8 el ihkfnnieaddMcagdthe changed II/ dMM MDCK and of kifunensine Treatment with 1988). al., cells et Wnt5aL68N (Rademacher complex-type and/or hybrid- glycan of formation the initiates vertebrates, iuesn n exmnoiiyi dM nii ER- inhibit (dMM) deoxymannojirimycin and Kifunensine ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal pcr eedcnoue osnl hre ions. charged singly to mass deconvoluted The were ESI-MS. spectra liquid by nano-flow analyzed from were (N326) chromatography 36.5 min (N114), 28.6 32.1 and Wnt5a. after (N312) of eluted profiles that glycan glycopeptides the Wnt5a of Determination 2. Fig. f Ed H (Endo a f mnoiaeI epciey(Elbein respectively I, -mannosidase rssatgya nrae Fg 3B), (Fig. increased glycan -resistant N aeyguoaietaseae in transferases -acetylglucosamine f ,wihcevshigh-mannose cleaves which ), a -

Journal of Cell Science Wta3N,adWta3Nwsadtoal oiidwith modified additionally was Wnt3aL35N and (Wnt3aL35N), secretion. apical 3E). its (Fig. impair would by manipulations Wnt5aL68N TfR these complex- on glycan or high-mannose-type the hybrid and from the glycan podocalyxin of type maturation the supplementary of of prevention distribution 3E; Therefore, affect (Fig. not polarized did manipulations Wnt5aL68N these the contrast, By of S1F). Fig. secretion material apical inhibited galectin-3 the and Gnt-I for siRNAs 3E). (Fig. secretion apical EERHARTICLE RESEARCH estvt fWta6Nt noH Endo to Wnt5aL68N of sensitivity were precipitates the and Blue–Sepharose, using precipitated was cells II MDCK from secretion. medium apical conditioned its H in induces Wnt5aL68N Endo Wnt5a or with of Wnt5a treated region (B) N-terminal shown. the are to Wnt11 glycans and of Addition 3. Fig. n5 rWta6Naesoni h ih ae.()MC IWta6Nclswr rae ihkfnnie(i)o exmnoiiyi dM,and (dMM), deoxymannojirimycin (20 or media (kif) Conditioned kifunensine panels). H with Endo two treated with (left were treated antibody were cells precipitates anti-Dvl2 II/Wnt5aL68N The with MDCK Blue–Sepharose. usin Wnt5aL68N-contai probed using (D) precipitation or precipitated then panel. B, Wnt5a- then were right 100%. was of lysates medium the to amounts conditioned and in set indicated in h, shown was Wnt5aL68N the 2 fraction are with for (Ap) Wnt5aL68N stimulated cells apical or were ba L control Wnt5a control cells from the the NIH3T3 (CM) in in (C) Blue–Sepharose Neutravidin–agarose medium using Neutravidin–agarose. using conditioned precipitated using precipitated was precipitation was Wnt5a N, Wnt5aL68N of signal of Sepharose; The signal panels). the lower and and (middle fraction assay sorting apical-basolateral the to niWtaatbd.()MC IWta6Nclswr rae ihkfo M,o rnfce ihGtIo aetn3(a3 iN,adteclswere cells the and siRNA, (Gal3) 3 galectin or means Gnt-I as with shown transfected are or Results dMM, control. or cont, kif assay. with sorting treated apical-basolateral were the cells to II/Wnt5aL68N subjected MDCK (E) antibody. anti-Wnt5a iia oWta e3 a uae oAni Wnt3a in Asn to mutated was Leu35 Wnt5a, to Similar f rPGs ,adte rbdwt niWtaatbd uprpnl) DKI/n5 rMC IWta6Nclswr subjected were cells II/Wnt5aL68N MDCK or II/Wnt5a MDCK panels). (upper antibody anti-Wnt5a with probed then and F, PNGase or f Fg D n niie its inhibited and 3D) (Fig. ih esfiin o nst hnetersrigdestination. Wnts sorting of their region change to appropriate Wnts the for sufficient at Therefore, be substitution activities. might their acid losing amino without Wnt3a one apically and Wnt5a secreted for be sufficient complex-type is to of region addition N-terminal the the the to that glycan suggest induced results These Wnt3aL35N 4C). apical (Fig. 4A; 4B). the (Fig. (Fig. inhibited of galectin-3 Wnt3aL35N accumulation apically and of Gnt-I kifunensine, secreted for secretion with siRNAs Treatment and and S1H). dMM Fig. glycan material supplementary complex-type the ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal 6 ...fo oridpnetexperiments. independent four from s.e.m. A lcnpoie fWta n5L8,Wta Wnt3aL35N Wnt3a, Wnt5aL68N, Wnt5a, of profiles Glycan (A) b ctnnt iia erea idtp Wnt3a wild-type as degree similar a to -catenin f rPGs n hnpoe with probed then and F PNGase or m )containing l) oaea (Bl) solateral Blue– g ning 1055

Journal of Cell Science EERHARTICLE RESEARCH lhuhtebnigseiiiybtenWt n z snot is Fzs 1056 and 2007). Wnts Bryja, between and Schulte specificity as 2011; binding function al., the which et members, Although (Kikuchi family receptors (Fz) Frizzled Wnt ten are receptors There Wnt of localization Polarized H Endo with treated precipitated were was precipitates cells the II and MDCK Blue–Sepharose, from is using Wnt3a. medium region of conditioned N-terminal secretion in the polarized Wnt3aL35N at the site change glycosylation to the sufficient of Addition 4. Fig. lf w aes.Wl-yeWtaadWta3Ni odtoe medium conditioned in Wnt3aL35N and Wnt3a (20 Wild-type panels). two (left yae eete rbdwt anti- with and probed h, wild- then 2 of for were amounts (CM) indicated lysates medium conditioned the Wnt3aL35N-containing with or stimulated Wnt3a- were type cells assay. means L sorting as (C) apical-basolateral siRNA, shown experiments. the (Gal3) are to galectin3 Results subjected or control. Gnt-I then cont, with were transfected cells or the dMM, and or were kif N, cells with Blue–Sepharose; II/Wnt3aL35N treated MDCK using (B) precipitation Neutravidin–agarose. (Ap) B, using apical 100%. precipitation control to the set in was Blue–Sepharose fraction signal using the precipitated or precipitated fraction Wnt3aL35N (Bl) Wnt3a of basolateral of control signal the apical- in The Neutravidin–agarose the panels). using to (right II subjected MDCK assay were panels). sorting Wnt3aL35N (left basolateral or antibody Wnt3a anti-Wnt3a expressing with probed cells then and F PNGase m )aesoni h ih ae.GSK-3 panel. right the in shown are l) b ctnnadanti-GSK-3 and -catenin 6 b ...fo orindependent four from s.e.m. a sda odn control. loading a as used was A n3 or Wnt3a (A) b antibodies f or itiue nbt h pcladbsltrlmembranes basolateral and apical the both exogenously basolateral in evenly the was and Ryk–GFP 5A,D,E), at distributed (Fig. LRP6 cells located II MDCK endogenous primarily Yamamoto of membranes Wnt11, 2010; were Although al., and Ror2–GFP 2006). et expressed Wnt5a Nishita al., Wnt3a, 2008; basolateral al., et of et the co-receptors (Kim respectively that as function and complex. AP-1 Ryk, and Wls, clathrin the contain plasma requires the Fz2 results not of to localization These destined do vesicles S1D,E). Fz7-loaded membranes or Fig. Fz2- material that suggest supplementary 5B; (Fig. both the of depleted supplementary affect cells and was and 5B,C; cells FLAG–Fz2 not clathrin-depleted of (Fig. in localization did impaired polarized Fz membranes The Wls S1D). either basolateral Fig. material of of and localization Knockdown apical polarized both 5A–C). (Fig. to evenly localized FLAG– whereas was membranes, basolateral Fz7 the to FLAG– localized of was majority Fz2 The expressing respectively). stably II/FLAG–Fz7, II/FLAG–Fz2 MDCK (MDCK cells and generated II (Sato were the FLAG–Fz7 MDCK receptor determine or Fzs, To FLAG–Fz2 Wnt11 Wnt5a of 2012). the and Kypta, localization and as Wnt3a polarized Uysal-Onganer mediates functions 2010; Fz2 al., Fz7 et that that shown and been signaling has it clear, aoaea iemr fiinl hnteaia ie(i.5G). (Fig. side the apical at the Dvl2 than of efficiently with shift more side mobility basolateral the cells and more phosphorylation Rac1 LRP6 Wnt3a Recombinant 5G). II/FLAG–Fz2 activated induced (Fig. side side apical the basolateral at MDCK that than the efficiently at polarized Wnt5a recombinant of stimulation Ror2–GFP and m and clathrin- LRP6 in of disturbed was localization membrane S1D). of Fig. basolateral localization material the The supplementary affect 5D–F; not (Fig. did co-receptors Wls these of Knockdown 5A,F). (Fig. oee,kokono n4o n7 i o fetapical as affect well S3A); As not S3B). did Fig. Fig. Wnt7a material material or (supplementary formation Wnt4 supplementary lumen of 6C; knockdown (Fig. however, Wnt5a- had cells in observed which diameter were depleted phenotypes lumens, Similar a narrow 6B). (Fig. and with increased cysts cells of decrease, multilayered number to lumens the dilated was with cysts whereas diameter of the number the which caused IWP2 of lined was and which cells, lumen, . monolayer of dilated polarized consisting a a showed cysts by cells of I 70% MDCK matrix Approximately control extracellular 6A,B). the with with (Fig. some treated contact the cysts make (ECM) 7, not of of day did space they luminal At formation and the IWP2, 6A). for in (Fig. observed required still cells were was monolayer cells so days of formation, 10–11 lumen consisting in of lumen delay period a a caused 2009), that al., acquiring et 6A). (Chen by (Fig. IWP2 6–7, surface 8–9 days days basal at at diameter and complete lumen was lateral the cystogenesis increased free, and cyst cell a the Each and 4. and cultured had day polarity, were cyst at synthesis cells observed the was I lumen in MDCK nascent Wnt5a a When Matrigel, was cells. 3D endogenous in cells I MDCK epithelial inhibiting in in secretion secretion by basolateral Wnt5a examined of role lumen The apical cysts for MDCK required of is formation Wnt5a of secretion Basolateral -eltdcls(i.5,;splmnaymtra i.S1D,E). Fig. material supplementary 5D,E; (Fig. cells 4-depleted 0 hto h yt(i.6) ycnrs,tetetwith treatment contrast, By 6B). (Fig. cyst the of that 50% igetasebaercpos nldn R6 o2and Ror2 LRP6, including receptors, transmembrane Single ossetwt h oaie oaiaino h receptors, the of localization polarized the with Consistent ramn fMC el ihaWtsceininhibitor secretion Wnt a with cells I MDCK of Treatment m B( 1B ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal m 1A/ m Bdpee) u o in not but 1B-depleted), m 1A/ m Bdpee el u o in not but cells 1B-depleted , 0 hto h cyst, the of that 50% m -eltdcells 4-depleted m 1A

Journal of Cell Science EERHARTICLE RESEARCH nplrzdMC /n5L8 el,adbasolateral and I/ cells, MDCK in observed I/Wnt5aL68N was Ror2–GFP of MDCK observed localization was membrane Wnt5aL68N polarized of secretion apical in cells, II MDCK in xrsino idtp n5,btntb Wnt5aL68N by by not rescued but were The Wnt5a, siRNA S3C). wild-type Wnt5a Fig. of by material expression induced (supplementary phenotypes cells Ror2–GFP ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal a ciainasyo the or assay the activation to Rac subjected then and were sides, lysates basolateral or from apical panel) the (right ng/ml) with (100 stimulated Wnt3a were cells or II panel) MDCK (left ng/ml) (50 Wnt5a with stimulated were cells II/ FLAG–Fz2 MDCK (G) 100%. control. cont, to apical; set Ap, was which fraction, basolateral (Bl) control the from agarose Neutravidin– using receptors precipitated the was of signal The assay. sorting apical-basolateral the to m (Cla), clathrin Wntless (Wls), for siRNA with were transfected cells (F) II/Ryk–GFP or MDCK (E) II/Ror2–GFP MDCK II (D), MDCK (C), II/FLAG–Fz7 MDCK (B), II/FLAG–Fz2 MDCK (B–F) 10 bar: Scale indicated antibodies. the with cysts stained the were and Matrigel, 3D in cultured were cells II/Ryk–GFP MDCK or II/Ror2–GFP MDCK FLAG–Fz7, II/ MDCK II/FLAG–Fz2, MDCK receptors. Wnt localization of polarized The 5. Fig. needn experiments. independent as means shown are Results and Dvl2. LRP6 of assay phosphorylation ,adteclswr hnsubjected then were cells the and 4, 6 ...fo four from s.e.m. A DKII, MDCK (A) m Aand 1A m m. m B or 1B, 1057

Journal of Cell Science EERHARTICLE RESEARCH eurdfrefcetaia ue omto nMC I MDCK is in 1058 side formation basolateral lumen results the apical These from efficient cells. 20 S3A). signaling bars: for Scale types. Fig. Wnt5a luminal required their material that by sho classified are were and indicate supplementary 7 types, luminal day of at 6C; ratio cysts the (Fig. The as DRAQ5. expressed and day are at phalloidin Results cysts with The counted. stained shown. was are then type (2) were lumen each narrow cysts of or the cysts (1) and of lumen dilated siRNA, number a the with means and cyst (%) I the types, MDCK ratio as luminal of diameter their images 5 formula: Schematic by without nucleus. following classified the or the were visualize with to using treated (blue) calculated DRAQ5 were was and Matrigel (green) cyst 3D whole in the line) cultured (red and cells cyst lumen the apical of line)/diameter the cyst. (blue between MDCK lumen of ratio formation diameter lumen The apical the F-actin. for required is 5 Wnt5a without of or secretion Basolateral 6. Fig. m W2frteidctddy,adtecsswr hnsandwt anti- with stained then were cysts the and days, indicated the for IWP2 M 6 ...fo oridpneteprmns C DKI DKIWta rMC /n5L8 el eetasetdwt oto rWnt5a or control with transfected were cells I/Wnt5aL68N MDCK or I/Wnt5a, MDCK I, MDCK (C) experiments. independent four from s.e.m. 6 0.Tema ftetodaee aisi h yt tteidctddy eepotd(ih ae) B DKI MDCK (B) panel). (right plotted were days indicated the at cysts the in ratios diameter two the of mean The 100. m W2fr7dy,adtecsswr hnsandwt lx-lo-8-ojgtdphalloidin Alexa-Fluor-488-conjugated with stained then were cysts the and days, 7 for IWP2 M ramn fMC el ihIP rdpeino Wnt5a 5–7 days of at depletion paxillin or and IWP2 FAK of with phosphorylation cells the I suppressed MDCK of Wnt5a- Treatment in formation lumen cells apical depleted for required is Apoptosis b ctnnatbd n lx-lo-8-ojgtdpalii ovisualize to phalloidin Alexa-Fluor-488-conjugated and antibody -catenin ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal A DKIclsclue n3 arglwr rae with treated were Matrigel 3D in cultured cells I MDCK (A) 5 imtro central of diameter m m. wn 7

Journal of Cell Science ntelmnlrgo tdy –,rsligi ea flumen of delay by a in not resulting treated 5–9, days but were at observed region cells were Wnt5a, paxillin cells luminal some I the inhibitor), in wild-type GEF MDCK activity, Rac (a When of NSC23766 with FAK were 7A,B). (Fig. expression depletion Wnt5a Wnt5aL68N in to by due Decreases activity Rac rescued Rac reduced and 7B). also phosphorylation cell- (Fig. in manipulations reduction a These activity suggesting adhesion. 7A), (Fig. to-substrate culture Matrigel 3D in ARTICLE RESEARCH o h aoaea otn fmmrn rtis(oh and (Boehm proteins membrane of sorting basolateral machineries the common because are which 2013), for AP-1, al., and et clathrin requires (Yamamoto sorting Wnt3a its of that as its mechanism activity. same change lose to to able is it Wnt causing of noteworthy without substitution is route acid It trafficking amino galectin-3. single by a N-terminal apically secretion that the secreted at be apical glycan might complex-type their region the Wnts with Therefore, and modified Wnt11. are of region site, that that to similar N-terminal glycosylation galectin-3, was on their the depended glycan complex-type at to The site attached Wnt3aL35N). glycosylation and the (Wnt5aL68N of unknown. remains addition site Wnt5b, this and at Wnt5a glycosylation in of unique role is the site but glycosylation wild- The of Wnt5a. that type to the comparable because secretion secretion for displayed hybrid-type essential mutant not Wnt5aN312Q with was it modified although also N312, at was glycan impaired, are Wnt5a was structure. Wnt11 glycans folded Wnt11 high-mannose-type and overall and these both Wnt5a Wnt3a that Wnt3a, removing of suggesting of By secretion residues 2013). the Asn glycans, al., also conserved et was most glycan the (Yamamoto in high-mannose-type at conserved with are observed high- modification residues with and modified Asn by Wnts, were Both Wnt5a Wnt5a to glycans. of attached mannose-type N326 glycans and of N114 profiles ESI-MS/MS. the determined we Fo study, 2008; al., with Bonifacino, et and signals Deborde (Boehm APs 2001; trafficking and clathrin cytoplasmic as such of machineries sorting interaction the are proteins by sorted is delivered basolaterally proteins whereas sorted signals, apically glycan-mediated or some of trafficking glycosylphosphatidylinos on the dependent that believed is It Wnt5a of secretion Polarized DISCUSSION integrins. through adhesion cell-to-substrate by of formation enhancement lumen the promotes signaling Wnt5a that (Myllyma integrins be suggest formation of results knockdown might lumen that apical report apoptosis the the impairs signaling-deficient with in Wnt5a Thus, together in Taken 11 formation cells. 7E). day lumen (Fig. apical at for QVD-Oph required of cells formation monolayer of decreased by presence a surrounded showed lumen cysts apical I or presence Wnt5a-depleted the MDCK but in QVD-Oph, IWP2-treated even cysts inhibitor cells, caspase few I general a the MDCK with of lumen Control apical apoptotic clear 7C,D). the a (Fig. formed with caspase-3 underwent staining by cleaved cells determined marker, luminal as cells, few suppressed apoptotic was signaling very had Wnt5a which cysts, and in cysts control whereas al., trafficking apoptosis, et the (Datta proposed In vesicle been 2011). have directional respectively, death, cell require luminal which cavitation, siRNA Wnt5a and IWP2 7C). with treatment (Fig. the to similar formation, oelmnfrainmdl,icuighloigand hollowing including models, formation lumen Some h n5 aoaea erto slkl ob eitdb the by mediated be to likely is secretion basolateral Wnt5a The the following apically secreted were Wnt3a and Wnt5a lc,20;Vgne l,20) nthis In 2009). al., et Vagin 2005; ¨lsch, eesr o anann the maintaining for necessary tlaco-neatn signals itol-anchor-interacting k ta. 01,these 2011), al., et ¨ki oddi h aevscecnann l n htteei a is there that and Wls binding. containing their are inhibit vesicle receptors to same Wnt mechanism the and Wnts in which An that vesicles. loaded by be same receptors, the might signaling in possibility receptors Wnt their alternative and of ligands of containing activation co-existence the intracellular vesicles could prevents and from vesicles probably (Port basolateral Wnt-loaded distinct and Wnts Therefore, apical of the receptors. be for Wnt trafficking required of not the is sorting it for but essential 2010), Basler, is Wls 2014). Pr n alr 00 aaooe l,21) l should Wls 2013), al., et vesicles. Wnt3a-loaded Yamamoto and Wnt5a- 2010; to localize Basler, Wnts and palmitoleic-acid-modified by basolateral (Port recognized the to is trafficked and also is membrane, Wls that Given 2001). Bonifacino, 00,adF7fntosa eetrfrWt1(i tal., et are (Kim which al., Wnt11 Ror2, et and for (Sato LRP6 receptor 2007). Wnt5a a Nishida, and and as Yamanaka Wnt3a functions 2008; for Fz7 receptor and common 2010), a Fz2 membranes. as basolateral evenly acts and relatively apical was the Fz7 throughout whereas distributed cells, II the MDCK in of membranes apical localization PCP the the the specificity. is of pathway it and determines activation that that the junctions, suggest receptors for results apical necessary These it is pathway. the dFz1 prevents of dFz2 at C- localization of The accumulating sequence 2004). acid al., from et amino throughout (Wu distributed cytoplasmic membranes is terminal basolateral dFz2 and whereas apical disc, the in junctions (PCP) imaginal apical wing polarity at concentrated the cell is the dFz1 planar 2003). in (Strutt, the functions pathway especially also pathway, dFz1 independent and pathway, dependent Drosophila receptors Drosophila Wnt of trafficking Polarized Yg ta. 02,adta ncdw fitgisimpairs integrins of knockdown (Myllyma cells that MDCK cysts the and in MDCK formation at 2012), of lumen apical polarization activation al., the Rac1 et for 2010). (Yagi required that is al., demonstrated side et basolateral have (Matsumoto cells studies cell- through cancer promotes Other cells Wnt5a in that IEC6 and adhesion 2013), epithelial to-substrate al., single- et intestinal the (Gon activation rat in Rac1 involved of is polarization Wnt5a that cell previously shown have We basolateral Wnt5a by of formation secretion lumen apical of Regulation basolateral the in present epithelium is midgut of LRP6 that embryonic region and the 2010), of al., sides et (Yamada basal the to localized of activation the efficient as fractions to polarized signals. leading same the their thereby the to Wnts, appropriate to the coupled sorted corresponding that are likely functionally is receptors it is Wnt Thus, that Wnts. receptors of indicating secretion side, their polarized basolateral of the activated stimulated localization strongly Wnts were both Wnt5a and The when understandable. Wnt3a is of of cells pathways localization II signaling the MDCK polarized apically, in secreted receptors secreted their is are and Wnt11 Wnt5a apical and the and both basolaterally Wnt3a to Because localized membranes. was basolateral 2008), basolateral Wnt11 al., the mediates et to which (Kim Ryk, trafficked signaling AP-1. also and clathrin were using 2006), membranes 2010; al., al., et et (Nishita respectively Yamamoto Wnt5a, and Wnt3a for receptors u td hwdta z anylclzdt h basolateral the to localized mainly Fz2 that showed study Our u eut r ossetwt h idnsta o2is Ror2 that findings the with consistent are results Our ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal Xenopus n ooo.Bt eetr eit the mediate receptors Both homolog. Wnt z dz)addz c srcposfrwnls,a wingless, for receptors as act dFz2 and (dFz1) Fz1 ue coemlcls(un n Niehrs, and (Huang cells ectodermal outer k ta. 2011). al., et ¨ki b b -catenin- -catenin- 1059

Journal of Cell Science EERHARTICLE RESEARCH Dtae l,21) hs elahso oEMi h is event first the 1060 is ECM to adhesion expansion cell Thus, lumen 2011). and al., polarization, et (Datta basal and to apical linked recognition, are cells. events epithelial cellular polarized these in how lumen clear apical form not is it However, 7. Fig. ue omto eiso h oriaino cell-to-substrate of coordination the on relies formation Lumen e etpg o legend. for page next See OBine l,20) eas u Dcluewsperformed was culture 3D our of laminin Because orientation basolateral 2001). for the al., I of et necessary type assembly (O’Brien are in through activities cystogenesis polarization apical MDCK Rac1 During gel, cell-to-substrate culture. collagen enhances 3D secretion in adhesion Wnt5a luminogenesis. for ora fCl cec 21)18 0116 doi:10.1242/jcs.163683 1051–1063 128, (2015) Science Cell of Journal

Journal of Cell Science ujce oteRcatvto sa.()MC el n3 utr were culture 3D then in were cells 7 I day MDCK at (C) lysates assay. cell activation with and Rac transfected days, the were 7 to or for subjected IWP2, siRNA of cells Wnt5a absence I/Wnt5aL68N or or MDCK control presence anti-pY397- or the with I/Wnt5a in probed MDCK as cultured then I, used were were MDCK were 7 (B) paxillin or controls. and 5 loading FAK 5 day for antibodies. at anti-pY118-paxillin panels) lysates and three two cell FAK (right (left and siRNA IWP2 days, Wnt5a of 7 or absence or control or with presence transfected the or in Wnt5a- panels) in Matrigel in formation cultured lumen were apical the for cells. required depleted is Apoptosis 7. Fig. ARTICLE RESEARCH rvddb rSij aaa(ainlIsiue fNtrlSciences, Natural were of Institutes Fz7 (National pRK5/mouse Takada Shinji and Dr Fz2 by provided is pRK5/rat Wnt5a, Wnt5a pPGK-neo/mouse chemicals different and of Materials by METHODS secretion AND the MATERIALS apoptosis. cells without to basolateral formation epithelial lumen trafficked the apical are for polarized necessary and receptors of its mechanisms, side and Wnt5a basolateral 2010). are al., summary, cells, clumps, et of (Yamada In gut cell orientation embryonic random Ror2-knockout epithelial and in observed aberrant loss basal polarity epithelial midgut; suggest and which embryonic mesenchyme Wnt5a the of example, receive in region For expressed might are cells. membranes Ror2 mesenchymal and basolateral from express an the secreted that cells Wnt5a at in the epithelial receptors secretion, polarization the in Wnt5a Wnt5a are of basolateral involved cells source mesenchymal primary and be because However, might apical manner. these autocrine In axis of cells. 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Cary, data Inc., the Institute, (SAS software StatView oyabnt itrfra diinl3days. 3 additional an for filter polycarbonate asspectrometry mass ionization chromatography/electrospray liquid Nano-flow 2010). al., then et (Awada Applied previously described analyzer, as was system proteomics MA) Framingham, Biosystems, (4700 identification MALDI-TOF/TOF nano-LC peptide a Ultimate using overall an out carried and into Germany), injected Idstein, were (Dionex, Wnt5a of digests Tryptic spectrometry mass desorption/ionization laser chromatography/matrix-assisted liquid Nano-flow ARTICLE RESEARCH 1062 (Komekado previously 2007). al., described et as Kurayoshi 2007; Wnt- performed conditioned al., from of et lysates were Preparation and cells fraction 2013). previously (ECM) al., producing matrix described et extracellular as Yamamoto the 2010; medium, were al., PCR et real-time (Sato quantitative for Methods methods Additional significant. are Two-tailed results the and times, four means least as at expressed performed were Experiments analysis Statistical (Life Opti-MEM in suspended were 10 receptors, cells its at or Technologies) Wnt5a MDCK expressing cells MDCK trypsinized siRNA into by siRNA cells transfect MDCK To in mRNA of Knockdown 1 at of CA) density Jose, San a Biosciences, (BD Matrigel in suspended were cells cells MDCK MDCK of culture 3D (2 cells II or I MDCK 2013). described al., as examined et was (Yamamoto receptors Wnt previously and Wnt5a of sorting Polarized assay sorting Apical-basolateral High- 2013). al., Hitachi et (Kimura LD, previously described NanoFrontier as nano- (Hitachi Japan) a Tokyo, a source Technology, by with ion analyzed equipped electrospray continuously spectrometer a flow then mass to were ion-trap/time-of-flight applied eluates linear were the digests mm and tryptic (0.075 NewObjective), above column glycoform the C18 each site, of PicoFrit abundance glycosylation relative each and chains at glycan the examine To ne h odtos DKclsfre yt.Frtetetwith treatment For cysts. formed cells cells). MDCK II 5 (MDCK inhibitors, days conditions, 3 or the cells) DMEM I of (MDCK Under days ml 7 1 for in incubated FBS 10% and containing plate culture 24-well a to transferred were narudcvrlpa ecie rvosy(aaooe l,21) After 2013). 37 al., at et incubation (Yamamoto previously described as coverslip round a on itcnlg,Rcfr,I)fr3 i t4 at min 30 for cells (Pierce sulfo-NHS-LC-biotin the IL) mg/ml of Rockford, 0.5 membranes Biotechnology, or with surface incubated Wnts basolateral selectively cell-surface-associated or were membrane apical II detect the (MDCK To receptors, figures). their days in 3 with (indicated ‘B’ precipitated or medium culture were as the cells) sites into secreted Biological, basolateral Wnts I detect and to apical Blue–Sepharose Quality (MDCK the days from Costar Media 7 cells). for (Corning MD) filters on Gaithersburg, seeded were polycarbonate receptors Wnt or transwell mutants their Wnt3a, Wnt5a, expressing rbduigec nioy(niae s‘’i figures). in ‘N’ as were using (indicated antibody precipitates precipitated each the were using probed and proteins Biotechnology), Biotinylated (Pierce 2013). 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NEPA21 pulses al., square et using five electroporated with Japan) were cells and hnpaigi argl n eimwscagdeey2days. 2 every changed was medium and Matrigel, in plating when m P 6 MIWP2,5 vle esta .5wr osdrdstatistically considered were 0.05 than less -values 10 ˚ o 0mnt oiiytegl h el ntecoverslip the on cells the gel, the solidify to min 30 for C 4 el/l n 80 and cells/ml, 6 6 el e 100 per cells ...Saitclaayi a efre using performed was analysis Statistical s.e.m. m MNSC23766or10 6 10 6 5 0 m nrsl3- Inertsil mm, 100 m ftecl upninwsmounted was suspension cell the of l el)wr eddo Transwell a on seeded were cells) m ,sRAwsadd(6 pmol), (160 added was siRNA l, m ˚ V-p eeadded were QVD-Oph M Skn ta. 2012; al., et (Sakane C m atcesize, particle m 6 10 5 t cells) -test. rvta . avjlGnae,J . atr,R,Dbre . afle,J R., J. J. Banfelder, S., Deborde, M. R., Mattera, M., J. Caplan, Carvajal-Gonzalez, D., Gravotta, and A. L. Dunbar, J., A. C. Kikuchi, and Gottardi, S. Matsumoto, Y., Ku, K., Fumoto, H., Gon, S. Tsukita, and H. Sasaki, K., Furuse, M., Furuse, uoo . iuh,K,Gn .adKkci A. Kikuchi, and H. Gon, K., Kikuchi, K., Fumoto, uran . as,E,Lge,G n leh H. Ploegh, and G. Legler, E., Bause, U., Fuhrmann, D. A. Chalmers, and P. Whitley, D., J. R. Dukes, Schreiner, S., Salvarezza, A., Deora, D., Gravotta, E., Perret, S., Deborde, Fo P. G. Kaushal, and M. Mitchell, E., J. Tropea, D., A. Elbein, E. K. Mostov, and M. D. Bryant, A., Datta, atrn,M n osae,H C. H. Korswagen, and M. Harterink, upeetr aeilaalbeoln at online available material Supplementary material Supplementary Medicine of Faculty University Program. Osaka Training the Science Scientist by of Doctor supported Promotion Medical is the T.S. for A.K. (Japan) number in to Foundation grant [grant (2013) NOVARTIS research (2013–2014) the a Foundation by by Mitsubishi and and the 25117]; Japan; from of the sciences Culture from natural H.Y. and the to Science 26460365] Education, number of [grant (2011–2013) [grant Ministry Research (2014–2016) Scientific and (2011–2013) for 23590333] and Areas number A.K., Priority to on 23112004] (2009–2011) Research number Research Scientific [grant Scientific for for 21249017], Grants-in-Aid number by [grant supported was work This Funding manuscript. the wrote and experiments the designed A.K. advice. and spectrometry provided mass by Wnt5a of profiles lipidation and glycosylation the analyzed T.T. and C.A. assay. fractionation the performed T.S. the assay. apical-basolateral sorting out carried T.K. analyses. immunofluorescence performed the S.M. manuscript. wrote and analyses, immunofluorescence the and performed proteins, assay sorting Wnt apical-basolateral of purification out carried experiments, designed H.Y. contributions Author interests. financial or competing no declare authors The interests Competing Fo H. and Kohsaka, S. Ohsawa, K. Minami, plasmids. Y. Takada, donating S. Drs thank We Acknowledgements akk,T,Ca . u,X,Tagc,S,Xe . uia . ioa . Lydon, Y., Hirota, T., Fujita, H., Xie, S., Tranguch, X., Sun, W., J., Hao, Cha, T., S., Daikoku, Wei, W., C. Fan, Z., Ma, J., Lu, W., Tang, E., M. Dodge, B., Chen, E. K. Mostov, and M. D. Bryant, S. J. Bonifacino, and M. T. Boehm, Takao, and T. Sato, C., Awada, References http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.163683/-/DC1 sh H. lsch, ¨ oiaio .S n orge-oln E. Rodriguez-Boulan, and S. J. 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