Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation

38 Pages·2014·4.72 MB·English

(cid:2)2015.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2015)128,1051–1063doi:10.1242/jcs.163683
RESEARCH ARTICLE
Basolateral secretion of Wnt5a in polarized epithelial cells is
required for apical lumen formation
Hideki Yamamoto1, Chihiro Awada2,*, Shinji Matsumoto1, Tomoyuki Kaneiwa1, Takayuki Sugimoto1,
Toshifumi Takao2 and Akira Kikuchi1,`
ABSTRACT processes;defectiveWntsignalinginpostnatallifecausesdisease
inhumans(Kikuchietal.,2011;LoganandNusse,2004;Polakis,
Wnt5aregulatesplanarcellpolarityinepithelialcells,butitremains
2007). Our previous study showed that when IEC6 cells were
to be determined whether Wnt5a and its receptors are sorted
seeded on thick Matrigel containing basement membrane
apically or basolaterally, and how Wnt5a signaling is involved in
proteins, a single cell was polarized cell autonomously, with an
apical and basolateral polarization. We found that Wnt5a was
F-actincapformingontheoppositesidetoMatrigel;knockdown
secreted basolaterally in polarized kidney epithelial cells. The
of Wnt5a inhibited this phenotype (Gon et al., 2013). In cancer
basolateralsecretionofWnt5arequiredWntless(Wls),clathrinand cells, Frizzled-2 (Fz2), a Wnt5a receptor, forms a complex with
adaptorprotein1(AP-1).Wnt5areceptorswerealsolocalizedtothe integrin a2, and Dvl, a cytoplasmic protein that mediates Wnt5a
basolateral membranes, but their sorting did not require Wls. signaling, interacts with focal adhesion kinase (FAK), thereby
Wnt5a-induced signaling was stimulated more efficiently at the enhancing cell-to-substrate adhesion (Matsumoto et al., 2010).
basolateralsidethantheapicalsideofepithelialcells.Knockdown Thus, Wnt5a and its receptors appear to sort to the basolateral
ofWnt5adelayedapicallumenformationoftheepithelialcyst,and membranesofpolarizedepithelialcellsandmightberequiredfor
these phenotypes were rescued by wild-type Wnt5a, but not by a the formation of apical-basolateral polarity.
Wnt5amutantthatissecretedapically.Althoughapoptosiswasnot Wntproteinsarepost-translationallymodifiedwitholigosaccharides
required for apical lumen formation in a wild-type cyst, apoptosis andlipidsduringtheirsynthesis(HarterinkandKorswagen,2012;
was necessary for eliminating luminal cells in a Wnt5a-depleted Komekadoetal.,2007;Kurayoshietal.,2007;Takadaetal.,2006;
cyst.TheseresultssuggestthatWnt5aanditsreceptorsaresorted Willert et al., 2003). Our previous study also revealed that the
to their correct destination by different mechanisms and that the majority of Wnt3a and Wnt11 are secreted basolaterally and
basolateral secretion of Wnt5a is necessary for apical lumen apically, respectively, in polarized Madin-Darby canine kidney
(MDCK)epithelialcells(Yamamotoetal.,2013).Thebasolateral
formationintheepithelialcyst.
traffic of Wnt3a requires adaptor protein 1 (AP-1) and clathrin,
KEYWORDS:Epithelialcells,Glycosylation,Polarity,Lumen, which are known to be important for the sorting of basolateral
Wnt5a membraneproteins(BoehmandBonifacino,2001;Debordeetal.,
2008; Fo¨lsch, 2005). It is generally thought that glycosylation of
INTRODUCTION secretaryproteinsisnecessaryfortheirfoldingintheendoplasmic
The generation and maintenance of epithelial cell polarity reticulum (ER) or exit from the ER (Spiro, 2002). Wnt3a and
requires the correct delivery of newly synthesized and Wnt11 were glycosylated at multiple Asn residues, and their
endocytosed proteins to apical or basolateral membrane glycosylation profiles differed (Yamamoto et al., 2013). The
domains (Yeaman et al., 1999). To establish apical-basolateral complex-typeglycanattachedtoWnt11attheN-terminalregionis
polarity, cells must sense their environment, which can be unique, and glycosylation is necessary for the apical secretion of
mediatedbytheinteractionofcellswiththeextracellularmatrix, Wnt11.
including basement membrane proteins. Cells also recognize In addition, Wnt3a and Wnt11 are modified with palmitoleic
soluble factors, such as morphogens and growth factors, and acid at a conserved Ser residue (Takada et al., 2006; Yamamoto
activate signaling pathways to support the orientation of their et al., 2013). The lipidation of Wnt3a and Wnt11 occurs in the
polarity (Bryant and Mostov, 2008). As an example of a soluble ER,iscatalyzedbyporcupineandisessentialfortheirexitfrom
factor, Wnt5a promotes the induction of single-cell polarization the ER. Wntless (Wls), which plays a crucial role in the
in rat intestinal epithelial cells (IEC6 cells) (Gon et al., 2013). traffickingofWnt,recognizespalmitoleic-acid-modifiedWntsin
Wnt5aisamemberoftheWntfamily,whichisalargefamilyof the ER and cycles between the plasma membranes and the ER
e
secreted molecules that play an important role in developmental through the Golgi (Port and Basler, 2010; Yu et al., 2014). Wls c
n
itselfistraffickedtothebasolateralmembranesanditsrecycling e
1DepartmentofMolecularBiologyandBiochemistry,GraduateSchoolof is not required for the apical sorting of Wnt11 from the trans- ci
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MR2Leeasdbeiocarirnacetho,,rOyOsosafakkPaaroUUtenniinivveePrrrssoiitftyiyl,i,n23g--22anYYdaammFauadndcaatooiokknaaa,,lSSPuuriiottaate,,oOOmssaiackksaa,I55n66s55ti-t-0u08t8e771f1o,,rJJPaaprpoaatenni..n Greovlegaile(dYathmaatmthoetolipeitdaml.,od2i0f1ic3a)t.ioCnryosftaXlesntorupcutsurWalnat8nailnysseerstshianvtoe Cell
*Presentaddress:R&DDept.BioMedicalProject,PharmaFoodsInternational thegrooveof the extracellular domainof mouseFz8, suggesting
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Co.,Ltd.,1-49Goryo-Ohara,Nishikyo-ku,Kyoto615-8245,Japan. the necessity of lipid modification of Wnts for the recognition o

`Authorforcorrespondence([email protected]) and activation of receptors (Janda et al., 2012). Thus, post- nal
translational modifications have important roles in the secretion ur
Received21September2014;Accepted17December2014 and action of Wnts, and determination of Wnt5a modifications Jo
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using mass spectrometry is necessary for the understanding of Wls is essential for the secretion of Wnts, and is trafficked to
Wnt5a signaling. the basolateral membranes by AP-1 and clathrin from the
Low-density lipoprotein receptor-related protein 6 (LRP6), trans-Golgi network (Port and Basler, 2010; Yamamoto et al.,
receptor tyrosine kinase-like orphan receptor 2 (Ror2) and 2013). Consistent with these observations, knockdown of Wls
receptor-like tyrosine kinase (Ryk) function as co-receptors for completely suppressed Wnt5a secretion in polarized MDCK II/
Wnt3a,Wnt5aandWnt11,respectively(Kimetal.,2008;Nishita Wnt5a cells, although the polarized distribution of podocalyxin
et al., 2010; Yamamoto et al., 2006), although their cross- andTfRwasnotimpaired(Fig. 1D;supplementarymaterialFig.
reactivitywithotherWntshasalsobeenreported(Luetal.,2004; S1D). Clathrin and AP-1A, AP-1B and AP-4 sort cargo proteins
Yoshikawa et al., 2003). To transmit signals efficiently, these to the basolateral membranes in epithelial cells (Boehm and
receptors need to be sorted to the same location as their specific Bonifacino, 2001; Deborde et al., 2008; Fo¨lsch, 2005; Gravotta
Wnts.However,themechanismunderlyinghowtheyaresortedto etal.,2012;Simmenetal.,2002).Knockdownofclathrinorm1A
their specific destinations is largely unknown. To elucidate the and m1B (also known as AP1M1 and AP1M2, respectively),
common and unique mechanisms in the polarized secretion of which are medium subunits of AP-1A and AP-1B, respectively,
Wnt family members, we examined the polarized sorting of disturbedthepolarizedsortingofTfRbutnotthatofpodocalyxin
Wnt5aanditsreceptors.Here,weshowthebasolateraltrafficking (Fig. 1E; supplementary material Fig. S1D,E). Under these
mechanisms of Wnt5a and its receptors. Furthermore, we conditions, the basolateral secretion of Wnt5a was severely
examine the roles of Wnt5a polarized secretion in apical lumen suppressed (Fig. 1E). However, the depletion of m4 (also known
formation of the epithelial cyst, and we demonstrate that as AP4M1; a medium subunit of AP-4) did not affect the
basolaterally but not apically secreted Wnt5a promotes cell-to- polarized distribution of Wnt5a, podocalyxin and TfR (Fig. 1F;
substrate adhesion and activates Rac, thereby enhancing apical supplementary material Fig. S1D). m1B–HA expression
lumen formation without apoptosis. rescued the phenotypes observed in m1A- and m1B-depeleted
cells (Fig. 1G), excluding small interfering (si)RNA off-target
RESULTS effects. These results suggest that Wls, clathrin and AP-1
PolarizedsecretionofWnt5a are important for the sorting of Wnt5a to the basolateral
MDCK cells are commonly used in apical and basolateral membrane.
polarization experiments with two-dimensional (2D) and 3D
cultures. There are two types of MDCK cells: MDCK type I Post-translationalmodificationofWnt5a
(MDCK I) and MDCK type II (MDCK II). The difference Because post-translational modifications of secretory proteins are
betweenthetwotypesofMDCKcellsisbasedonlow(MDCKI) importantfortheirtrafficking,wedeterminedtheglycanprofilesof
and high (MDCK II) passages of parent cell lines (Dukes et al., Wnt5a.Wnt5ahasfourpossibleN-glycosylationsites;namely,Asn-
2011). In addition, MDCK I cells have higher trans-epithelial X-Ser/Thr, including N114, N120, N312 and N326 (Kurayoshi
electricresistancethanMDCKIIcellsbecausetheylackclaudin- etal.,2007).TrypticpeptidesofpurifiedWnt5aweresubjectedto
2expression(Furuseetal.,2001).InMDCKIcells,Wnt4,Wnt5a nano-flow liquid chromatography followed by matrix-associated
and Wnt7a mRNAs were expressed at higher levels than Wnt1, laserdesorption/ionizationmassspectrometry(MALDI-MS).About
Wnt3, Wnt5b and Wnt11 mRNAs, whereas Wnt5a mRNA 74.3%oftheentireWnt5aaminoacidsequencewascovered;N114,
expression was lower than Wnt4 and Wnt7a mRNA expression N312andN326wereglycosylatedwithhigh-mannose,hybridand
in MDCK II cells (supplementary material Fig. S1A). Western high-mannose-type oligosaccharides, respectively, but N120 was
blot analysis revealed that endogenous Wnt5a was detected in not glycosylated (supplementary material Table S1). Further
MDCKIcells,butnotinMDCKIIcells(supplementarymaterial analysesoftrypticpeptidesusingnano-flowliquidchromatography/
Fig.S1B).WhenMDCKcellswereculturedasamonolayerona electrosprayionizationmassspectrometry(LC/ESI-MS)confirmed
filtersupport(2Dculture)(Gottardietal.,1995),podocalyxin(an that glycans were attached to N114, N312 and N326 of Wnt5a
apical membrane marker) and transferrin receptor (TfR, a (Fig. 2;supplementarymaterialTableS2).
basolateral membrane receptor) were clearly sorted to apical When all glycosylation sites were mutated, Wnt5a was never
and basolateral membranes, respectively (Fig. 1A). Under these secreted (Kurayoshiet al., 2007).Toinvestigate the roleof each
conditions, the majority of endogenous Wnt5a in MDCK I cells glycanofWnt5ainsecretion,variouscombinationsofmutations
was secreted basolaterally (Fig. 1A). To show the trafficking of were added to the Wnt5a glycosylation sites (supplementary
Wnt5a more clearly, Wnt5a was stably expressed in MDCK I material Fig. S2A). Wnt5aN114Q/N120Q (N120 is glycosylated
and MDCK II cells (MDCK I/Wnt5a and MDCK II/Wnt5a; when N114 is mutated), Wnt5aN312Q, Wnt5aN326Q or
supplementarymaterialFig.S1C).ExogenouslyexpressedWnt5a Wnt5aN312Q/N326Q showed similar secretion levels to wild-
was secreted basolaterally in both cell lines (Fig. 1B). type Wnt5a. By contrast, Wnt5aN114Q/N120Q/N312Q or
These findings were confirmed using MDCK II cysts in 3D Wnt5aN114Q/N120Q/N326Q completely impaired secretion.
Matrigel culture. When Wnt5a was transiently expressed in These results show that glycosylation at N114 alone, but
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MDCK II cells and the cells were permeabilized, Wnt5a was not glycosylation at N312 or N326 alone, is sufficient for c
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detected in the cytoplasm but not in the apical lumens, whereas Wnt5a secretion, thereby suggesting that glycosylation at N114 e
whenthecellswerestainedwithoutpermeabilization,Wnt5awas is important for maintaining the overall folded structure of ci
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detected on the basal membranes (Fig. 1C). In MDCK II/Wnt5a Wnt5a.
cells with permeabilization, Wnt5a was observed in the Alipid-modifiedpeptidewasalsoobservedamongthepeptides ell
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immediate vicinity of basal membranes but not in the apical identified by MALDI-tandem mass spectrometry (MALDI-MS/
f
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lumen (Fig. 1C). In subsequent experiments, we overexpressed MS) (supplementary material Table S1). The peptide containing
Wnt5a and Wnt receptors in MDCK II cells to examine its S244,observedatm/z1455.9(supplementarymaterialFig.S2B), al
n
traffickingmechanisms,andknockeddownendogenousWnt5ain wasidentifiedasthepeptidewiththemonounsaturatedfattyacid ur
MDCK I cells to examine its function. (C16:1), palmitoleic acid. The site of lipidation was clearly o
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e
c
Fig.1.Seenextpageforlegend. n
e
ci
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identified as S244 by MALDI-MS/MS (supplementary AdditionofglycanstotheN-terminalregionofWnt5ainduces
material Fig. S2C), indicating that Wnt5a is modified with itsapicalsecretion ell
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palmitoleic acid as are Wnt3a and Wnt11 (Takada et al., 2006; Wehavepreviouslyshownthatthecomplex-typeglycanattached
f
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Yamamoto et al., 2013). In addition, Wnt5aS244A failed to to N40 of Wnt11 is essential for its apical secretion, and that
be secreted (supplementary material Fig. S2D), confirming that galectin-3, a galactose-binding lectin, is required for the apical al
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the modification with palmitoleic acid is essential for Wnt secretion of Wnt11 (Yamamoto et al., 2013). Given that Wnt5a ur
secretion. lacks a glycosylation site in the N-terminal region, Leu68 was o
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Fig.1. PolarizedsecretionofWnt5a.(A)MDCKIcellsseededona patternsbySDS-PAGEupontreatmentwithpeptide:N-glycosidase
Transwellpolycarbonatefilterweresubjectedtotheapical-basolateral F(PNGaseF),whichcleavesN-linkedglycans,andupontreatment
sortingassay.SolubleWnt5awasdetectedintheprecipitatesusingBlue– with endoglycosidase H (Endo H), which cleaves high-mannose
f f
Sepharosefromtheapicalmedia,andmembrane-associatedWnt5awas andhybrid-typeglycans,butnotthecomplex-typeglycan(Fig. 3B).
observedintheprecipitatesusingNeutravidin–agarosefromthebasolateral
This indicates that Wnt5a is heterogeneously glycosylated,
membranes.Wnt5asignalswerequantifiedusingNIHImagingandthe
which is consistent with the results of mass spectrometric
resultswereexpressedastheratioofWntsbetweenapical(Ap)and
basolateral(Bl)fractions.Podocalyxinandtransferrinreceptor(TfR)were analyses. Wnt5aL68N secreted from MDCK II/Wnt5aL68N cells
usedasapicalandbasolateralmembraneproteinmarkers,respectively. (supplementary material Fig. S1G) exhibited lower mobility by
Resultsareshownasmeans6s.e.m.fromfourindependentexperiments.B, SDS-PAGEthanwild-typeWnt5aowingtotheadditionofglycan,
precipitationusingBlue–Sepharose;N,precipitationusingNeutravidin– and the amount of Endo H-resistant glycan increased (Fig. 3B),
agarose.(B)MDCKI/Wnt5aandMDCKII/Wnt5acellsweresubjectedtothe f
indicating that the complex type was attached to Wnt5aL68N. In
apical-basolateralsortingassay.(C)MDCKIIcellstransientlyorstably
contrasttowild-typeWnt5a,Wnt5aL68Nwassecretedapicallyin
expressingWnt5awereculturedin3DMatrigel,andthecystswith(+)or
without(2)permeabilizationwerestainedwiththeindicatedantibodiesor polarized MDCK II/Wnt5aL68N cells (Fig. 3B). Basally secreted
Alexa-Fluor-633-conjugatedphalloidintovisualizeF-actin,orobservedby Wnt5a was associated with membranes (see ‘N’ in Fig. 3B),
relief-contrastmicroscopy(DIC).F-actinandb-cateninwereusedasapical whereas apically secreted Wnt5a was present in the medium (see
andbasolateralmarkers,respectively.Scalebar:10mm.(D)MDCKII/Wnt5a ‘B’ in Fig. 3B), suggesting that Wnt5a might interact with the
cellsweretransfectedwithcontrol(cont)orWlssiRNA,andthecellswere
basement membranes. Wnt5aL68N in the conditioned medium
thensubjectedtotheapical-basolateralsortingassay.TheWnt5asignalwas
induced a mobility shift of Dvl2 in NIH3T3 cells at a similar
precipitatedusingNeutravidin–agaroseinthecontrolbasolateralfraction,
efficiencytothatofcontrolWnt5aconditionedmedium(Fig. 3C),
whichwassetto100%.(E,F)MDCKII/Wnt5acellsweretransfectedwith
siRNAsforclathrin(Cla)(E),m1Aandm1B(E)orm4(F),andthecellswere suggestingthatWnt5aL68Nisactive.
subjectedtotheapical-basolateralsortingassay.(G)MDCKII/Wnt5acells Kifunensine and deoxymannojirimycin (dMM) inhibit ER-a-
stablyexpressingm1B–HAweretransfectedwithsiRNAsform1Aandm1B, mannosidase I and Golgi-a-mannosidase I, respectively (Elbein
andthecellswerethensubjectedtotheapical-basolateralsortingassay. et al., 1990; Fuhrmann et al., 1984). Gnt-I (also known as
MGAT1),whichisoneofsixN-acetylglucosaminetransferasesin
mutated to Asn to generate the amino acid sequence N68-X-S70, vertebrates,initiatestheformationofhybrid- and/orcomplex-type
whichisapossibleglycosylationsite(Fig. 3A).Wild-typeWnt5a glycan (Rademacher et al., 1988). Treatment of MDCK II/
secreted from MDCK II/Wnt5a cells showed different migration Wnt5aL68N cells with kifunensine and dMM changed the
Fig.2. DeterminationoftheglycanprofilesofWnt5a.
Wnt5aglycopeptidesthatelutedafter32.1(N114),36.5
(N312)and28.6min(N326)fromnano-flowliquid
chromatographywereanalyzedbyESI-MS.Themass
spectraweredeconvolutedtosinglychargedions.
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Fig.3. AdditionofglycanstotheN-terminalregionofWnt5ainducesitsapicalsecretion.(A)GlycanprofilesofWnt5a,Wnt5aL68N,Wnt3a,Wnt3aL35N
andWnt11areshown.(B)Wnt5aorWnt5aL68NinconditionedmediumfromMDCKIIcellswasprecipitatedusingBlue–Sepharose,andtheprecipitateswere
treatedwithEndoH orPNGaseF,andthenprobedwithanti-Wnt5aantibody(upperpanels).MDCKII/Wnt5aorMDCKII/Wnt5aL68Ncellsweresubjected
f
totheapical-basolateralsortingassay(middleandlowerpanels).ThesignalofWnt5awasprecipitatedusingNeutravidin–agaroseinthecontrolbasolateral(Bl)
fractionandthesignalofWnt5aL68NwasprecipitatedusingBlue–Sepharoseinthecontrolapical(Ap)fractionwassetto100%.B,precipitationusingBlue–
Sepharose;N,precipitationusingNeutravidin–agarose.(C)NIH3T3cellswerestimulatedwiththeindicatedamountsofWnt5a-orWnt5aL68N-containing
conditionedmedium(CM)fromLcellsfor2h,andlysateswerethenprobedwithanti-Dvl2antibody(lefttwopanels).Conditionedmedia(20ml)containing
Wnt5aorWnt5aL68Nareshownintherightpanel.(D)MDCKII/Wnt5aL68Ncellsweretreatedwithkifunensine(kif)ordeoxymannojirimycin(dMM),and
Wnt5aL68NinconditionedmediumwasthenprecipitatedusingBlue–Sepharose.TheprecipitatesweretreatedwithEndoH orPNGaseFandthenprobedwith
f
anti-Wnt5aantibody.(E)MDCKII/Wnt5aL68NcellsweretreatedwithkifordMM,ortransfectedwithGnt-Iorgalectin3(Gal3)siRNA,andthecellswere
subjectedtotheapical-basolateralsortingassay.cont,control.Resultsareshownasmeans6s.e.m.fromfourindependentexperiments.
e
sensitivity of Wnt5aL68N to Endo H (Fig. 3D) and inhibited its the complex-type glycan and secreted apically (Fig. 4A; c
f n
apicalsecretion(Fig. 3E).siRNAsforGnt-Iandgalectin-3inhibited supplementary material Fig. S1H). Treatment with kifunensine, e
the apical secretion of Wnt5aL68N (Fig. 3E; supplementary dMM and siRNAs for Gnt-I and galectin-3 inhibited the apical ci
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materialFig.S1F).Bycontrast,thesemanipulationsdidnotaffect secretion of Wnt3aL35N (Fig. 4B). Wnt3aL35N induced the
the polarized distribution of podocalyxin and TfR (Fig. 3E). accumulationofb-catenintoasimilardegreeaswild-typeWnt3a ell
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Therefore,preventionofthematurationofthehybridorcomplex- (Fig. 4C).Theseresultssuggestthattheadditionofcomplex-type
f
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typeglycanfromthehigh-mannose-typeglycanonWnt5aL68Nby glycantotheN-terminalregionissufficientforWnt5aandWnt3a
thesemanipulationswouldimpairitsapicalsecretion. to be secreted apically without losing their activities. Therefore, al
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Similar to Wnt5a, Leu35 was mutated to Asn in Wnt3a one amino acid substitution at the appropriate region of Wnts ur
(Wnt3aL35N), and Wnt3aL35N was additionally modified with might be sufficient for Wnts to change their sorting destination. o
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clear, it has been shown that Fz2 mediates Wnt3a and Wnt5a
signaling and that Fz7 functions as the Wnt11 receptor (Sato
et al., 2010; Uysal-Onganer and Kypta, 2012). To determine the
polarized localization of Fzs, MDCK II cells stably expressing
FLAG–Fz2orFLAG–Fz7weregenerated(MDCKII/FLAG–Fz2
andMDCKII/FLAG–Fz7,respectively).ThemajorityofFLAG–
Fz2waslocalizedtothebasolateralmembranes,whereasFLAG–
Fz7 was localized to both apical and basolateral membranes
evenly (Fig. 5A–C). Knockdown of Wls did not affect the
polarized localization of either Fz (Fig. 5B,C; supplementary
materialFig.S1D).ThepolarizedlocalizationofFLAG–Fz2was
impairedinclathrin-depletedcellsandcellsdepletedofbothm1A
and m1B (m1A/m1B-depleted), but not in m4-depleted cells
(Fig. 5B; supplementary material Fig. S1D,E). These results
suggest that Fz2- or Fz7-loaded vesicles destined to the plasma
membranes do not contain Wls, and that the basolateral
localization of Fz2 requires the clathrin and AP-1 complex.
Single transmembrane receptors, including LRP6, Ror2 and
Ryk, function as co-receptors of Wnt3a, Wnt5a and Wnt11,
respectively (Kim et al., 2008; Nishita et al., 2010; Yamamoto
et al., 2006). Although endogenous LRP6 and exogenously
expressed Ror2–GFP were primarily located at the basolateral
membranesofMDCKIIcells(Fig. 5A,D,E),Ryk–GFPwasevenly
distributed in both the apical and basolateral membranes
(Fig. 5A,F).KnockdownofWlsdidnotaffectthelocalizationof
theseco-receptors(Fig. 5D–F;supplementarymaterialFig.S1D).
The basolateral membrane localization of LRP6 and Ror2–GFP
was disturbed in clathrin- and m1A/m1B-depleted cells but not in
m4-depletedcells(Fig. 5D,E;supplementarymaterialFig.S1D,E).
Consistent with the polarized localization of the receptors,
stimulation of polarized MDCK II/FLAG–Fz2 cells with
recombinant Wnt5a at the basolateral side activated Rac1 more
efficientlythanthatattheapicalside(Fig. 5G).RecombinantWnt3a
inducedLRP6phosphorylationandthemobilityshiftofDvl2atthe
basolateralsidemoreefficientlythantheapicalside(Fig. 5G).
BasolateralsecretionofWnt5aisrequiredforapicallumen
formationofMDCKcysts
The role of Wnt5a basolateral secretion in epithelial cells was
examined by inhibiting endogenous Wnt5a synthesis and
Fig.4. AdditionoftheglycosylationsiteattheN-terminalregionis secretion in MDCK I cells. When MDCK I cells were cultured
sufficienttochangethepolarizedsecretionofWnt3a.(A)Wnt3aor
in3DMatrigel,anascentlumenwasobservedatday4.Eachcell
Wnt3aL35NinconditionedmediumfromMDCKIIcellswasprecipitated
in the cyst had a free, lateral and basal surface by acquiring
usingBlue–Sepharose,andtheprecipitatesweretreatedwithEndoH or
f
PNGaseFandthenprobedwithanti-Wnt3aantibody(leftpanels).MDCKII polarity, and the cyst increased the lumen diameter at days 6–7,
cellsexpressingWnt3aorWnt3aL35Nweresubjectedtotheapical- and cystogenesis was complete at days 8–9 (Fig. 6A).
basolateralsortingassay(rightpanels).ThesignalofWnt3aprecipitated Treatment of MDCK I cells with a Wnt secretion inhibitor
usingNeutravidin–agaroseinthecontrolbasolateral(Bl)fractionorthesignal IWP2(Chenetal.,2009),causedadelayinlumenformation,so
ofWnt3aL35NprecipitatedusingBlue–Sepharoseinthecontrolapical(Ap)
that a period of 10–11 days was required for formation of the
fractionwassetto100%.B,precipitationusingBlue–Sepharose;N,
lumen consisting of monolayer cells (Fig. 6A). At day 7, some
precipitationusingNeutravidin–agarose.(B)MDCKII/Wnt3aL35Ncellswere
cellswerestillobservedintheluminalspaceofcyststreatedwith
treatedwithkifordMM,ortransfectedwithGnt-Iorgalectin3(Gal3)siRNA,
andthecellswerethensubjectedtotheapical-basolateralsortingassay. IWP2,andtheydidnotmakecontactwiththeextracellularmatrix
cont,control.Resultsareshownasmeans6s.e.m.fromfourindependent (ECM) (Fig. 6A,B). Approximately 70% of cysts consisting of
e
experiments.(C)Lcellswerestimulatedwiththeindicatedamountsofwild- control MDCK I cells showed a dilated lumen, which was lined c
typeWnt3a-orWnt3aL35N-containingconditionedmedium(CM)for2h,and by a polarized monolayer cells, and of which the diameter was en
lysateswerethenprobedwithanti-b-cateninandanti-GSK-3bantibodies .50% that of the cyst (Fig. 6B). By contrast, treatment with ci
(lefttwopanels).Wild-typeWnt3aandWnt3aL35Ninconditionedmedium S
(20ml)areshownintherightpanel.GSK-3bwasusedasaloadingcontrol. IwWhePr2eacsautsheednthuemnbuemrboefrocfysctysstwsiwthithnadrirlaotwedlluummeennss,towdheicchreahsaed, ell
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multilayered cells and a diameter ,50% that of the cyst,
f
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PolarizedlocalizationofWntreceptors increased(Fig. 6B).SimilarphenotypeswereobservedinWnt5a-
There are ten Frizzled (Fz) family members, which function as depleted cells (Fig. 6C; supplementary material Fig. S3A); al
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Wnt receptors (Kikuchi et al., 2011; Schulte and Bryja, 2007). however, knockdown of Wnt4 or Wnt7a did not affect apical ur
Although the binding specificity between Wnts and Fzs is not lumen formation (supplementary material Fig. S3B). As well as o
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RESEARCHARTICLE JournalofCellScience(2015)128,1051–1063doi:10.1242/jcs.163683
Fig.5. Thepolarizedlocalization
ofWntreceptors.(A)MDCKII,
MDCKII/FLAG–Fz2,MDCKII/
FLAG–Fz7,MDCKII/Ror2–GFPor
MDCKII/Ryk–GFPcellswere
culturedin3DMatrigel,andthecysts
werestainedwiththeindicated
antibodies.Scalebar:10mm.
(B–F)MDCKII/FLAG–Fz2(B),
MDCKII/FLAG–Fz7(C),MDCKII
(D),MDCKII/Ror2–GFP(E)or
MDCKII/Ryk–GFP(F)cellswere
transfectedwithsiRNAforWntless
(Wls),clathrin(Cla),m1Aandm1B,or
m4,andthecellswerethensubjected
totheapical-basolateralsorting
assay.Thesignalofthereceptors
wasprecipitatedusingNeutravidin–
agarosefromthecontrolbasolateral
(Bl)fraction,whichwassetto100%.
Ap,apical;cont,control.(G)MDCKII/
FLAG–Fz2cellswerestimulatedwith
Wnt5a(50ng/ml)(leftpanel)or
MDCKIIcellswerestimulatedwith
Wnt3a(100ng/ml)(rightpanel)from
theapicalorbasolateralsides,and
lysateswerethensubjectedtothe
Racactivationassayorthe
phosphorylationassayofLRP6and
Dvl2.Resultsareshownas
means6s.e.m.fromfour
independentexperiments.
e
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ci
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ell
C

f
o

in MDCK IIcells, apical secretion of Wnt5aL68N was observed Ror2–GFP cells (supplementary material Fig. S3C). The al
n
in polarized MDCK I/Wnt5aL68N cells, and basolateral phenotypes induced by Wnt5a siRNA were rescued by ur
membrane localization of Ror2–GFP was observed in MDCK I/ expression of wild-type Wnt5a, but not by Wnt5aL68N o
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RESEARCHARTICLE JournalofCellScience(2015)128,1051–1063doi:10.1242/jcs.163683
Fig.6. BasolateralsecretionofWnt5aisrequiredfortheapicallumenformationofMDCKcyst.(A)MDCKIcellsculturedin3DMatrigelweretreatedwith
orwithout5mMIWP2fortheindicateddays,andthecystswerethenstainedwithanti-b-cateninantibodyandAlexa-Fluor-488-conjugatedphalloidintovisualize
F-actin.Thediameterratiobetweentheapicallumenandthewholecystwascalculatedusingthefollowingformula:diameterratio(%)5diameterofcentral
lumen(blueline)/diameterofthecyst(redline)6100.Themeanofthetwodiameterratiosinthecystsattheindicateddayswereplotted(rightpanel).(B)MDCKI e
cellsculturedin3DMatrigelweretreatedwithorwithout5mMIWP2for7days,andthecystswerethenstainedwithAlexa-Fluor-488-conjugatedphalloidin c
n
(green)andDRAQ5(blue)tovisualizethenucleus.SchematicimagesofMDCKIcystwithadilatedlumen(1)ornarrowlumen(2)areshown.Thecystsatday7 e
wereclassifiedbytheirluminaltypes,andthenumberofcystsofeachtypewascounted.Resultsareexpressedastheratioofluminaltypes,andareshown ci
S
asthemeans6s.e.m.fromfourindependentexperiments.(C)MDCKI,MDCKI/Wnt5a,orMDCKI/Wnt5aL68NcellsweretransfectedwithcontrolorWnt5a

siRNA,andthecystswerethenstainedwithphalloidinandDRAQ5.Thecystsatday7wereclassifiedbytheirluminaltypes.Scalebars:20mm. ell
C

f
o
(Fig. 6C; supplementary material Fig. S3A). These results ApoptosisisrequiredforapicallumenformationinWnt5a-
indicate that Wnt5a signaling from the basolateral side is depletedcells nal
required for efficient apical lumen formation in MDCK I Treatment of MDCK I cells with IWP2 or depletion of Wnt5a ur
cells. suppressed the phosphorylation of FAK and paxillin at days 5–7 o
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RESEARCHARTICLE JournalofCellScience(2015)128,1051–1063doi:10.1242/jcs.163683
in 3D Matrigel culture (Fig. 7A), suggesting a reduction in cell- Bonifacino,2001).GiventhatWlsisalsotraffickedtothebasolateral
to-substrate adhesion. These manipulations also reduced Rac membrane, and is recognized by palmitoleic-acid-modified Wnts
activity (Fig. 7B). Decreases in FAK activity, paxillin (Port and Basler, 2010; Yamamoto et al., 2013), Wls should
phosphorylation and Rac activity due to Wnt5a depletion were localizetoWnt5a-andWnt3a-loadedvesicles.
rescued by expression of wild-type Wnt5a, but not by
Wnt5aL68N (Fig. 7A,B). When MDCK I cells were treated PolarizedtraffickingofWntreceptors
withNSC23766(aRacGEFinhibitor),somecellswereobserved Drosophila Fz1 (dFz1) and dFz2 act as receptors for wingless, a
in the luminal region at days 5–9, resulting in a delay of lumen DrosophilaWnthomolog.Bothreceptorsmediatetheb-catenin-
formation,similartothetreatmentwithIWP2andWnt5asiRNA dependent pathway, and dFz1 also functions in the b-catenin-
(Fig. 7C). independent pathway, especially the planar cell polarity (PCP)
Some lumen formation models, including hollowing and pathway(Strutt,2003).dFz1isconcentratedatapicaljunctionsin
cavitation, which require directional vesicle trafficking and the wing imaginal disc, whereas dFz2 is distributed throughout
luminalcelldeath,respectively,havebeenproposed(Dattaetal., the apical and basolateral membranes (Wu et al., 2004). The C-
2011). In the control cysts, very few luminal cells underwent terminal cytoplasmic amino acid sequence of dFz2 prevents it
apoptosis,whereascystsinwhichWnt5asignalingwassuppressed from accumulating at the apical junctions, and the apical
had apoptotic cells, as determined by staining with the apoptotic localization of dFz1 is necessary for the activation of the PCP
marker, cleaved caspase-3 (Fig. 7C,D). Control MDCK I cysts pathway. These results suggest that it is the localization of the
formedaclearapicallumenwithafewcells,eveninthepresence receptors that determines pathway specificity.
ofthegeneralcaspaseinhibitorQVD-Oph,butWnt5a-depletedor Our studyshowedthat Fz2mainly localizedto the basolateral
IWP2-treated MDCK I cysts showed a decreased formation of membranesinMDCKIIcells,whereasFz7wasrelativelyevenly
apical lumen surrounded by monolayer cells at day 11 in the distributedthroughouttheapicalandbasolateralmembranes.Fz2
presence of QVD-Oph (Fig. 7E). Thus, apoptosis might be acts as a common receptor for Wnt3a and Wnt5a (Sato et al.,
required for apical lumen formation in Wnt5a signaling-deficient 2010), and Fz7 functions as a receptor for Wnt11 (Kim et al.,
cells.Takentogetherwiththereportthatknockdownofintegrins 2008;YamanakaandNishida,2007).LRP6andRor2,whichare
impairs apical lumen formation (Myllyma¨ki et al., 2011), these receptorsforWnt3aandWnt5a,respectively(Nishitaetal.,2010;
resultssuggestthatWnt5asignalingpromoteslumenformationby Yamamoto et al., 2006), were also trafficked to the basolateral
theenhancementofcell-to-substrateadhesionthroughintegrins. membranesusingclathrinandAP-1.Ryk,whichmediatesWnt11
signaling(Kimetal.,2008),waslocalizedtoboththeapicaland
DISCUSSION basolateral membranes. Because Wnt3a and Wnt5a are secreted
PolarizedsecretionofWnt5a basolaterally and Wnt11 is secreted apically, the localization of
Itisbelievedthatthetraffickingofsomeapicallysortedproteinsis theirreceptorsinpolarizedMDCKIIcellsisunderstandable.The
dependentonglycosylphosphatidylinositol-anchor-interactingsignals signalingpathwaysofWnt3aandWnt5awerestronglyactivated
orglycan-mediatedsignals,whereasbasolaterallysortedproteinsare when both Wnts stimulated the basolateral side, indicating that
delivered by the interaction of cytoplasmic trafficking signals with localization of their receptors is functionally coupled to the
sortingmachineriessuchasclathrinandAPs(BoehmandBonifacino, polarizedsecretionofWnts.Thus,itislikelythattheappropriate
2001;Debordeetal.,2008;Fo¨lsch,2005;Vaginetal.,2009).Inthis Wnt receptors are sorted to the same polarized fractions as the
study, we determined the profiles of glycans attached to Wnt5a by corresponding Wnts, thereby leading to efficient activation of
ESI-MS/MS. N114 and N326 of Wnt5a were modified with high- their signals.
mannose-type glycans. Both Asn residues are conserved in most Our results are consistent with the findings that Ror2 is
Wnts, and modification with high-mannose-type glycan was also localized to the basal sides of the embryonic midgut epithelium
observed at the conserved Asn residues of Wnt3a and Wnt11 (Yamadaetal.,2010),andthatLRP6ispresentinthebasolateral
(Yamamoto et al., 2013). By removing both high-mannose-type region of Xenopus outer ectodermal cells (Huang and Niehrs,
glycans, the secretion of Wnt3a, Wnt5a and Wnt11 was impaired, 2014). Wls is essential for the trafficking of Wnts (Port and
suggesting that these glycans are necessary for maintaining the Basler,2010),butitisnotrequiredfortheapicalandbasolateral
overallfoldedstructure.Wnt5awasalsomodifiedwithhybrid-type sorting of Wnt receptors. Therefore, Wnt-loaded vesicles could
glycanatN312,althoughitwasnotessentialforsecretionbecausethe be distinct from vesicles containing their receptors, which
Wnt5aN312Qmutantdisplayedsecretioncomparabletothatofwild- probably prevents intracellular activation of Wnt signaling by
typeWnt5a.TheglycosylationsiteisuniqueinWnt5aandWnt5b, theco-existenceofligandsandreceptorsinthesamevesicles.An
buttheroleofglycosylationatthissiteremainsunknown. alternative possibility might be that Wnts and Wnt receptors are
Wnt5a and Wnt3a were secreted apically following the loaded in the same vesicle containing Wls and that there is a
addition of the glycosylation site at their N-terminal region mechanism to inhibit their binding.
(Wnt5aL68N and Wnt3aL35N). The complex-type glycan was
e
attached to the glycosylation site, and their apical secretion Regulationofapicallumenformationbybasolateral c
n
dependedongalectin-3,similartothatofWnt11.Therefore,Wnts secretionofWnt5a e
thataremodifiedwiththecomplex-typeglycanattheN-terminal We have shown previously that Wnt5a isinvolved inthe single- ci
S
region might be secreted apically by galectin-3. It is noteworthy cell polarization of rat intestinal epithelial IEC6 cells through
thatasingleaminoacidsubstitutionofWntisabletochangeits Rac1activation(Gonetal.,2013),andthatWnt5apromotescell- ell
C
trafficking route without causing it to lose activity. to-substrate adhesion in cancer cells (Matsumoto et al., 2010).
f
o
TheWnt5abasolateralsecretionislikelytobemediatedbythe Other studies have demonstrated that Rac1 activation at the
samemechanismasthatofWnt3a(Yamamotoetal.,2013),because basolateral side is required for the polarization of MDCK cysts al
n
itssortingrequiresclathrinandAP-1,whicharecommonmachineries (Yagi et al., 2012), and that knockdown of integrins impairs ur
for the basolateral sorting of membrane proteins (Boehm and apicallumenformationinMDCKcells(Myllyma¨kietal.,2011). o
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RESEARCHARTICLE JournalofCellScience(2015)128,1051–1063doi:10.1242/jcs.163683
e
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Fig.7.Seenextpageforlegend. ell
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However, it is not clear how these cellular events are linked to for luminogenesis. Wnt5a secretion enhances cell-to-substrate
f
o
form apical lumen in polarized epithelial cells. adhesion in 3D culture. During MDCK cystogenesis in type I

Lumenformationreliesonthecoordinationofcell-to-substrate collagen gel, Rac1 activities are necessary for orientation of al
n
recognition, apical and basal polarization, and lumen expansion apical polarization through assembly of the basolateral laminin ur
(Dattaetal.,2011).Thus,celladhesiontoECMisthefirstevent (O’Brien et al., 2001). Because our 3D culture was performed o
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