Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation ...

16 Pages·2017·10.56 MB·English

©2018.PublishedbyTheCompanyofBiologistsLtd|BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
RESEARCHARTICLE
Nonmuscle myosin IIA is involved in recruitment of apical junction
α
components through activation of -catenin
MasayukiOzawa
ABSTRACT cells and human tumors (Vleminckx et al., 1991; Schipper et al.,
1991;Berxetal.,1995).
MDCKdogkidneyepithelialcellsexpresstwoisoformsofnonmuscle
Cadherinsaretransmembranereceptorsthatmediatehomophilic
myosinheavychainII,IIAandIIB.UsingtheCRISPR/Cas9system,
intercellular adhesion via their extracellular domains and recruit
weestablishedcellsinwhichtheIIAgenewasablated.Thesecells
werethentransfectedwithavectorthatexpressesGFP–IIAchimeric proteinpartnerstotheircytoplasmicdomains.Intracellularly,they
interact with a group of proteins, collectively termed catenins
moleculeunderthecontrolofatetracycline-responsibleelement.In
theabsenceofDox(doxycyclin),whenGFP–IIAisexpressed(GFP– (Ozawa et al., 1989). α-catenin (Herrenknecht et al., 1991;
Nagafuchi et al., 1991) links cadherin/β-catenin or cadherin/
IIA+),thecellsexhibitepithelialcellmorphology,butinthepresence
ofDox, when expression ofGFP–IIA is repressed(GFP–IIA−), the plakoglobin complexes to F-actin, either directly (Rimm et al.,
cells lose epithelial morphology and strong cell–cell adhesion. 1995) or indirectly via α-actinin (Knudsen et al., 1995), vinculin
Consistent with these observations, GFP–IIA− cells failed to (Watabe-Uchida et al., 1998), formin (Kobielak et al., 2004), or
EPLIN (epithelial protein lost in neoplasm) (Abe and Takeichi,
assemble junction components such as E-cadherin, desmoplakin,
andoccludinatcell–cellcontactsites.Therefore,IIAisrequiredfor 2008).Althoughbiochemicalstudieshadchallengedthenotionthat
thecadherin–catenincomplexbindsdirectlytoF-actin(Dreesetal.,
assemblyofjunctioncomplexes.MDCKcellswithanablationofthe
α-cateningenealsoexhibitedthesamephenotype.However,whenin 2005;Yamadaetal.,2005),recentstudiesrevealedstrongandstable
GFP–IIA−cellsexpressedα-cateninlackingtheinhibitoryregionor bondsbetweenthecadherin–catenincomplexandanactinfilament
E-cadherin/α-catenin chimeras, the cells acquired the ability to formunderforce(Buckleyetal.,2014).Thus,monomericα-catenin
bound to the cadherin/β-catenin complex mediates the physical
establish the junction complex. These experiments reveal that IIA
actsasanactivatorofα-catenininjunctionassembly. linkagebetweenAJsandtheactincytoskeleton(Desaietal.,2013),
and cadherin is constitutively underactomyosin-generated tension
KEYWORDS:Adhesion,α-Catenin,Cadherin,Celljunctions, (Smutnyetal.,2010;Borghietal.,2012).Moreover,α-cateninhas
NonmusclemyosinIIA been proposed to act as a mechanosensor in AJ formation by
facilitatingtherecruitmentofvinculintoAJsinamannerdependent
INTRODUCTION upon actomyosin-derived tension (Yonemura et al., 2010). Force-
Theestablishmentandregulationofcelladhesionarefundamental dependentconformationalchangesinα-cateninregulateitsbinding
to the development and maintenance of tissue organization in to vinculin, an actin-binding protein, and reinforce intercellular
multicellularorganisms.Inepithelia,cell–celladhesionismediated adhesion(Yonemuraetal.,2010;leDucetal.,2010;Ishiyamaetal.,
primarily through adherens junctions (AJs) and desmosomes. 2013; Yao et al., 2014; Kim et al., 2015). Moreover, as
Because of the importance of cell–cell adhesion in development demonstrated using a fluorescence resonance energy transfer
and tissue function in adults, it is critical to understand the (FRET)tensionsensor,theactomyosincytoskeletonexertstensile
mechanismsbywhichcellsregulatetheassemblyanddisassembly forces on E-cadherin in an α-catenin-dependent manner (Borghi
ofadhesivejunctions. etal.,2012).Takentogether,theseobservationsshowthatα-catenin
Cadherins comprise a family of transmembrane cell-surface is a key mechanosensory protein involved in transmitting
glycoproteins that mediate Ca2+-dependent cell–cell adhesion actomyosincytoskeletaltensiontothecellmembrane.
(Takeichi, 2014). In epithelial cells with well-developed NonmusclemyosinII(NMII)isamyosinthatconstitutesseveral
intercellular junctions, E-cadherin is concentrated in the AJs but percentoftotalcellularprotein(Vicente-Manzanaresetal.,2009),
appears to influence other intercellular junctions such as tight and NMII isoforms are widely expressed in embryonic and adult
junctions (TJs) and desmosomes (Gumbiner et al., 1988; Watabe tissues(Golombetal.,2004).Theyplaydistinctbutrelatedrolesin
et al., 1994; Amagai et al., 1995). Dysfunction of E-cadherin has cell biology, including processes such as motility (Maupin et al.,
been implicated in the invasiveness and carcinogenesis of tumor 1994; Wylie and Chantler, 2001; Kolega, 2003), adhesion (Conti
and Adelstein, 2008; Wang et al., 2010), and vesicle trafficking
(TogoandSteinhardt,2004).Experimentsusingahighlyselective
DepartmentofBiochemistryandMolecularBiology,GraduateSchoolofMedical
andDentalSciences,KagoshimaUniversity,8-35-1Sakuragaoka,Kagoshima inhibitor, blebbistatin (Straight et al., 2003), or siRNA-mediated
890-8544,Japan. knockdown, have shown that NMII activity is required for
*Authorforcorrespondence([email protected]) accumulation and stability of cadherins at junctions (Shewan en
et al., 2005; Miyake et al., 2006; Lambert et al., 2007). Genetic p
M.O.,0000-0002-7887-8441 ablation of NM heavy chain IIA (IIA) from embryonic stem cells O
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution and mouse embryos results in a loss of cell–cell adhesion, which gy
License(http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse, correlateswithadecreaseinE-cadherinandβ-cateninlocalizationat o
distributionandreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed. cell–cell contacts (Conti et al., 2004). Knockdown of NMIIA by ol
Received20November2017;Accepted8February2018 siRNA decreased both the amount of E-cadherin at cell–cell Bi
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
contactsandcadherin-basedadhesion(Smutnyetal.,2010).Thus, cells, fibroblast-like elongation of cells in the basal layer, and
NMIIA activity is needed for E-cadherin localization to the roundingofcellsinthetoplayer(Fig.1E).
junctions. These studies suggestthat tension on actin provided by Concomitantwiththesemorphologicalchanges,cellsculturedin
NMIIisnecessaryformaintainingtheproperlocalizationofthese the presence of Dox lost the integrity of the epithelial sheet. Cell
celladhesionproteins.Ontheotherhand,NMIIhasbeenreportedto sheetintegritywasdeterminedusingtheDispase-baseddissociation
be involved in disassembly of AJs, and blebbistatin prevents assay,whichwasoriginallydevelopedtodeterminethemechanical
disassemblyofAJsincalcium-depletedcells(Ivanovetal.,2004). integrity of epidermal keratinocytes, cells that endure mechanical
To address the functional role of NMIIA in the assembly of stress (Ishii et al., 2005; Simpson et al., 2010). Although cells
junction complex components, we ablated its heavy chain gene cultured in the absence of Dox were resistant to mechanical
usingtheCRISPR/Cas9system,andthenintroducedanexpression dissociation,cellsculturedinthepresenceofDoxweredissociated
vector for GFP-tagged IIA (GFP–IIA) under the control of the intosinglecellsorsmallaggregatesofcellsafterdetachmentfrom
tetracycline (Tet) promoter. We analyzed the assembly of the the dishes by Dispase and subsequent mechanical dissociation by
components of AJs, as well as other junctions, in the presence or pipetting(Fig.1F).Thus,lossofGFP–IIAexpressiondisruptedthe
absence of Dox. In the absence of Dox, when GFP–IIA was mechanicalintegrityofthecellsheets.
expressed (GFP–IIA+ cells), the cells exhibited epithelial cell In the absence of Dox, when GFP–IIA was expressed, the
morphology,butinthepresenceofDox,whenexpressionofGFP– components of cell–cell junctions (E-cadherin, β-catenin,
IIAwasrepressed(GFP–IIA−cells),thecellslostthismorphology. plakoglobin, desmoplakin, and occludin) were distributed
Consistent with these observations, GFP–IIA− cells failed to predominantly at plasma membrane sites involved in lateral cell–
assemble junction components such as E-cadherin, desmoplakin, cell contacts (Fig. 2A, left panels). Addition of Dox not only
andoccludinatcell–cellcontactsites.Therefore,IIAisrequiredfor reducedGFP–IIAexpressionandinducedmorphologicalchanges,
assembly of junction complexes. However, when α-catenin but also caused these components to move from cell–cell contact
constructs with deletions were expressed in GFP–IIA− cells, the sitesontheplasmamembranetoanevendistributionthroughoutthe
cellswererescued,andacquiredtheabilitytoestablishthejunction cell(Fig.2A,rightpanels).
complex, indicating that these deletions activate α-catenin. These To monitor the presence of E-cadherin on the outer side of
experiments reveal that (1) α-catenin is, indeed, the downstream the plasma membrane, we selectively biotinylated the cell surface
targetofIIAinjunctionassembly,and(2)IIAactsasanactivatorof of GFP–IIA/IIAKO cells cultured in the absence or presence
α-catenininjunctionassembly. of Dox with a membrane-impermeable reagent, sulfoNHS-biotin.
Biotinylatedproteinswererecoveredwithimmobilizedavidinand
RESULTS subjected to immunoblotting with antibodies against E-cadherin
EstablishmentofMDCKcellslackingtheIIAgeneand (Fig.2B).E-cadherinwasexpressedatthecellsurfaceirrespective
expressingaGFP–IIAchimericmoleculeunderthecontrolof of the addition of Dox (Fig. 2B). These data demonstrate that the
thetetracycline-responsiveelement establishedcelllinefailedtoassemblejunctioncomponentsdueto
Inthisstudy,weusedaMDCK(typeII)cloneT23expressingthe theabsenceofGFP–IIA.
Tetrepressor(Barthetal.,1997).WefirstestablishedMDCKcells Complex formation of E-cadherin with catenins, β-catenin,
inwhichtheIIAgenewasablated,usingtheCRISPR/Cas9system plakoglobin,andα-cateninisrequiredfortheassemblyofjunction
(Jineket al.,2012; Conget al.,2013; Mali et al.,2013), and then complexes.Therefore,weinvestigatedtheassociationofE-cadherin
introducedtheGFP-taggedIIAconstruct(WeiandAndersen,2000) with catenins. E-cadherin was collected by immunoprecipitation
expressedunderthecontroloftheTet-repressibleelement(TRE);in using E-cadherin antibodies, and after separation and transfer to
the presence of either Tet or doxycycline (Dox), expression of nitrocellulosemembranetheco-precipitatedmaterialswereprobed
exogenous protein was completely repressed (Gossen and Bujard, with antibodies against catenins. E-cadherin isolated from cells
1992). cultured in the absence and presence of Dox collected similar
Immunoblotting (Fig. 1A) and immunofluorescence staining amountsofβ-catenin,plakoglobin,andα-catenin(Fig.2C).Thus,
(Fig. 1B) with antibodies against IIA confirmed that a selected thepresenceorabsenceofGFP–IIAhadnoeffectontheassociation
MDCK clone in which the IIA gene was ablated (IIAKO) lost ofE-cadherinwithcatenins.
expressionofIIA.Consistentwiththeseresults,sequencingofthe
genomic DNA isolated from the clone revealed either a 1-bp Correctlocalizationofjunctioncomponentsrequiresα-
deletionor1-bpaddition,resultinginaframe-shiftmutationinthe catenin
IIA gene (Fig. 1C). The MDCK clone was then transduced with The failure of junction complex formation in IIAKO cells is also
theGFP–IIAconstructunderthecontroloftheTRE;theresultant observedincellslackingα-cateninexpression,suchashumancolon
clonesweretermedGFP–IIA/IIAKO.Thecellswereculturedfor4 cancer cells DLD-1/Δα (Taniguchi et al., 2005), raising the
days without or with Dox (−/+ Dox), and then analyzed for possibility that these molecules functionally interact. Therefore,
expressionofGFP–IIA(Fig.1D).GFP–IIAwasdetectedinprotein wefirstestablishedα-cateninKOMDCKcellsusingtheCRISPR/Cas9
extractsfromthecellsculturedwithoutDox,butnotinthosefrom system; the resultant cells were termed αKO cells (Fig. 3A).
thecellsculturedwithDox. Sequencing of genomic DNA isolated from the clone revealed
either a 5-bp deletion or 1-bp insertion, resulting in a frame-shift
GFP–IIA-dependentlocalizationofjunctioncomponentsto mutationintheα-cateningene(Fig.3B). en
cell–cellcontactsitesinIIAKOcells The MDCK clone was then transduced with FLAG-tagged α- p
GFP–IIA/IIAKO cells cultured in the absence of Dox expressing cateninconstructunderthecontrolofTRE,yieldingα-catFLAG/αKO O
GFP–IIA grew in monolayer cultures as epithelial clusters with a cells. The cells were cultured for 4days without or with Dox gy
typicalcobblestonemorphology(Fig.1E).Culturingthecellsinthe (−/+Dox),andthenanalyzedbywesternblotting(Fig.3C).FLAG- o
presenceofDox,causingalmostcompleterepressionofGFP–IIA taggedα-cateninwasdetectedinproteinextractsfromcellscultured ol
i
expression, induced dramatic changes, including multilayering of without Dox, but not with Dox. When the same extracts were B
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Fig.1.EstablishmentofMDCKcellslackingtheNMHCIIAgeneandexpressingaGFP-NMHCIIAchimericmoleculeunderthecontrolofthe
tetracycline-responsiveelement.UsingtheCRISPR/Cas9system,NMHCIIA(IIA)geneofMDCKcellswasablated,yieldingIIAKOcells,whichwere
transfectedwithavectorencodingGFP-taggedNMHCIIA(GFP–IIA)underthecontroloftheTRE,yieldingGFP–IIA/IIAKOcells.(A)Immunoblotdetectionof
IIAandvinculin(vinc)inIIAWTandIIAKOcells.Vinculinwasusedasaloadingcontrol.(B)Cellswerestainedwithanti-IIAorDAPI.(C)Genomic
sequencingrevealedmutationsinthetargetregionsoftheNMHCIIAgenethatinducedframe-shiftmutations.Ofsixindependentclonessequenced,a1-bp
deletionwasdetectedintwoclones,anda1-bpinsertionwasidentifiedinfourclones.(D,E)Dox-repressibleexpressionofGFP–IIAinGFP–IIA/IIAKOcells. n
Cellswereculturedfor4dayswith(+)orwithout(−)Dox.(D)Thecellsweresubjectedtoimmunoblotanalysiswiththeindicatedantibodies.GFP–IIAwas pe
detectedwithananti-GFPantibody.Vinculinwasusedasaloadingcontrol.(E)GFP–IIAwasdetectedasGFPfluorescenceandcellswerestainedwithanti- O
IIBorDAPI.(F)Cellmorphology.Cellsculturedwith(+)orwithout(−)Doxwereobservedusingaphase-contrastmicroscope.(G)Dissociationassays.Cells
y
wereculturedinthepresence(+)orabsence(−)ofDoxpriortotheassay.(Upperpanels)Phase-contrastmicrographs.CellstreatedwithDoxlostthe g
mechanicalintegrityoftheircellsheets,butuntreatedcellsdidnot.(Bottompanels)Quantificationofcelldissociationassays.Theextentofcelldissociation o
isrepresentedbytheindexNp/Nc,whereNpandNcarethetotalnumbersofparticlesandcellsperdish,respectively.Valuesrepresentthemean±s.e.;n=at ol
least3times.*P<0.01comparedwithcontrol(−Dox).Scalebars:25µm. Bi
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Fig.2.GFP–IIA-dependent
localizationofthejunction
componentstothecell–cell
contactsitesinIIAKOcells.
(A)Immunofluorescencestainingof
junctioncomponents.GFP–IIA/
IIAKOcellswereculturedfor4days
with(+)orwithout(−)Dox,and
stainedwithantibodiesagainstthe
followingjunctionproteins:E-
cadherin(E-cad),β-catenin(β-cat),
plakoglobin(PG),desmoplakin(DP),
oroccludin(OCLN).Intheabsence
ofDox[(−)Dox],thesecomponents
weredetectedatthecell–cell
contactsites.Inthepresenceof
Dox,(+)Dox,noneofthese
componentswasdetectedatcontact
sites.Scalebar:25µm.(B)Cell
surfaceexpressionofE-cadherin
doesnotrequireGFP–IIA.Cell
surfaceproteinswerebiotinylated,
andthebiotinylatedproteinswere
immunoprecipitated(IP)with
streptavidin–agarose(avidin).The
collectedmaterialswereseparated
onagel,transferredtoamembrane,
andsubsequentlyprobedwithanti-
E-cadherinantibodies.Totalcell
lysateswereusedasloading
controls.(C)NMIIAdoesnotaffect
complexformationofE-cadherin
withcatenins.Afterlysis,E-cadherin
wascollectedwithanti-E-cadherin
antibodies.Thecollectedmaterials
wereseparatedonagel,transferred
toamembrane,andsubsequently
probedwiththeindicatedantibodies.
analyzedforexpressionofE-cadherin,β-catenin,andvinculin,we components to move from cell–cell contact sites of the plasma
observednosignificantdifferenceintheamountsofthesejunction membrane to an even distribution throughout the cell (Fig. 3G).
complex components, irrespective of the presence or absence of Surface biotinylation analysis, however, revealed that E-cadherin
Dox(Fig.3C).FLAG-taggedα-cateninwasobservedincomplexes was exposed on the plasma membrane of α-catFLAG/αKO cells,
ofE-cadherincontainingβ-catenin(Fig.3D).IntheabsenceofDox, irrespective of the presence of Dox (Fig. 3H). These data
thecellsexhibitedepithelialmorphology,butthismorphologywas demonstrate that the established cell line lost cell–cell adhesion
disrupted by thepresence of Dox (Fig. 3E). Cell–cell adhesion of activityduetoablationofα-catenin.
α-catFLAG/αKO cells, assessed by the dissociation assays, was
restoredintheabsence,butnotthepresence,ofDox(Fig.3F).Thus α-cateninactsdownstreamofIIA
the expression of FLAG-tagged α-catenin is required forcell–cell IIAKOandαKOcellsexhibithighlysimilarpropertiesinregardto n
e
adhesionactivity. assembly of the adhesion complex. To elucidate the molecular p
In the absence of Dox, the components of cell–cell junctions interaction of IIA and α-catenin, we ablated the α-cateningenein O
(E-cadherin,β-catenin,desmoplakin,andoccludin)weredistributed GFP–IIA/IIAKO cells. The resultant cells, termed GFP–IIA/ gy
predominantly at plasma membrane sites involved in lateral cell– IIAKO-αKO, did not express α-catenin (Fig. 4A). Sequencing of o
l
cell contacts (Fig. 3G). Addition of Dox not only decreased genomicDNAisolatedfromtheclonerevealedan8-bpdeletionin o
i
α-cateninexpressionandcell–celladhesion,butalsoinducedthese the α-catenin gene, which results in a frame-shift mutation B
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
n
e
p
O
y
g
o
Fig.3.Seenextpageforlegend. l
o
i
B
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Fig.3.Mislocalizationofjunctioncomponentsintheabsenceofα- molecules in cells lacking IIA expression. GFP–IIA/IIAKO cells
catenin.UsingtheCRISPR/Cas9system,theα-cateningeneofMDCKcells weretransfectedwithexpressionvectorsfortheseconstructs;stable
wasablated,yieldingαKOcells,whichweretransfectedwithavector transfectants were isolated, cultured in the presence orabsence of
encodingFLAG-taggedα-catenin(α-catFLAG)underthecontroloftheTRE,
Dox for 4days, and then analyzed. Immunoblot analysis revealed
yieldingα-catFLAG/αKOcells.(A)Immunoblotdetectionofα-catenin(α-cat),
E-cadherin(E-cad),β-catenin(β-cat),andvinculin(vinc)inαWTandαKO that although the level of GFP–IIAwas decreased by addition of
cells.Vinculinwasusedasaloadingcontrol.(B)Genomicsequencing Dox,thelevelsofthedeletionconstructswerenot (Fig.5B).Cells
revealedmutationsinthetargetregionsofα-cateningenethatinduced expressingtheseconstructsexhibitedepithelialmorphologyinthe
frame-shiftmutations.Ofthreeindependentclonessequenced,a5-bp absence of Dox (not shown), and the constructs were detected at
deletionwasdetectedintwoclonesanda1-bpinsertionwasidentifiedin cell–cell contact sites (Fig. 5C). Cells expressing α1–302 lost
oneclone.(C)Dox-repressibleexpressionofFLAG-taggedα-catenininα-
epithelial morphology in the presence of Dox, whereas cells
catFLAG/αKOcells.Thecellswereculturedfor4dayswith(+)orwithout(−)
Dox,andsubjectedtoimmunoblotanalysiswiththeindicatedantibodies.α- expressingeitherα1–381orαΔ203–612retainedthismorphology
catFLAGwasdetectedwithananti-FLAGantibody.TheadditionofDox (Fig. 5D). Dissociation assays of cells cultured in the presence of
repressedα-catFLAGexpression,butdidnotaffecttheexpressionofother Dox revealed that cells expressing α1–302 were sensitive to
proteins.Vinculinwasusedasaloadingcontrol.(D)α-catFLAGco- mechanicaldissociation,whereascellsexpressingeitherα1–381or
immunoprecipitatedwithE-cadherin,alongwithβ-catenin.(E)Cell αΔ203–612 were resistant (Fig. 5E). Consistent with these
morphology.Cellsonthedishwereobservedusingaphase-contrast observations, α1–302 and the components of the junction
microscope.(F)Dissociationassays.(Upperpanels)Phase-contrast complex were no longer detected at cell–cell contact sites in cells
micrographs.CellstreatedwithDoxlostthemechanicalintegrityoftheircell
expressing α1–302 after culture with Dox, whereas in the cells
sheets,whereasuntreatedcellsdidnot.(Bottompanels)Quantificationof
celldissociationassays.Valuesrepresentthemean±s.e.;n=atleast3times. expressing either α1–381 or αΔ203–612, these constructs and the
*P<0.01comparedwithcontrol(−Dox).(G)Immunostaining.Cellswere junction components remained at cell–cell contact sites even after
culturedandstainedwithantibodiesagainstFLAG(α-catFLAG),E-cadherin culture with Dox (Fig. 5F). Of note, the distribution of vinculin
(E-cad),β-catenin(β-cat),desmoplakin(DP),oroccludin(OCLN).The differedamongcellsexpressingdifferentconstructs.Vinculinwas
additionofDoxdisruptedlocalizationofjunctioncomponentsatcell–cell detectedatcell–cellcontactsitesincellsexpressingα1–381,butin
contactsites.Scalebars:25µm.(H)Cellsurfaceproteinswerebiotinylated,
andthebiotinylatedproteinswereimmunoprecipitated(IP)withstreptavidin– the cytoplasm in cells expressing αΔ203–612 (Fig. 5F, bottom
agarose(avidin).Thecollectedmaterialswereseparatedonagel, panels).Thusα1-381mutantproteinrestoresα-cateninfunctionby
transferredtoamembrane,andsubsequentlyprobedwithanti–E-cadherin recruitingvinculinintheabsenceofNMIIAactivity.
antibodies.Totalcelllysateswereusedasloadingcontrols. The recruitment of vinculin to cell–cell contact sites in cells
expressing α1–381 strongly suggests that the action of α1–381
requiresvinculin.Toinvestigatethispossibility,thevinculingene
(Fig.4B).Whenthesecellswereculturedintheabsenceorpresence was ablated using the CRISPR/Cas9 system in GFP–IIA/IIAKO
of Dox, the cell morphology was not observed irrespective of the cells expressing α1–381. Immunoblot analysis revealed that the
presenceofDox(Fig.4C).Theintegrityofcelljunctionswasalso vinculin knockout (vincKO) cells did not express vinculin
lost,asdeterminedbydissociationassays(Fig.4D).Moreover,the (Fig. 6A). Genomic sequencing revealed a mutation in the target
components of the adhesion complex were not assembled at cell– regionofvinculingenethatinducedaframe-shift(Fig.6B).When
cellcontactsitesofthecellsurface(Fig.4E).Theseresultsshowthat the cells were cultured in the presence of Dox to suppress the
α-catenin acts downstream of IIA in the assembly of junction expressionofGFP–IIA,controlwild-typevinculin(vincWT)cells
components. exhibited epithelial morphology, whereas vincKO cells lost tight
cell–cell contacts, as revealed by morphological observation
Expressionofα-catenindeletionconstructsinIIAKOcells (Fig. 6C). Dissociation assays confirmed that vincKO cells lost
inducesjunctionassemblyandrestorescell–celladhesion tight cell–cell contact (Fig. 6D). Under these conditions, the
α-cateninconsistsofthreedomains(Fig.5A)(Ishiyamaetal.,2013; componentsofcelljunctionswerenotassembledatcell–cellcontact
Yao et al., 2014; Escobar et al., 2015; Kang et al., 2017). The sites (Fig. 6E, right panels). Thus, the presence of vinculin is
N-terminal(N)domainbindstoβ-catenin.Themiddle(M)domain requiredfortheactionofα1–381.
comprises three regions, MI, MII, and MIII. MI contains binding
sites for vinculin, and MII and MIII are involved in regulation of ExpressionofanE-cadherin–α-cateninchimericmolecule
force-dependent vinculin binding and are collectively termed the restorescell–celladhesionofcellslackingIIAexpression
inhibitory region (Yonemura et al., 2010). The C-terminal (C) ChimericproteinscomposedofE-cadherinandα-cateninfunction
domainbindsactin.Wepreviouslyreportedthatexpressionofnot as cell adhesion molecules and can rescue the loss of E-cadherin,
only full-length α-catenin, but also α-catenin constructs with β-catenin, or α-catenin (Nagafuchi et al., 1994; Ozawa, 1998;
internalorC-terminaldeletions,inα-catenin-deficientDLD-1cells OzawaandKobayashi,2014).Toobtainfurtherinsightintotherole
results in acquisition of cell–cell adhesion and formation of both of IIA in cadherin-mediated cell adhesion, we expressed mutant
adherensjunctionanddesmosomes(Ozawa,1998;Matsubaraand E-cadherin (ELA) or an E-cadherin–α-catenin chimera (ELAαC,
Ozawa, 2001; Taniguchi et al., 2005). α1–381 is a construct in EWALAαC,orELAαM)inGFP–IIA/IIAKOcells(Fig.7A).ELAis
whichthecarboxy-terminal525residuesareablatedbyatruncation amutantE-cadherininwhichthetwoleucineresiduesatpositions587
thatremovestheactin-bindingsiteandinhibitoryregionbutretains and588werereplacedwithtwoalanineresidues(LAsubstitution).
thevinculin-bindingsite(Fig.5A).α1–302,whichhasanadditional Thissubstitutionincreasesthecell-surfacelocalizationofE-cadherin n
e
deletioninthevinculin-bindingsite,wasusedasanegativecontrol. when β-catenin is not available (Ozawa and Kobayashi, 2014). p
αΔ203–612 has an internal deletion of residues 203–612, and ELAαC,achimeracomposedofC-terminallytruncatedE-cadherin O
thereforelackstheentireMdomainand thusthevinculin-binding andtheC-terminalone-thirdoftheα-cateninpolypeptide(residues gy
site, but still retains the actin-binding site. We assumed that if 612–906),doesnotinteractwithβ-catenin,butstillfunctionsasacell o
NMIIA induces conformational changes in α-catenin and thereby adhesion molecule (Ozawa and Kobayashi, 2014). The second ol
i
activatesit,thesedeletionconstructscouldactasactivatedα-catenin chimera(EWALAαC)hasanadditionalmutationofTrp2toalanine B
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
Fig.4.KOofα-cateningeneinGFP–IIA/IIAKOcellsresultsinmislocalizationofadhesionjunctioncomponentsandinactivationofcell–cell
adhesionirrespectiveofGFP–IIAexpression.UsingtheCRISPR/Cas9system,theα-cateningeneofGFP–IIA/IIAKOcellswasablated,yieldingGFP–IIA/
IIAKO-αKOdouble-knockoutcells.(A)Immunoblotdetectionofα-catenin(α-cat)andvinculin(vinc).GFP–IIA/IIAKOcellswithwild-typeα-catenin(αWT)were
usedasapositivecontrol,andvinculinwasusedasaloadingcontrol.(B)Genomicsequencingrevealedadeletioninthetargetregionoftheα-catenin
genethatinducedaframe-shiftmutation.An8-bpdeletionwasidentifiedinallsixclonessequenced.(C)MorphologyofGFP–IIA/IIAKO-αKOcells.
(D)Dissociationassays.CellsculturedinthepresenceorabsenceofDoxweresubjectedtodissociationassays,anddissociationwasquantified.
Valuesrepresentthemean±s.e.;n=atleast3times.(E)Distributionofadhesioncomponents.CellswerestainedwithantibodiesagainstE-cadherin(E-cad),
β-catenin(β-cat),desmoplakin(DP),orTRITC-labeledphalloidin(actin).Scalebars:25µm.
(W2A) in the E-cadherin extracellular domain, which abolishes thepresenceofDoxdidnotrescuecellsundergoingmorphological
lateral dimerization and the adhesive interactions of E-cadherin changes,expressionofELAαC,butnotthatofEWALAαC,amutant
(Ozawa,2002).ELAαMisanotherELA–α-cateninchimericprotein chimera without adhesion activity, prevented the morphological
withα-cateninregionsencompassingaminoacids157–381,which changesinducedbytheadditionofDox(Fig.7C).Thethirdchimera
contain the domains necessary forassociation with vinculin. Like (ELAαM)alsopreventedthemorphologicalchangesinducedbythe
ELAαC,ELAαMfunctionsasacelladhesionmolecule(Ozawaand addition of Dox (Fig. 7C). We then analyzed the integrity of cell
Kobayashi, 2014). GFP–IIA/IIAKO cells were transfected with junctions in dissociation assays. GFP–IIA/IIAKO cells expressing
expression vectors for these constructs; stable transfectants were ELAαC or ELAαM exhibited a significant degree of integrity,
isolated,culturedinthepresenceorabsenceofDoxfor4days,and whereasexpressionofELAorEWALAαChadnoeffect(Fig.7D). n
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then subjected to analysis. Immunoblot analysis revealed that Thus, ELAαCor ELAαM expression in GFP–IIA− cells restored p
althoughthelevelofGFP–IIAwasdecreasedbyadditionofDox,the assemblyofcelljunctionsintheabsenceofNMIIA. O
levels of the constructs were not (Fig. 7B). Expression of these When GFP–IIA/IIAKO cells expressing ELAαC or ELAαM gy
constructsinthecellsculturedintheabsenceofDox,whenGFP–IIA werestainedwithantibodiesagainstHA,β-catenin,ordesmoplakin, o
wasexpressed,didnotinduceanymorphologicalchanges(datanot these proteins were detected at the cell–cell contact sites of the ol
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shown).AlthoughexpressionofELAinGFP–IIA−cellsculturedin membrane, whereas in GFP–IIA/IIAKO cells expressing ELA or B
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
Fig.5.Expressionofα-catenindeletionconstructsinIIAKOcells panels).AlthoughvincKOcellsculturedfor2daysintheabsence
inducesjunctionassemblyandrestorescell–celladhesion. of Dox exhibited almost identical morphology (Fig. 8C) and
(A)Schematicrepresentationofα-catenin(top)andmutantderivatives distribution of junction components in comparison with vincWT
(bottom).TheN-terminal(N)domainbindstoβ-catenin.Themiddle(M)
cells(Fig.8E),thekineticsofjunctionassemblyweremuchslower
domaincomprisesthreeregions,MI,MII,andMIII.MIcontainsbindingsites
invincKOthaninvincWT(Fig.8F).Thus,thepresenceofvinculin
forvinculin,andMIIandMIIIareinvolvedinregulationofforce-dependent
vinculinbinding.TheC-terminal(C)domainbindsactin.α1–302doesnot acceleratesjunctionformation.CellsheetsformedbyvincKOcells
containthevinculin-andactin-bindingsites.α1–381isaconstructinwhich dissociated more easily than those formed by vincWT cells, as
thecarboxy-terminal525residueshavebeentruncated,therebyremoving confirmedbydetachmentofcellsheetswithDispase(Fig.8G).
theactin-bindingsitebutretainingthevinculin-bindingsite.αΔ203–612has
aninternaldeletionofresidues203–612,andthereforelacksthevinculin-
DISCUSSION
bindingsitebutretainstheactin-bindingsite.Allconstructsweretaggedwith
HA.(B)Immunoblotdetectionofα-cateninpolypeptides.GFP–IIA/IIAKO In this study, we ablated the NMHCIIA (IIA) gene (MYH9) in
cellsexpressingα1–302,α1–381,orαΔ203–612wereculturedinthe MDCK cells, and then introduced GFP-tagged IIA (GFP–IIA)
presenceorabsenceofDoxfor4days,andthenanalyzed.Theblots under the control of the Tet promoter. Thus, we could control the
wereprobedwithanti-HA,anti-GFP,andanti-vinculinantibodies. expressionofGFP–IIAbyculturingcellsinthepresenceorabsence
(C)Immunofluorescencestainingwithanti-HArevealedthattheseconstructs ofDox.Specifically,thecellsexpressedGFP–IIAintheabsenceof
werelocalizedatcell–cellcontactsitesofcellsculturedintheabsenceof
Dox but not in the presence of Dox, and could assemble the
Dox.(D)MorphologyofGFP–IIA/IIAKOcellsexpressingα1–302,α1–381,or
epithelialjunctioncomplexonlyintheformercondition.Therefore,
αΔ203–612.CellsculturedinthepresenceofDoxtosuppressGFP–IIA
weconcludethatexpressionofGFP–IIAisrequiredforassemblyof
expression,andwereobservedusingaphasecontrastmicroscope.
(E)Dissociationassays.CellswereincubatedwithDispase,andthen thejunctioncomplex.Whentheα-cateningenewasablatedinthe
detachedcellsweresubjectedtomechanicalstressbypipettingand samecells,theybecameunabletoformthejunctionirrespectiveof
quantified.Valuesrepresentthemean±s.e.;n=atleast3times.*P<0.01 theexpressionofGFP–IIA,stronglysuggestingthatα-cateninacts
comparedwithcontrol(cellsexpressingα1–302).(F)Distributionofthe downstreamofNMIIA.
junctioncomponents.Dox-treatedcellswerestainedwithantibodiesagainst Vinculin has been implicated in the anchoring of cadherin–
HA,E-cadherin(E-cad),β-catenin(β-cat),desmoplakin(DP),occludin
catenincomplexestoactin(Miyakeetal.,2006;Maddugodaetal.,
(OCLN),orvinculin(vinc).TRITC-phalloidinwasusedtodetectactin.
Scalebars:25µm. 2007). Vinculin is a component of integrin-associated complexes
(Geiger et al., 1980) and plays a role in mechanotransduction at
focaladhesionsRivelineetal.(2001).VinculinisalsofoundatAJs
EWALAαC, these proteins were not localized at these sites (Geigeretal.,1985)andactsasabindingpartnerforα-cateninin
(Fig. 7E). Actin stained with TRITC-labeled phalloidin was maturecadherin-basedAJs(Watabe-Uchidaetal.,1998).Vinculin
detected at cell–cell contact sites of cells expressing ELAαC or binds to α-catenin in response to conformational changes in both
ELAαM, but not those of cells expressing ELA or EWALAαC. proteins; this, in turn, leads to the formation of a putative
Consistent with the results obtained with the α-catenin deletion heterotetramer in which both the vinculin and α-catenin C-
constructs, vinculin was detected at cell–cell contact sites in cells terminal domains bind F-actin with high affinity (Rangarajan and
expressing ELAαM, but not those expressing other constructs Izard,2012;Choietal.,2012).Structuralandbiochemicalanalyses
(Fig.7E,bottompanels).ThustheELAαMchimera,whichcontains have suggested that α-catenin undergoes aconformational change
only the vinculin-binding site of α-catenin, recruited vinculin to from the closed to open conformation, thereby unmasking the
cell–cellcontactsitesandrescuedthedefectsresultingfromNMIIA vinculin-bindingdomain(Yangetal.,2001;Pokuttaetal.,2002).
deficiency. This conformational change of α-catenin, controlled by a force-
dependentmechanism,mayberesponsibleformyosinII-dependent
GFP–IIA/IIAKOcellslackingthevinculingeneretainthe recruitment of vinculin in mature AJs (Miyake et al., 2006;
abilitytoassemblejunctioncomplexes Maddugodaetal.,2007;leDucetal.,2010;Yonemuraetal.,2010).
Although an α-catenin construct containing only the vinculin- Indeed, recent experimental data suggest that cadherin is
bindingsitecouldestablishthejunctioncomplexesincellswithout constitutively under actomyosin-generated tension (Smutny et al.,
IIAexpression,anα-cateninconstructwithonlytheactin-binding 2010;Borghietal.,2012).ItisgenerallyassumedthatmyosinII-
sitealsohadthisability,raisingthepossibilitythatvinculinisnotan generated tension induces a conformational change in α-catenin,
absolute requirement for junction formation. To investigate this uncoveringavinculin-bindingsite.VinculinisthenrecruitedtoAJs
possibility,weablatedthevinculingeneinGFP–IIA/IIAKOusing andbecomesassociatedwithmoreactinfilaments,thusreinforcing
the CRISPR/Cas9 system, yielding GFP–IIA/IIAKO-vincKO cell–celladhesion(Yonemuraetal.,2010;leDucetal.,2010;Yao
double-knockout cells. Immunoblot analysis revealed that the et al., 2014; Kim et al., 2015). According to this paradigm, wild-
cellsdidnotexpressvinculin(Fig.8A).GenomicDNAsequencing type(WT)α-cateninincellslackingmyosin-IIactivitywouldnotbe
revealed a mutation in the target region of the vinculin gene that stretched to expose the vinculin-binding site, and thereforewould
inducedaframe-shift(Fig.8B).Whenthecellswereculturedinthe not interact with vinculin. By contrast, α-catenin mutant proteins
absenceofDox,theyexhibitedepithelialmorphology(Fig.8C),and with truncations that remove the putative auto-inhibitory domain
dissociation assays revealed that they could form cell sheets that would be predicted to have exposed vinculin-binding sites, and
were resistant to mechanical dissociation by pipetting (Fig. 8D). wouldthusinteractwithvinculininaconstitutivemanner (Jurado
n
Immunostaining of cells revealed that the components of cell et al., 2016). Consistent with this idea, we found that NMIIA KO
e
junctions were assembled at cell–cell contact sites (Fig. 7E, left cells expressing endogenous WT α-catenin failed to assemble p
panels).WhenexpressionofGFP–IIAwassuppressedbyculturing junction components, but expression of not only GFP-tagged O
in the presence of Dox, the cells lost tight cell–cell contact, as NMIIA, but also α1–381 mutant protein (which contains the gy
revealed by morphological observation (Fig. 8C) and dissociation vinculin-binding site), induced assembly of the complex. The o
assays (Fig. 8D). Under these conditions, the components of cell C-terminallytruncatedformsofα-cateninthatbindconstitutivelyto ol
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junctionswerenotassembledatcell–cellcontactsites(Fig.8E,right vinculin strongly stabilize AJ dynamics (Chen et al., 2015; B
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RESEARCHARTICLE BiologyOpen(2018)7,bio031369.doi:10.1242/bio.031369
Fig.6.Theα-cateninconstructα1–381requiresvinculinforitsabilitytorescuejunctionformation.UsingtheCRISPR/Cas9system,thevinculingene
wasablatedinα1–381/GFP–IIA/IIAKOcells,yieldingα1–381/GFP–IIA/IIAKO-vincKOdouble-knockoutcells.(A)Immunoblotdetectionofvinculin(vinc)and
actin.α1–381/GFP–IIA/IIAKOcellswithwild-typevinculin(vincWT)wereusedasapositivecontrol,andactinwasusedasaloadingcontrol.(B)Genomic
sequencingrevealedamutationinthetargetregionofvinculingenethatinducedaframe-shift.All13independentclonessequencedharboredthesame
insertionof1bp.(C)Morphologyofα1–381/GFP–IIA/IIAKO-vincKOcells.CellsculturedinthepresenceofDoxwereobservedusingaphase-contrast
microscope.(D)CellsculturedinthepresenceofDoxwereincubatedwithDispase,andthendetachedcellsweresubjectedtodissociationassaysand
quantified.Valuesrepresentthemean±s.e.;n=atleast3times.*P<0.01comparedwithcontrol(vincWTcells).(E)Distributionofadhesioncomponents.
CellswereculturedinthepresenceofDox,andthenstainedwithantibodiesagainstHA,E-cadherin(E-cad),β-catenin(β-cat),ordesmoplakin(DP).
Scalebars:25µm.
Yonemuraetal.,2010;Thomasetal.,2013).Consistentwiththese this construct in α-catenin–deficient cells induces assembly of
observations, we observed accumulation of vinculin at cell–cell junctions (Taniguchi et al., 2005). α-catenin mutants lacking the
contact sites of cells expressing α1–381. Thus, α1-381 mutant vinculin binding site can form actin-associated junctions in
protein restores α-catenin function by recruiting vinculin in the α-catenin–deficient cells (Huveneers et al., 2012; Twiss et al.,
absenceofNMIIAactivity.Therefore,NMIIAactsasanactivatorof 2012;Desaietal.,2013;Chenetal.,2015),whicharecompletely
α-cateninbyexposingitsvinculin-bindingsite.Insupportofthese devoidofvinculin(Chenetal.,2015).BecauseWTα-cateninisnot n
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results, the ELAαM chimera, which contains only the vinculin- activated without NMIIA activity, the truncation introduced in p
bindingsiteofα-catenin,alsorecruitedvinculintocell–cellcontact αΔ203–612 activated its actin-binding activity. Recent studies O
sitesandrescuedthedefectsresultingfromNMIIAdeficiency. revealed strong and stable bonds between the cadherin–catenin gy
Another construct, αΔ203–612, contains the actin-binding site complex and an actin filament form under force (Buckley et al., o
butlacksthevinculin-bindingsite.αΔ203–612couldalsorestoreα- 2014). Therefore, the region deleted in the construct, aa 203–612 ol
i
catenin function in the absence of NMIIA activity. Expression of mayalsobeinvolvedinmodulationoftheactin-bindingsite.Thus, B
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