Drosophila vinculin is more harmful when hyperactive than absent, and can circumvent integrin to ...

12 Pages·2016·3.72 MB·English

©2016.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
RESEARCHARTICLE
Drosophila vinculin is more harmful when hyperactive than absent,
and can circumvent integrin to form adhesion complexes
AidanP.Maartens,JuttaWellmann*,EmmaWictome,BenjaminKlapholz,HannahGreenandNicholasH.Brown‡
ABSTRACT recruitment of numerous cytoplasmic adhesion proteins (Zhang
etal.,2008;Zervasetal.,2011).Ofparticularinterestistheforce-
Vinculinisahighlyconservedproteininvolvedincelladhesionand
dependentrecruitmentofvinculin.Invitroworkhasestablishedthat
mechanotransduction,andbothgainandlossofitsactivitycauses
stretching the rod of talin exposes previously hidden vinculin-
defective cell behaviour. Here, we examine how altering vinculin
bindingsites(VBSs,singleheliceswithintheα-helicalbundlesthat
activity perturbs integrin function within the context of Drosophila
make up the rod; Bass et al., 1999) that can then bind vinculin
development. Whereas loss of vinculin produced relatively minor
phenotypes, gain of vinculin activity, through a loss of head–tail (Papagrigoriou et al., 2004; del Rio et al., 2009). Consistent with
thismodel,therecruitmentofvinculintoadhesionsincellcultureis
autoinhibition, caused lethality. The minimal domain capable of
particularlysensitivetomyosinIIinhibition(Rivelineetal.,2001;
inducinglethalityisthetalin-bindingD1domain,andthisappearsto
Pasaperaetal.,2010;Cariseyetal.,2013).
require talin-binding activity, as lethality was suppressed by
A series of four-helical bundles (seven in vertebrates, six in
competition with single vinculin-binding sites from talin. Activated
invertebrates)makeuptheheaddomainofvinculin,whichislinked
Drosophilavinculintriggeredtheformationofcytoplasmicadhesion
by a partially disordered proline-rich region to the five-helical
complexes through the rod of talin, but independently of integrin.
bundle of the tail (Bakolitsa et al., 2004; Borgon et al., 2004).
Thesecomplexescontainasubsetofadhesionproteinsbutnolonger
Interactionsitesforvinculinligandshavebeenmapped acrossthe
link the membrane to actin. The negative effects of hyperactive
protein (reviewed in Ziegler et al., 2006). A key ligand is talin
vinculinweresegregatedintomorphogeneticdefectscausedbyits
(Burridge and Mangeat, 1984), and the interaction has been
whole head domain and lethality caused by its D1 domain. These
narrowed to the first two four-helical bundles of the head, the D1
findingsdemonstratethecrucialimportanceofthetightcontrolofthe
domain(alsoknownasVh1;Boisetal.,2006):theVBSs intalin
activityofvinculin.
bindtothefirstfour-helicalbundleoftheD1domain,transforming
KEYWORDS:Adhesion,Drosophila,Vinculin,Integrin,Talin,Rhea, itintoafive-helicalbundle(Izardetal.,2004).Thisfirstbundleof
Proteincomplex D1retainsmostoftheVBS-bindingactivityoftheD1domainina
two-hybrid assay (Bass et al., 2002), suggesting it is the minimal
INTRODUCTION talin-bindingsite,butthesecondbundleisalsocapableofbinding
Cell adhesion is mediated by multiprotein complexes that link someligands(NhieuandIzard,2007),andtheentireD1domainis
transmembranereceptorstothecytoskeleton.Thesecomplexesare generallyusedasaminimalheaddomain(Humphriesetal.,2007;
assembledatdiscretesitesofthemembrane,andbothlossandgain Cariseyetal.,2013).Vinculinisnotableamongintegrin-associated
of adhesion protein activity causes cellular and developmental proteins for also localising to cell–cell adhesions (Geiger et al.,
defects (Wickstrom et al., 2011; Maartens and Brown, 2015a), 1980),andthisismediatedthroughaninteractionoftheheadwith
which have pathological consequences (Winograd-Katz et al., either α- or β-catenin (Hazan et al., 1997; Watabe-Uchida et al.,
2014). 1998).TheflexibleneckofvinculinbindsproteinsoftheCAPand
Thefirststepinbuildingacell–matrixadhesionisthebindingof vinexinfamily(Kiokaetal.,1999;Bharadwajetal.,2013),among
transmembrane integrin receptors to extracellular matrix (ECM) other ligands, and the tail binds to actin (Jockusch and Isenberg,
components. This is followed by recruitment of cytoplasmic 1981; Johnson and Craig, 1995b), the scaffolding protein paxillin
adhesionproteins,forexampletalin(alsoknownasRheainflies), (Turner et al., 1990; Wood et al., 1994), and the membrane lipid
which occurs through the cytoplasmic tail of integrin. Talin is a phosphatidylinositol 4,5-bisphosphate (PIP ) (Johnson and Craig,
2
crucialcomponentofthelinkasitcansimultaneouslybindintegrins 1995a; Gilmore and Burridge, 1996), and also promotes
(withitsFERM-domainhead)andactin(withanactin-bindingsite dimerisation (Johnson and Craig, 2000; Janssen et al., 2006;
at the C-terminus of its long rod domain; Critchley, 2009). Talin Chinthalapudi et al., 2014). By simultaneously binding talin and
feeds back to promote integrin activation and is required for the actin, vinculin provides an additional link to the cytoskeleton,
e
giving extra mechanical support to the adhesion. A strengthening c
n
DepartmentofPhysiology,DevelopmentandNeuroscience,andtheGurdon roleisconsistentwiththerelativelymildereffectsoflosingvinculin e
i
Institute,UniversityofCambridge,DowningSt.,CambridgeCB21DY,UK. comparedtolosingtalinincellsinculture(Xuetal.,1998b;Zhang c
*Deceased S
etal.,2008)andindevelopinganimals(Alatortsevetal.,1997;Xu l
l
‡Authorforcorrespondence([email protected]) etal.,1998a;Monkleyetal.,2000;Brownetal.,2002;Bharadwaj Ce
etal.,2013).
f
N.H.B.,0000-0002-8958-7017 Although vinculin has many binding partners, the full-length o
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution protein has little binding activity due to a head–tail interaction al
License(http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse, stabilising the inactive conformation (Johnson and Craig, 1994; n
distributionandreproductioninanymediumprovidedthattheoriginalworkisproperlyattributed. r
Bakolitsa et al., 2004; Borgon et al., 2004; Cohen et al., 2005). u
o
Received24March2016;Accepted7October2016 Constructsthatrelievethishead–tailautoinhibitionarehyperactive, J
4354
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
and dramatically increase the size and stabilityof focal adhesions the effect of force on talin. Finally, we dissect the negative
associatedwithactivatedintegrins(Cohenetal.,2005;Humphries effects of hyperactive vinculin into two discrete activities:
etal.,2007),aswellasmakingtherecruitmentofadhesionproteins morphogenetic defects caused by its head domain, and lethality
nolongersensitivetomyosinIIinhibition(Cariseyetal.,2013).The causedbyitsD1domain.
talin-bindingD1domainaloneissufficienttoproducetheseeffects
(Cohen et al., 2006; Humphries et al., 2007), and, reciprocally, RESULTS
reducingtheabilityoftheD1domaintobindtalineliminatesthem Althoughabsenceofvinculinhasminorconsequences,
(Cohenetal.,2006;Humphriesetal.,2007;Cariseyetal.,2013). hyperactivevinculincauseslethalitythroughitsD1domain
The vinculin tail adds additional activity: it is required for FlieshomozygousforaninversionthatdisruptstheVinculin(Vinc)
hyperactive vinculin to produce traction forces (Dumbauld et al., locusareviableandfertile(Alatortsevetal.,1997),andwerecently
2013) and reorient adhesions in response to polarised forces reported that a deletion removing the coding sequence, ΔVinc, is
(Cariseyetal.,2013).Akeyaspectofvinculinfunctionistherefore alsohomozygousviableandfertile (Klapholzetal.,2015).ΔVinc
its activation status, and its effects on cell behaviour might be flies had normal embryonic muscles (Fig. 1A,B), without the
causedbyitsactionontalinaswellasdirectorindirectrecruitment detachmentsthat typicallyarise from loss of integrin function. To
of proteins to adhesions. Although the impact of hyperactive examinewhether thereareanychangestotheadhesionsitein the
vinculin on cellular behaviour has been well documented, the absenceofvinculin,weexaminedadhesioncomponentlevels.The
impactsofthesechangesoncellswithintheorganismhaveyettobe αPS2βPS integrin (αPS2 is also known as Inflated and βPS as
addressed. A mutant that produces hyperactive vinculin in mouse Myospheroid), talin and paxillin were still recruited to the muscle
has a milder version of the defects caused byabsence of vinculin attachmentsites(MASs;Fig.1C).Levelsofrecruitmentwerethen
(Marg et al., 2010), but vinculin levels are also strongly reduced, quantifiedusingrescueconstructstaggedwithfluorescentproteins
makingitdifficulttoseparateloss-andgain-of-functioneffects. inliveembryos.LevelsoftheintegrinβPSsubunit,talin,ILKand
Toprobefurtherhowvinculincontributestoadhesion,wehave PINCH(alsoknownasSteamerduckinflies)werenotchangedin
used Drosophila to compare loss- and gain-of-function effects ΔVincanimals,whereasthelevelsofpaxillinwasreducedtoabout
during development. We find that vinculin hyperactivity is far half, and those of tensin (also known as Blistery) were increased
more deleterious to the organism than inactivity, and discover a (Fig. 1D). Thus, in addition to paxillin contributing to vinculin
new function for vinculin in bringing adhesion proteinstogether recruitment (Pasaperaetal.,2010),theconverseisalsofound,and
independently of the usual integrin cue. The D1–talin-rod theelevationoftensinsuggeststhattensinandvinculincompetefor
interaction is crucial for the formation of these cytoplasmic limitingrecruitmentsites.Aspreviouslyreported(Bharadwajetal.,
adhesion subcomplexes, supporting a model where hyperactive 2013), CAP (the single fly orthologue of the CAP and vinexin
vinculinectopicallyactivatestalininthecytoplasmbymimicking family)islostfromMASsinΔVinclarvae,butwefounditwasstill
A B C Fig.1.Lossofvinculinhasminimaleffectson
embryonicmuscles.Musclemorphologyoflatestage
16wild-type(A)andΔVinc(B)embryos,stainedfor
musclemyosin.(C)Recruitmentofintegrin(stainingfor
theαPS2subunit),talin–mCherryandpaxillin–GFPto
ΔVincMASs.Scalebars:10µm.(D)GFP-orYFP-tagged
proteinaveragefluorescenceintensitiesforindividual
αPS2 integrin talin MASscomparedtothewild-typeaverage,usinglive
stage17embryos.Theboxrepresentsthe25–75th
percentiles,andthemedianisindicated.Thewhiskers
extendtodatapointsthatarelessthan1.5×the
interquartilerange(IQR)awayfrom1stand3rdquartile.
Circlesrepresentoutliers.n=28MASmeasurementsin
≥5embryospergenotype.Darkblue,wild-typeMASs,
showingextentofvariation;paleblue,ΔVincMASs.
myosin paxillin merge ***P≤0.001;****P≤0.0001;ns,notsignificant(Student’s
t-test).
wild type ∆Vinc ∆Vinc
D ns ns **** ns ns ***
2
ASs age e
ndividual Md type aver 11.5 wild type Scienc
vel at id to wil ∆Vinc Cell
n leare 0.5 of
Proteicomp nal
0 βPS ur
integrin talin paxillin ILK PINCH tensin o
J
4355
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
recruited normally in the embryo (Fig. S1), demonstrating a stage induction of lethality, head–tail autoinhibition is required to limit
specificity of this interaction. Thus, although there are some the activity of the head. As vinc-CO and vinc-Head behave
alterations in adhesion protein levels in the absence of vinculin, identically in this assay, and in others described below, the ‘T12’
embryonicmuscledevelopmentproceedsnormally. changeappearsequivalenttodeletingthetail.
Totestwhetherthemusclesfunctionednormallyinfullygrown Within head of vinculin, the D1 domain contains all known
larvae (third larval instar) lacking vinculin, we used time-lapse binding sites for integrin-associated ligands. Expression of a
imaging to measure their crawling, but did not detect any construct encompassing this domain (UAS vinc-D1) was also
impairment in crawling velocity (Fig. S1E). We then examined lethal (Fig. 2), whereas expression of constructs lacking the D1
adult tissues; wings appeared normal (data not shown), but the domain(UASvinc-HeadΔD1andUASvinc-FLΔD1),werenotlethal
indirect flight muscles showed a defect in the distribution of the (Fig.2).Thus,thelethalityoftheheaddomainmapssolelytothe
actinatthemuscle termini. Inthesemuscles,actinaccumulatesat D1domain.
theMAS,butinΔVincmutantstheactinwasfrayedandexpanded Although the eight-helical D1 domain is generally used as the
(Fig.S1F).Theexceptionalsensitivityofthistissuetotheabsence minimal functional domain of the head, VBSs bind the first four-
of vinculin might reflect its exceptional biomechanical activity helicalbundle(Izardetal.,2004),andD1truncationsretainVBS-
during flight. This defect shares some similarity with the mild binding activity (Bass et al., 2002). We thustested the activityof
muscle phenotype in larvae reported by Bharadwaj et al. (2013). smallerconstructs, reducing the eight helices of D1 to six (amino
Thus,Drosophilavinculindoeshavefunctionalroles,albeitminor acids1–184,UASvinc-D1184),five(aminoacids1–152,UASvinc-
onesunderlaboratoryconditions. D1152)andfour(aminoacids1–127,UASvinc-D1127).Thesecond
Wethenaddressedtheconsequencesoftoomuchvinculinactivity bundle of D1 also has ligand-binding activity (Nhieu and Izard,
by using the UAS-Gal4 system (Brand and Perrimon, 1993) to 2007), so we also made a construct with this bundle on its own
express vinculin constructs with differing capacity for (amino acids 128–257, UAS vinc-D1128-257). However, when
autoinhibition,eachC-terminallytaggedwithTagRFP.Expression expressed with Mef2::Gal4 or Act5C::Gal4, none of these D1
of wild-type, full-length vinculin (UAS vinc-FL) either in the truncations caused lethality (Fig. 2), and hence the whole D1
muscles(withMef2::Gal4)orubiquitously(withAct5C::Gal4)did domainistheminimallethaldomain.
notaffectviability(Fig. 2;calculatedasrelativeeclosionofadults Finally, we tested whether the lethal effect of hyperactive
compared to heterozygous siblings). In contrast, expression of a vinculinwasconservedbyexpressingtheD1domainfromchicken
constitutivelyopenform(vinc-CO),whereautoinhibitionhasbeen (Gallusvinc-D1;onlyfiveresiduesdifferentfromhumanvinc-D1)
reducedbymutationofthe‘T12’clusterofchargedresiduesinthe in flies. Gallus vinc-D1 was completely lethal with Act5C::Gal4
tail to alanine residues(Cohenetal.,2005), was lethal with either andalmostentirelylethalwithMef2::Gal4(Fig.2),consistentwith
driver(noadultseclosed;Fig.2).Thus,withinthewholeanimal,too the high level of conservation of the D1 domain between
muchvinculinactivityismoredeleteriousthaneitheritsabsenceor invertebratesandvertebrates(55%identical).Insummary,theD1
anexcessofthewild-typeprotein. domainmediatesthelethalityofhyperactivevinculininvivo.
To address which portions of vinc-CO mediated lethality, we
designedasetofconstructstoexpressthedifferentdomains.Like Hyperactivevinculinformscytoplasmicaggregatesthatare
vinc-CO, expression of the head was lethal (Fig. 2). In contrast, adhesionsubcomplexes
expressionofthetail,aloneorwhenconnectedtotheflexibleneck Todiscoverwhyvinculinhyperactivitywasdeleterious,weexamined
region, had no effect on viability (Fig. 2). Thus, as assayed by thedistributionoftheproteinsinvivo.Inlatestage16/earlystage17
Vinculin domains Fig.2.Expressionofhyperactivevinculincauses
lethalitythroughitsD1domain.Vinculindomainsare
% Observed/
shown,witheachblockrepresentingasingleα-helix.
D1 Head Tail expected (n)
Neck Thetableshowsdetailsofregionsofvinculinexpressed
throughaUASpromoter,withaminoacidnumbers.
aa Act5C::Gal4 Mef2::Gal4
Viabilityisshownaspercentageobservedcomparedto
FL 1-961 118 91 expected,calculatedbycomparingthenumberof
(213) (298)
CO 1-961* >D ARARAIRIA 0 (73) 0 (93) Gflieasl4c>oUuAnStefdlieissgtoivtehneirinbpaalarnecnethressibelisn.gs.Thenumberof
Head 1-724 0 0
(78) (328)
D1 1-257 0 0
(208) (201)
cts D1184 1-184 101(191) 68(314) e
stru D1152 1-152 69 (209) 93(503) nc
Con D1127 1-127 68(337) 84(444) cie
AS D1128-257 128-257 109(96) 100(214) lS
U FL∆D1 257-961 64(246) 95 (228) Cel
Head∆D1 257-724 103 119 f
(161) (106) o
Neck+Tail 726-961 91 (283) 96(328) al
n
Tail 788-961 91(405) 87(197) ur
Gallus D1 1-258 0 (74) 12(131) Jo
4356
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
A A’ B B’ Fig.3.Distributionofvinculinconstructs
inmuscles.Vinculinconstructswere
expressedusingMef2::Gal4inembryonic
latestage16/earlystage17muscles(A–F),
ornewlyhatchedL1larvae(H–Q).(A,A′)
Vinc-FL,stainedformusclemyosinand
αPS2integrin;thearrowheadshowsMAS
enrichment.(B,B′)Vinc-CO,stainedfor
myo (cid:1)PS2 Vinc-FL vinc-FL myo (cid:1)PS2 vinc-CO vinc-CO musclemyosin(myo)andαPS2integrin;
C C’ D D’ thearrowheadshowsMASenrichment,the
arrowshowscytoplasmicaggregates.
(C,C′)Vinc-COexpressedwithGFP–talin;
arrowsshowenrichmentoftalinin
aggregates.(D,D′)AsinC,withpaxillin–
GFP.(E,E′)Vinc-CO-expressingembryos
stainedwithphalloidintodetectfilamentous
actin;thearrowshighlightthatthereisno
actinenrichmentintheaggregates.
talin vinc-CO talin paxillin Vinc-CO paxillin (F,F′)Vinc-COcoexpressedwith
E E’ F F’
myristolated(myr-)GFP;thearrows
highlightthatthereisnomyr-GFP
enrichmentintheaggregates.
(G)Enrichmentofadhesioncomponentsin
theaggregatescalculatedbycomparing
GFPsignalinindividualaggregatestothe
averagesignalincytoplasm,andtakingan
averageoftheseratios,withthestandard
actin vinc-CO actin myr-GFP vinc-CO myr-GFP deviationshownbelow.n=40aggregates
measuredperembryo,4–5embryosper
G βinPtSegrin talin fit1 vinculin paxillin ILK PINCH tensin CAP actin myr-GFP gvmienuncsouctllyienpsec;.oa(nrHrso–twrQusc).tisnSdeinixcgpalrteeecschsyeatodnpninlealstihmmeaicgesof
1.3 3.8 1.1 1.2 3.7 2.8 2.6 2.1 1.3 1.0 1.1
aggregates.ThearrowheadinJshows
±0.3 ±1.3 ±0.2 ±0.3 ±1.7 ±0.8 ±1 ±0.7 ±0.4 ±0.3 ±0.2
associationofvinc-Tailwithsarcomeric
Enrichment in vinc-CO aggregates compared to cytoplasm
actin.Scalebars:10µm.
H I J K L
vinc-Head vinc- Neck+Tail vinc-Tail vinc-D1 vinc-D1184
M N O P Q
vinc-D1152 vinc-D1127 vinc-D1128-257 vinc-FL∆D1 vinc-Head∆D1
embryos (15–18hafteregglaying),vinc-FLwasrecruitedtoMASs (LAMP1 in Fig. S3A,A′), autophagy (HuLC3, Fig. S3B,B′), or
inmuscleswhenexpressedwithMef2::Gal4(Fig.3A,arrowheadin early,lateorrecyclingendosomes(Fig.S3C–E′).Aggregationwas
Fig.3A′)andshowedalow,uniformlevelinthecytoplasm.Thiswas notcausedbyTagRFP,asotherTagRFP-taggedconstructsdidnot
similar tothelocalisationofwild-typevinculin expressed under its form aggregates (for example, vinc-FL in Fig. 3A, and other
e
ownpromoter(Fig.S2A–A″),althoughGal4-drivenvinc-FLhada constructs below), and aggregates were also formed by vinc-CO c
n
highercytoplasmic level. In contrast, Gal4-driven vinc-CO formed taggedwithGFP(Fig.S3F).TagRFPismodifiedtobemonomeric e
i
aggregatesthroughoutthecytoplasmofthemuscle(Fig.3B,arrowin (Merzlyaketal.,2007). c
S
Fig. 3B′), in addition to being enriched at the MAS (arrowhead in We next tested the effects of vinc-CO on the distribution of l
Fig. 3B′). These distributions were not tissue-specific: wild-type adhesion proteins, by examining the products of GFP-tagged el
C
vinculin was recruited to adhesions in epithelial cells of the pupal genomic rescue constructs. We started with the well-established
f
wingandfollicularepitheliumoftheovary(Fig.S2B–E″),whereas vinculin ligands talin (Fig. 3C) and paxillin (Fig. 3D). In the o
vinc-COadditionallyformedcytoplasmicaggregates(Fig.S2F–G′). presence of vinc-CO, these proteins showed a dual localisation: at al
n
Toinvestigatethenatureofthecytoplasmicaggregates,wefirst their usual site of function at MASs, and ectopically in the
r
examined whether they were caused by protein misfolding and cytoplasmicaggregates(Fig.3C′,D′;comparetoFig.1C)atalevel u
o
degradation,buttheydidnotcolocalisewithmarkersoflysosomes >3× background levels in the cytoplasm (Fig. 3G). Among other J
4357
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
adhesion proteins, ILK, PINCH and tensin were recruited to the (Fig. 4I; statistical comparison of aggregate-forming capacity in
aggregates,whereasendogenousvinculin,CAPandfermitin1(fit1, TableS1;notethattheaggregatesizewastoovariabletobeauseful
an orthologue of kindlin) were not (Fig. 3G). A key function of measure and achange in aggregate number did not correlatewith
cytoplasmic adhesion complexes is to link integrins to actin, but changeincellsize).Lossofpaxillinalsohadlittleeffectonvinc-CO
the aggregates formed by vinc-CO contained neither integrins aggregates (Fig. 4D,I). In contrast, removal of talin (null talin62
(antibodystainingforαPS2integrininFig.3B,βPS-integrin–GFPin truncation;Klapholzetal.,2015)ledtoanalmostcompletelossof
Fig.3G)noractin(asdetectedwithphalloidin,Fig.3E,E′,G).The aggregates,withvinc-CObeingmoreuniforminthecytoplasm,and
aggregateswerenotclearlyassociatedwithmembranesasdetected enrichedatcell–celljunctions(Fig.4E,I).Aggregatesofvinc-Head
with myristolated GFP (Fig. 3F,F′,G). Thus, vinc-CO-stimulated and vinc-D1 were also much reduced in the absence of talin
aggregates represent cytoplasmic adhesion subcomplexes that (Fig. S4A,B), suggesting that a D1–talin interaction is the initial
appear to be uncoupled from both the membrane and the eventingeneratingaggregates.Weconfirmedthattalinwasrequired
cytoskeleton.This contrastswith experimentsinmammaliancells, for aggregate formation in embryos (Fig. S4C). Talin is therefore
wherevinc-COexpandsfocaladhesionsatthemembranecontaining required for hyperactive vinculin to generate cytoplasmic
bothintegrinandactin(Cohenetal.,2005;Humphriesetal.,2007). aggregates. The increased association of vinc-CO with cell–cell
Among recruited components of the subcomplex are proteins not junctions in the absence of talin is consistent with an interaction
known to be vinculin ligands (for example, ILK), suggesting a with α- or β-catenin, but the aggregate pathway appears to be
combinationofdirectandindirectrecruitmentevents. mediatedexclusivelythroughtalin.
To define the minimal vinculin fragment capable of forming We then mapped the regions of talin required for vinc-CO to
aggregates,weexaminedthedistributionoftruncatedvinculins(see induceaggregates.Theheaddomainoftalininteractswithintegrins
diagramsinFig.2)innewlyhatchedL1larvae.Likevinc-CO,vinc- and the membrane, and its rod domain is divided into R1–R13
Head formed aggregates (arrow in Fig. 3H). In contrast, tail domains containing 11 VBSs and a C-terminal dimerisation helix
constructs did not (Fig. 3I,J), ruling out acontribution of tail–tail (diagram in Fig. 4I; Critchley, 2009). We utilised a series of talin
dimerisationtoaggregation.TheD1domainalsoformedaggregates deletionmutations(Klapholzetal.,2015),andassayedthecapacity
(Fig. 3K). Of the non-lethal constructs, only vinc-D1184 formed ofvinc-COtomakeaggregatesinclonesofcellsthatexpressonly
cytoplasmic aggregates (Fig. 3L–Q). However, vinc-D1184 thetruncatedtalin.Removingtheheaddidnoteliminateaggregates
aggregates did not recruit talin or paxillin (Fig. S3G–H′), (Fig. 4F,I), showing that the rod alone is a sufficient platform for
suggesting that they are distinct from those formed by other aggregate formation. We next investigated how much of the rod
constructs,nordidtheycolocalisewithlysosomes(Fig.S3I,I′).As domainwasrequired.Thesmallestdeletion,talin2509,retainsallof
vinc-D1184 contains only half of the second four-helical bundle itsVBSsbuthasthedimerisationdomainpartlydeleted(Klapholz
domain in D1, the exposed hydrophobic surface might promote etal.,2015). Thiscausedareduction inthenumberofaggregates
aggregationthroughhomo-oligomerisation.TherecruitmentofD1 formedbyvinc-CO(Fig.4I;Fig.S4C),presumablycausedbythe
subfragments to the MAS indicates that they still bind adhesion reduction of 22 to 11 VBSs following loss of dimerisation.
proteins,andthusarelikelytobefoldedproperly.Wethusdefine Decreasing the rod length with progressively larger deletions
D1 as the minimal domain of vinculin capable of forming resultedinacorrelateddecreaseinthenumberofaggregatespercell
cytoplasmic aggregates of adhesion proteins, in addition to being (Fig.4G,I).However,C-terminaldeletionscontainingonlythefirst
theminimallethaldomain. twoVBSs,talin759,orjustthefirst,talin646,hadthesameeffectas
thenull:analmostcompletelossofaggregates(Fig.4H,I).Thus,a
Activevinculinbypassesintegrinstoformaggregates talinwithoneortwoVBSsintherodisnotasufficientplatformfor
throughthetalinrod aggregates. The next truncation, talin865, which has four VBSs,
The formation of cell–matrix adhesions involves integrin supportsaggregateformation(Fig.4I),albeittoalowlevel.Totest
recruitment of cytoplasmic adhesion proteins. Although the vinc- whether the N-terminal region of the rod specifically is necessary
COaggregatesdidnotcontainintegrins,integrinsmightstillplaya foraggregation,wegeneratedacomplementaryconstruct,talinΔ486-
role in seeding the complexes. We tested this by genetically 1937, which retains three C-terminal VBSs and the dimerisation
removingintegrins,andfoundnoeffectonaggregateformation:in helix. This supported aggregate formation to the same extent as
integrin mutant embryos (mysXG43, a null allele of the gene talin865 (Fig. 4I), showing that no single region of the rod is
encoding the βPS integrin subunit; orthologous to the β1 integrin required. Thus, the rod domain of talin is required for aggregate
subunit), vinc-CO still formed aggregates in the detached and formation,andmultiplesitesalongtherodappeartocontributeto
rounded up muscles (Fig. 4A). Furthermore, vinc-CO was still theprocess.
capable of recruiting talin and paxillin in the absence of integrins Consideringtalinwasrequiredforvinc-COtomakeaggregates,
(Fig. 4B). Thus, surprisingly, vinc-CO can induce adhesion weinvestigatedwhetherthisrequiredinteractionbetweentalinand
subcomplexes without integrins. This suggests that vinculin is the talin-binding site in vinculin. To outcompete binding to
e
providing an adhesion complex trigger that functions in parallel endogenous talin, we coexpressed VBSs, α-helices from the rod c
n
withintegrins,and,sincevinculinisnotneededtomakeadhesions, domainoftalinthatbindtotheheadofvinculinwithhighaffinity e
i
itmustbeactingredundantlywithintegrins. (Bois et al., 2006). We designed GFP- or mCherry-tagged UAS c
S
To further define the molecular requirements for aggregate constructs for VBS1 and VBS2 (helices 4 and 12, respectively; l
l
formation,weexaminedadifferentcelltype,thefolliclecellsofthe Gingras et al., 2005), and used a previously generated construct e
C
ovary,sothatwecouldusetheMARCMtechnique(LeeandLuo, encoding four helices of the R11 bundle (helices 47–50) that
f
2001) to generate clones of cells which lack a given adhesion containaVBS(helix50),knownasGFP–IBS2(Ellisetal.,2011). o
proteinandexpressvinc-CO.Vinc-COformedaggregatesinfollicle We observed an almost complete suppression of aggregates when al
n
cellswhenexpressedinwild-typecells(Fig.S2G).Aswefoundin vinc-CO was coexpressed with VBSs in embryonic muscles
r
theembryo,vinc-COformedaggregatesintheabsenceofintegrins (Fig. 4J–M). This result confirms the talin646 result (Fig. 4G) in u
o
(Fig.4C),althoughthenumberofaggregatespercellwasdecreased demonstrating that a single VBS is not a sufficient platform for J
4358
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
A BB C D Fig.4. Thetalinroddomainisrequiredfor
aggregateformation.(A)Integrinmutant
(mysXG43)embryoexpressingvinc-COinthe
M muscles,withactindetectedbyphalloidin.The
talin 4.35 C arrowshowsaggregates.(B)GFP–talinand
R
A paxillin–GFPinmysXG43embryosexpressing
M
vinc-CO;arrowsshowrecruitmentto
aggregates;theaverageenrichmentofprotein
myXsG43 actin vinc-CO mysXG43 vinc-CO Pax∆1 inaggregatesascomparedtocytoplasmis
paxillin 3.31
E F G H givenbelowtheimage.(C)MARCMcloneinthe
s follicularepithelium,mutantforβPSintegrinand
e
el expressingvinc-CO,vinc-COchannelshown.
all (D)MARCMcloneofcellsmutantforpaxillinand
a) expressingvinc-CO.(E–H)MARCMclones
e
h expressingvinc-COintalinmutantbackgrounds
rn ( indicatedinthepanel.(I)Numberofaggregates
ali percellcountedforMARCMclonesmutantfor
t 62 ∆Head 1968 646 integrin,paxillinortalin(variousalleles).The
insetshowstalindomainstructurewithVBS
I 100 Head Rod VBSs R7 dimerization helicesinred,thedimerisationhelixinpink,and
talin R1 R2 R3 R4 R5 R6 R8 R9 R10 R11 R12 R13 helix positionofthemutanttruncations.Significance
6 calculatedasinTableS1,*P≤0.05;ns,not
ell 80 R significant.Boxes,whiskersandcirclesareas
c
per 62 46 5965 ∆486-1937 68 09 dgeesncortiybpeed:iWnTF=ig3.91;.mNyusmXGbe43r=o5f4c;ePllsaxcΔo1u=n2t3e;dtapleinr
gates 60 6 78 19 25 a1l9le6l8e=s7:56,22=57009,=66446,=Δ6458,67-5197=3593=,5806,5Δ=H4e2a,d=38.
e
gr Overfourclonesinatleastfourindividualegg
g chamberspergenotype.(J–M)Vinc-CO
a
O 40 expressedonitsown(J),withonecopyof
nc-C * VorBoSn1e–cGoFpPy(oKf)G,tFwPo–cIBoSpi2es(Mo)f;VthBeSv2i–nGc-FCPO(L),
vi ns
of 20 ns channelisshown.Arrowsshowscytoplasmic
er aggregates;arrowheadsshowMAS
b localisation.Scalebars:10µm.
m
u
N 0
wild mysXG43 Pax∆1 62 646 759 865 1968 2509 ∆486- ∆Head
type 1739
talin (rhea) alleles
J K L M
UAS vinc-CO UAS vinc-CO UAS vinc-CO
UAS vinc-CO
+VBS1 +VBS2 +IBS2
vinc-COtomakeaggregates.Wefoundasimilarlossofaggregates proteins to the mitochondria with the C-terminal outer membrane
whenvinc-CO–GFPwascoexpressedwithmCherry-taggedVBS1 anchor from Listeria monocytogenes ActA (Pistor et al., 1994;
(Fig.S4D,E).Finally,vinc-Headandvinc-D1aggregateswerealso Bubecket al., 1997; Cohen et al., 2006), and testing whether they
lostuponVBScoexpression(Fig.S4F–I). could recruit talin. Most targeted vinculins formed discrete
Takentogether,theseresultssuggestthatVBSbindingisrequired aggregates on the mitochondria (Fig. 5A), whetheror not the non-
e
forvinc-COtomakeaggregates.Althoughthereareotherproteins targeted forms formed cytoplasmic aggregates, except for vinc- c
n
that contain VBSs, considering the loss of aggregates upon talin D1152-mito, which therefore was not used. While the lethal, e
i
removal,wefavourtalinasthekeybindingpartner. aggregate-forming vinculins recruited talin to the mitochondria c
S
(Fig.5B,B′,E),vinc-FL,vinc-D1184andvinc-D1127didnot(Fig.5C, l
Aggregateformationcorrespondswithabilityofvinculinto C′,E). Thus, the ability to form cytoplasmic aggregates with talin el
C
bindcytoplasmictalin correlateswithrecruitmentofcytoplasmictalintothemitochondria.
f
Our results suggest that vinc-CO binds to talin rod through its D1 Totestwhetherforce-dependentopeningoftalinwassufficient o
domain to form aggregates. Neither full-length vinculin nor D1 forittobindtoclosedvinculin,weexaminedwhetheranisolated al
truncationsformedtalin-containingaggregates,suggestingthatthey VBS, mimicking open talin, could bind to closed vinculin. GFP– n
r
cannotbindtalininthecytoplasm.Toinvestigatethisfurther,weused VBS1wasstronglyrecruitedtovinc-FL-mito(Fig.5D,E),and,as u
o
an alternative approach to assess talin binding, targeting vinculin expected, to open vinc-CO-mito (Fig. 5E). Thus, in this system, J
4359
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
A B C D Fig.5.Hyperactivevinculincanrecruittalintoan
ectopiclocation.(A,A′)CoexpressionofGFPwitha
mitochondrialimportsignal(mito-GFP)andvinc-D1127-
RFP-mito;thearrowshowsvinc-D1127-RFP-mito
aggregatesonthemitochondrialsurface.(B,B′)Expression
ofvinc-FL-RFP-mitoinaGFP–talinbackground;thearrow
showsfailureoftalinrecruitment.(C,C′)Expressionofvinc-
mito-GFP D1127-mito talin FL-mito talin D1-mito VBS1 FL-mito D1-RFP-mitoinaGFP–talinbackground;thearrowshows
A’ B’ C’ D’ talinrecruitment.(D,D′)Coexpressionofvinc-FL-RFP-mito
withGFP–VBS1;thearrowshowsGFP–VBS1recruitment.
(E)EnrichmentofGFP–talinorGFP–VBS1in
mitochondrialaggregatescomparedtobackground
cytoplasm,calculatedasinFig.3,withstandarddeviation
below.n=40measurementsperembryo,in4–5embryos
pergenotype.Scalebars:10µm.
D1127-mito talin talin VBS1
E
FL-mito C O-mito Head-mito D1-mito D1184-mito D1127-mito
1.1 3.4 3.0 4.6 1.1 1.1
talin
±0.2 ±1 ±0.8 ±1.5 ±0.2 ±0.1
VBS1 2.1 2.6
±0.5 0.8
bindingbetweenvinculinandtalinrequiresonlyonetobeactivated, activitiesoftheheaddomaininvivo.Giventhedifferencebetween
eitherbyexposureofatalinVBSorlossofvinculinautoinhibition. vinc-Head and vinc-D1,weanalysed whether the rest of the head
perturbed muscle development, but neither UAS-vinc-HeadΔD1-
Developmentaldefectsofhyperactivevinculin–decoupling RFP (localised to MASs) nor UAS-vinc-HeadΔD1-RFP-mito (in
aggregatesfromlethality aggregatesonmitochondria)causedmuscledefects(Fig.6B).The
Our data suggest that hyperactive vinculin generates aggregates wholeheadisthustheminimaldomaincapableofcausingmuscle
through the rod of talin and that avinculin–talin platform recruits phenotypes.Importantly,thesedatashowthataggregatesalonedo
othercytoplasmicproteins.Ashyperactivevinculinisalsorecruited notcorrelatewithmuscledefects.However,vinc-Headaggregates
to adhesions (Fig. 3B), it might exert its negative effects on could be different to those produced by vinc-D1. We therefore
developmentineitheroftheselocationsorboth.Weexaminedthe examinedrecruitmentoftalin,paxillinandILK,andfoundthatthe
relativecontributionofthetwopools,firstlookingattheeffectson levelsofpaxillinandILKwerehigherinthevinc-Headaggregates
muscle development. Expression of vinc-CO with Mef2::Gal4 comparedtothevinc-D1aggregates(Fig.6D).Asthefullheadis
caused a range of phenotypes, including misshapen muscles, moreeffectiveatsequestration,differencesinrecruitmentcapacity
misplaced muscles, gaps in the muscle pattern and ectopic to the aggregates might explain its muscle phenotypes.
attachments(Fig.6A,quantifiedinFig.6B;comparetowild-type Alternatively, it could have additional activities elsewhere in the
musclesinFig.1A),whereasexpressionofvinc-FLdidnotcause cell,suchasattheadhesionsites.
these defects (Fig. 6B). Phenotypes were suppressed when VBSs Finally,weinvestigatedwhetherlethalityofhyperactivevinculin
were coexpressed (Fig. 6B), indicating that binding to talin, or requiredaggregateformation.CoexpressionofVBSssuppressedthe
anotherVBS-containingprotein,mediatestheeffect. lethality of hyperactive vinculin (Fig. 6E), suggesting that
We wondered whether sequestration of proteins by vinc-CO interaction with talin (or another VBS-containing protein)
aggregates caused the muscle phenotypes. To test whether it is mediates lethality. Gallus vinc-D1 lethality was also suppressed
sequestrationoftalinandpaxillin,wecoexpressedthemalongwith byDrosophilaVBSs(Fig.6E),indicatingacommonmechanismof
hyperactive vinculin, but this did not suppress the muscle lethality.VBScoexpressionresultedinlossofGallusvinc-D1MAS
phenotypes (Fig. 6B) nor alter aggregate formation (Fig. S4J,K). localisation(Fig.6F,G),suggestingitisrecruitedbybindingaVBS.
Furthermore,levelsoftalinandpaxillinatMASswerenotreduced However,whenexpressedonitsown,Gallusvinc-D1didnotform
when vinc-CO was expressed (Fig. 6C; Fig. S4L–M). Thus, cytoplasmicaggregates(Fig.6F).Thus,aggregatesarenotrequired
sequestrationoftheseadhesionproteinsatleastdoesnotexplainthe tocauselethality.AsalllethalconstructswerelocalisedatMASs,it
e
muscle phenotypes, consistent with the defects not resembling seemslikelythatitistheiractionattheadhesionthatcauseslethality, c
n
typical integrin loss-of-function phenotypes such as muscle butwecannotruleoutactivityelsewhereinthecell.Thus,wecan e
i
detachment (seeFig.4A)oractindetachmentfromthemembrane distinguishtwoseparatedetrimentalactivitieswithinvinc-CO:one c
S
(Zervas et al., 2001). Either the aggregates sequester proteins causingmuscledefects,whichrequiresitswholeheaddomain,anda l
l
involved in other muscle developmental processes, or vinc-CO secondcausinglethality,whichisperformedbyitsD1domain. e
C
directlyimpairstheseprocesses,inaggregatesorattheadhesion.
f
AsVBSssuppressedbothmusclephenotypesandaggregates,we DISCUSSION o
expected the minimal aggregate-forming domain, D1, to induce Whereasfliescantoleratethelossofvinculin,wehavediscovered al
n
muscle phenotypes. However, although vinc-Head caused that excessive vinculin activity is lethal, and causes defects in
r
equivalent muscle defects to vinc-CO, vinc-D1 had only minor muscle development. Both of these deleterious effects appear to u
o
effects(Fig.6B).Thus,theD1domaindoesnotaccountforallthe requirebindingaVBS-containingproteinsuchastalin.Talinisalso J
4360
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
A Fig.6.Theeffectsofhyperactivevinculinonmuscledevelopmentand
viability.(A)Defectsinmusclemorphologyfollowingvinc-COexpression,as
revealedbymyosinandintegrinstaining;arrowsshowchangesinmuscle
pattern,thearrowheadshowsectopicattachment.(B)Quantificationofmuscle
defectsresultingfromexpressingdifferentvinculinconstructs,shownas
percentageofsegmentswithxnumberofdefectsascolourcoded.Numberof
segmentscounted:control=23,FL=40,CO=25,CO+IBS2=24,CO+talin=34,
CO+paxillin=32,Head=28,D1=42,HeadΔD1=32,HeadΔD1-mito=34.Stage16
embryos,≥5embryospergenotype.(C)Effectofvinc-COexpressiononlevels
oftalinandpaxillinattheMAS,shownasaveragefluorescenceintensitiesfor
Vinc-CO out there
individualMASscomparedtothewild-typeaverage.Boxes,whiskersand
myosin integrin
myosin integrin circlesareasdescribedinFig.1.Darkblue,wild-typeindividualMASs,toshow
B variation;paleblue,levelsinthepresenceofvinc-CO.n=34(talin)and26
(paxillin)measurementsinfournewlyhatchedL1larvae.ns,notsignificant
s100% (Student’st-test).(D)Levelofrecruitmentoftalin,paxillinandILKtovinc-Head
with defect 6800%% >43 5 gvdeeernvsioauttsyiopvnein.bc*e-*Dl*o*1wP.c≤yn0t=o.04p00la0ms1me(Saicstuaudgreegmnrete’gsnatt-tsetesps,etc)r.ae(lmEcu)blRaryteeosd,cianuse4i–on5fFleeigtmh.ab3lr,iytwyoisbthypsetrandard
nts 40% 2 coexpressionofVBSswithlethalvinculinproteins.Viabilityisshownas
e
% segm 200%% 10 pGineaprlc4ae>renUntaAthgSeesfoleiebsss.et(oFrv)tehNdeeicrwoblmyalhpaaantrcceehdrestdoibLeli1xnpglaesrc.vtTaehede,ecnxauplmrceubslesairtneogdfGfbliyeaslcluocsmovupinnatcrei-ndDgi1signivtheen
Control FL CO CO+IBS2 CO+talin CO+paxillinCO+pax Head D1 ∆D1HeadD1 Head ∆D1Headd D1 Mito -mito mchoiguehsxclpiglerhests.ssTtinhhgeeGraeradroluluwcstisovhninoicwn-sDMM1AAwSSitehlnoGrcicFahlPims–aeItBinoStn.2.S(icnGa)tlheNebemawrulsys:ch1la0etscµ;hmteh.deLa1rrolawrvae
C D ea
H
wild type Head D1 associated proteins do not induce lethality in Drosophila [for
Protein level at MAS compared to wild type 1.15 n+s vinc-COns paxGIilLlFiKnP---GGtaFFliPPn ±R±±332110F......654527P**n****s**±±±R312001F......752771P eaca2axnadu0uadht1omse2ieIsn)pLi.hollKeeinTb,thkith(taaZieilnloieinantrlys,ve(etTahwb(saaGuhnletierettannytbathzblti.ohaes,fpee2fdt0vweoa1itieln1al.sdc,l).u2-],t.nl0y2io0nTp0t4we0)r6soefa)eol,neiiretdnmmedtnpesagigoirsrnenvinn(ionT-evaaroe(saVrrslregoeaxldctkepoiuraarcleetoDettsigdsaorellnoo.dp,su:r2oooe0pftftoe0haic4iinllat)a.ssl,,
talin paxillin adhesionproteins:disruptionoftalinautoinhibitionhasmildeffects
F
E (Ellisetal.,2013),andwhereasexpressionoftensinfragmentsdoes
1
D cause some phenotypes (Torgler et al., 2004), expression of
% Observed/expected (n) c-
n fragments of other adhesion proteins does not (see above
Mef2::Gal4 Mef2::Gal4 Mef2::Gal4 s vi references). The severeeffects ofhyperactivevinculin fit with the
+mChVBS1 +mChVBS2 +GFP-IBS2 u
all very strong intermolecular interactions that keep it in the closed
CO 99 97 119 G
(307) (283) (378) state.Activatingmutationsofvinculinhavenot,tothebestofour
Head 96(495) 79 (322) 114(366) G knowledge,beenreportedinthehumanpopulation,asexpectedif,
D1 99(249) 89(186) 100(58) D1 2 asinflies,theycausedominantlethality.
GaDll1us 72(368) 80(259) 119(167) s vinc-FP-IBS ledExtopredsesvinelgopvminecn-CtaOl odrefveicntcs-.HTehadesientchoeudldevaerliospeinagsmausrecsuulalttuoref
allu+G hyperactivevinculin in theaggregates orattheadhesionsite. The
G defects are distinct from integrin loss phenotypes, and this might
reflect recruitment of additional proteins contributing to muscle
formationtotheaggregatesortheadhesion.Cytoskeletalmachinery
required for a new role of Drosophila vinculin: inducing the iscrucialformusclefusionandmusclepathfindingtotendoncell
formation of cytoplasmic aggregates that are adhesion targets (Maartens and Brown, 2015b), and an interaction between
subcomplexes. These subcomplexes are not linked to integrins or hyperactive vinculin complexes and more general cytoskeletal
thecytoskeleton,anddemonstratethatadhesionproteincomplexes factors might explain the muscle defects. Sequestration of Z-disc
canformwithoutanyinputfromintegrin. proteins to ectopic intracellular aggregates has been implicated in
Flieslackingvinculindisplayeddefectsintheadultmusculature, the muscle phenotypes associated with myofibrillar myopathy
e
similar to the mild defects in larval musculature reported by (Rupareliaetal.,2016),andasimilareffectmaybestimulatedby c
n
Bharadwaj et al., (2013). Other tissues appeared normal, and our hyperactivevinculin. e
i
attemptstoidentifyadditionalimpairmentintheathleticabilitiesof Cytoplasmic aggregate formation appears to be unique to c
S
theflieswerenotsuccessful,sotheflyphenotyperemainsweaker Drosophila vinculin. Gallus vinc-D1 did not generate cytoplasmic l
l
than the phenotypes observed in mice, zebrafish or nematodes aggregates,eventhoughitappearedtointeractwithDrosophilatalin e
C
lacking vinculin (Barstead and Waterson, 1991; Xu et al., 1998a; (coexpressing Drosophila VBSs blocked its recruitment and
f
Chengetal.,2016).Redundancywithotheradhesionproteins(such lethality).Invertebratecellculture,hyperactivevinculinisrecruited o
as talin; Klapholz et al., 2015) might explain the relatively mild to integrin adhesions at the membrane (Cohen et al., 2005; al
n
phenotypeofthishighlyconservedprotein. Humphriesetal.,2007;Cariseyetal.,2013),butcytoplasmictalin-
r
The contrast between the consequences of loss and gain of containing aggregates have not been reported. The interaction u
o
vinculin activity are striking. In general, overexpressed integrin- between vertebrate vinc-Head and talin rod in vitro requires prior J
4361
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
stretchingoftherodbyforce(delRioetal.,2009;Ciobanasuetal.,
2014;Yaoetal.,2014),consistentwiththeinvivointeractionrelying vinculin
?
onpriortalinstretchingattheadhesion.Incontrast,ourmitochondrial
targetingexperimentsindicatethatactivatedDrosophilavinculincan
bindtoun-stretchedtalininthecytoplasm.Apredictionfromthese
results is that vertebrate vinculin should not recruit talin to the
mitochondria, whether active or not. However, in vertebrate cells,
talinisrecruitedbymitochondriallytargetedvinc-CO(Cohenetal., actin actin
2006) and even full-length vinculin (albeit very weakly; Bubeck
mimicked by
etal.,1997).However,inthesecasestargetedvinculinconstructspull
Δtail or T12
themitochondriatothemembrane,sothatvinculinandtalinbecome mimicked by
associatedwithintegrinsandactin(nosuchassociationwasfoundin a free VBS
our targeting experiments). Thus, it seems feasible that, in these
eaxdpheersiimonenstsit,et,heraatshseorciathtiaonnowfivthincc-yCtoOpliasswmiitchsttarleitnchaesdtoalcicnuarstthine actin integrin
force
Drosophila.
Several lines of evidence show that hyperactive Drosophila
vinculinformedaggregatesbybindingtocytoplasmictalin.Inthe
absenceoftalin,noaggregateswereformed,andtherodoftalinwas
talin
a sufficient platform for aggregation, with longer sections integrin
supporting more aggregates, presumably due to an increase in the
number of VBSs available per talin molecule. VBS coexpression Fig.7.ModelfortheinteractionbetweenvinculinandtalininDrosophila.
blocked aggregate formation, suggesting that direct binding Interactionbetweenvinculinandtalincanbedriveneitherbylossofvinculin
betweenvinculinandtalinwasimportant,andindeedtheminimal autoinhibition,orbyexposureofVBSsintherodoftalin.Bundlesoftalinrodin
orange,andvinculininblue,basedonstructuresofhumanproteins(lightblue
vinculin fragment capable of forming aggregates was the talin-
bundle,vinculintaildomain;dottedbundle,singlebundlepresentDrosophila
binding D1 domain. Hyperactive, but not wild-type, vinculin was
vinculininsteadofthetwobundlesinhumanvinculin).
capable of recruiting talin to the mitochondrial surface. Vinc-CO
recruitmentoftalintotheaggregateswasnotalteredbythelossof
integrins, ruling out an alternative hypothesis whereby an initial morphogeneticevents.Elevatedintegrinexpressionhasbeenshown
stretchingoftalinattheadhesionisafirststepintheformationofthe to hindercell migration in the Drosophila ovary (Lewellyn et al.,
cytoplasmicaggregates. 2013), and vinculin stimulation of integrin activation might affect
An interesting feature of the vinculin–talin interaction is its similarprocesses.Thelethalitycausedbyvinc-D1constructsoccurs
reciprocity:justashyperactivevinculinappearstobindclosedtalin, without defects in muscle morphogenesis. Assessing whether the
isolated VBSs can bind closed vinculin on the mitochondria, muscle defects of vinc-Head and vinc-CO also contribute to
consistentwiththecapacityofvertebrateVBSstodislodgethehead lethality would requireamethod to block thelethalityof vinc-D1
fromthetailinvitro(Boisetal.,2006).Thus,theinteractionbetween withoutimpairingthemusclephenotypesofvinc-Headorvinc-CO,
vinculinandtalininDrosophilaneedonlyrequireactivationofone whichwecurrentlylack.
partner(Fig.7).Anopenquestioniswhethertherearenormalsignals, AlthoughwehaveonlyexaminedvinculinD1fromtwospecies,
mimicked by the ‘T12’ mutation, that open Drosophilavinculin so wespeculatethatvertebratevinculinhaslosttheabilitytobindto
that it can force talin into an extended conformation. Recently, closed talin, and might have become more tightly closed by the
Athertonetal.,(2015)foundthatexpressionofamutanttalinwitha addition of an eighth four-helix bundle that occurred during the
deletionofdomainsR2–R3,which containfourVBSs,causesvery evolution of the deuterostome lineage (our unpublished analysis).
similareffectsto expressing vinc-CO. Binding of activated-vinculin Thus, vertebrate cells might be even more sensitive to the
thusalleviatessomeformofinternalnegativeregulationwithintalin, consequencesofaberrantassociationbetweenvinculinandtalin.
whichmightinpartbeduetoregulationofthecentralactin-binding Ourresultssuggestthatcertainproteinshavetheabilitytoactasa
domainencompassingR4–R8(Athertonetal.,2015). switch, triggering assembly of an integrin adhesion complex.
Gallus vinc-D1 demonstrated that hyperactive vinculin could Integrins are well known to have this switch ability: engagement
inducelethalityinDrosophilawithoutformingaggregates.Howit withtheECMandclusteringtriggerstheformationofadhesionsites
doessoremainsanopenquestion,butwefavourtheideathatitis (Miyamoto et al., 1995). When Drosophila vinculin loses
caused by the action of the D1 domain of vinculin on talin at the autoinhibition, it triggers the assembly of an adhesion complex,
adhesion sites. The vinculin head stabilises talin into a stretched and this process can occur entirely independently of integrins. In
e
conformationincellsandinvitro(Margadantetal.,2011;Yaoetal., contrast to integrins, however, the full complement of adhesion c
n
2014),andthisreliesonpriorstretchingoftalin(Ciobanasuetal., proteinsisnotrecruited,suggestingthatadditionalmechanismsare e
i
2014;Yaoetal.,2014).Furthermore,vinculinisrequiredfortalinto required(forinstance,membraneproximity,applicationofforce,or c
S
extend fullyaway from the plasma membrane (Case, et al., 2015; signalling).Thisraisesthequestionofhowtheadditionalproteinsare l
l
Klapholzetal.,2015).Thus,lethalitycouldarisefromhyperactive recruited to the cytoplasmic aggregates, and whether the pathways e
C
vinculin binding to stretched talin and the failure of vinculin to involvedaresimilartothoseutilisedbyconstitutivelyactivevinculin
f
releasewhen force is reduced. Cycles of stretching and relaxation atadhesions(Cariseyetal.,2013)andbyintegrinsandtalininnormal o
might be crucial for normal talin function or relaxation of talin adhesions.Recruitmentrequirestalin,buttherelativecontributionsof al
n
might be required for its dynamic turnover. Alternatively, vinculinandtalinhaveyettobeestablished.
r
hyperactive vinculin might stimulate too much adhesion, Integrin-independent interactionsofadhesion proteinshave been u
o
stabilising integrin adhesions and reducing turnover in dynamic demonstratedbyfluorescencecorrelationanalysiswhereinadhesion J
4362
RESEARCHARTICLE JournalofCellScience(2016)129,4354-4365doi:10.1242/jcs.189878
components self-assembled inthe cytosol(Hoffmannet al., 2014). constructionofendogenoustaggedvinculinconstructs.TogenerateUAS
However, these ‘building blocks’ were composed of three or four controlledwild-typevinculinconstructs(UAS::vinc-FL),theVinccoding
protein species, never assembled into larger structures and did not sequencewasclonedintoUASpwithC-terminalGFPorTagRFPtags.To
include a talin–vinculin interaction. Nevertheless, the above work generate constitutively open vinculin (UAS vinc-CO), the ‘T12’ set of
charge-to-alaninemutations(DRRIR>AAAIA,aminoacids869–873;asin
showshowinteractionsbetweenthecomponentpartsoftheadhesion
Cohen et al., 2005) were introduced into the tail of genomic full-length
neednotnecessarilyrelyonadirectorevenindirectlinktointegrins,
vinculinandclonedintotheUASpvectorwithaC-terminalGFPorTagRFP
consistentwithourwork.Akeyroleofintegrinsmightbetotrigger
tag.BothUAS::vinc-FLandUAS::vinc-COwereintegratedbyuntargeted
theassemblyofthecytoplasmicadhesion-complex-specificsitesin
P-element insertion, and insertions with good expression levels were
themembrane,ratherthanbeinganecessarypartofthislink. selectedforanalysis.Togeneratevinculintruncationconstructs,therelevant
From an evolutionary perspective, certain cytoplasmic sequences were amplified by PCR from genomic DNA, and cloned into
componentslikevinculinandtalinpredatetheintegrins(Kreitmeier UASpAttPvector,andallinsertedintothegenomeinthesameposition
etal.,1995;Sebe-Pedrosetal.,2010).Itistemptingtoproposethat (landingsiteonchromsome2R:51D).Thecoordinatesoftheseconstructs
integrinsco-optedpre-existingcytoplasmiccomplexes,usingthemto inthegenomicDNA(where1=ATG)are:vinc-FL/CO,1–3135;vinc-Head,
strengthentheiradhesiontotheECMatdiscretesitesalongthecell 1–2172; vinc-D1, 1–771; vinc-D1184, 1–552; vinc-D1152, 1–456; vinc-
D1127, 1–381; vinc-D1128-257, 381–771; vinc-HeadΔD1, 769–2172; vinc-
surface. This evolutionary change may also have required
FLΔD1, 769–3135; vinc-Neck+Tail, 2176–3135; vinc-Tail, 2423–3135.
mechanisms to restrict the spontaneous formation of adhesion-like
Gallusgallusvinc-D1–RFP(aminoacids1–258)wasgeneratedsimilarly
complexes in the cytoplasm. The strong head–tail interaction of
from a cDNA clone (Carisey et al., 2013). To generate mitochondrially
vinculincouldbeonesuchmechanism.
targeted vinculin constructs, the C-terminal membrane anchor from the
Listeria ActA protein (Pistor et al., 1994) linked to TagRFP with a
MATERIALSANDMETHODS polyserine linker was synthesised (Mr Gene), and cloned with vinculin
Flygenetics fragmentsintotheUASpAttPvectorforinsertionintothe51Dlandingsite.
Drosophila melanogaster stocks usedin this study: Mef2::Gal4,Act5C:: TogeneratetalinVBSconstructs,weamplifiedsequencescodingforVBS1
Gal4, UAS::GFP-Lamp1, UAS::eGFP-huLC3, UAS::myr-GFP, UAS:: (helix4oftherod),VBS2(helix12oftherod)byPCR,andclonedthem
mito-GFP (all sourced from the Bloomington Drosophila Stock Center, intotheUASpvectorwithanN-terminalGFPormCherrytag.TheGFP–
Indiana University); ΔVinc, βPS-GFP, GFP-talin (all Klapholz et al., talinΔ486-1739genomicrescueconstructwasmadebythesamestrategyasfor
2015);mysXG43(Jannuzietal.,2002);paxΔ1(Batailleetal.,2010);talin- thewild-typeGFP-taggedconstructdescribedinKlapholzetal.(2015).
mCherry(Venkenetal.,2011);UAS::talin(Tanentzapfetal.,2006);UAS::
GFP-IBS2(Ellisetal.,2011);paxillin-GFP(Batailleetal.,2010);UAS:: Immunohistochemistryandimaging
paxillin(Vakaloglouetal.,2012);tensin-GFP(Torgleretal.,2004);ILK- Embryos,ovaries,wingsandadultflightmuscleswerestaged,fixed,stained
YFP(unpublished,madeasILK-GFPinZervasetal.,2001);PINCH-GFP andimagedusingstandardprotocols,withsodiumdeoxicholateaddedtothe
(Kadrmas et al., 2004); Fermitin1-GFP (a genomic fragment, −1579 to wash for the Rab protein stainings as in Gomez-Lamarca et al. (2015).
+3942relativetotheATG,withmGFP6insertedfollowingresidue228and Antibodies used were: paxillin (rabbit, 1:200; Chen et al., 2005); αPS2
with three-serine linkers on each side; a gift of Danelle Devenport, The integrin(rat,1:10;Bogaertetal.,1987);musclemyosinII(mouse,1:100;
Gurdon Institute, UniversityofCambridge, UK). To misexpress vinculin Kiehart et al., 1990); E-cadherin (rat, 1.100; Developmental Studies
constructs,theUAS-Gal4systemwasused(BrandandPerrimon,1993).To Hybridoma Bank, University of Iowa); CAP (rabbit, 1.1000; Bharadwaj
remove maternal and zygotic contributions of talin and paxillin, the etal.,2013);Rab5(rabbit,1:500;cat.no.ab31261,Abcam);Rab7(rabbit,
dominantfemalesteriletechniquewasused(ChouandPerrimon,1996).To 1:3000; Gomez-Lamarca, et al., 2015); Rab11 (rabbit, 1:8000; Gomez-
generatefolliclecellcloneslackingβPSandexpressingVinc-CO-RFP,the Lamarcaetal.,2015).PhalloidinconjugatedtoFITC,RhodamineandAlexa
MARCM system was used (Lee and Luo, 1999). hsFlp, Tub-Gal80ts, Fluor 647 (Life Technologies) was used at 1:50, 1:200 and 1:100,
FRT19A; Tub-Gal4, UAS cd8GFP/CyO flies were crossed to mysXG43, respectively, to image actin. Images were collected using an Olympus
FRT19A;UASVinc-CO-RFP/CyOflies,pupaewereheatshockedfor2hin Fluoview1000confocalmicroscopewitha60×1.35NAobjectivewith×1
a37°Cwaterbath,andnon-CyOadultfemalesselectedanddissected.rhea or×2zoom,andmeasurementsweretakenwithFiji(http://fiji.sc/Fiji).Data
and pax mutant clones expressing Vinc-CO were generated in the same was analysed and graphs produced using Microsoft Excel, Matlab and
mannerwithdifferentMARCMlinesforeachFRT(FRT2AandFRT40A, BoxPlotR (http://boxplot.tyerslab.com). For fluorescence intensity
respectively).Totestthedomainsoftalinrequiredforvinc-COaggregation, measurements, levels were modulated to avoid oversaturation, and the
weemployedaseriesofrheadeletionsaswellasanullallelerescuedby same laser intensities were used for each genotype or experimental
construct lacking the head or having the internal deletion in the rod condition.AggregatesandmuscledefectswerecountedmanuallyusingFiji.
(Klapholz et al., 2015). For controls, wild-type FRT chromosomes were
usedinplaceofmutantchromosomes.TogenerateFlp-outclones(Struhl
Acknowledgements
andBasler,1993)expressingVinc-COorVinc-Headinthefolliclecellsand WethankChristophBallestrem(UniversityofManchester)forchickenvinculin
wing, hsFlp; arm::FRTstopf+FRTGal4 (details available upon request) cDNA,AlexKolodkin(JohnsHopkinsSchoolofMedicine,Baltimore,MD)forCAP
flieswerecrossedtoUAS-vinc-XfliesandprogenyheatshockedeitherasL1 allelesandantibody,DanielKiehart(DukeUniversity,Durham,NC)formyosin
larvae(forclonesinthewing)orpupae(forclonesinthefollicle cells). antibody,SarahBray(UniversityofCambridge)forRabantibodies,andthe
Fluorescencefromthevinculinconstructswasusedtomarkclones. BloomingtonStockCenterforflystocks.WethankJohnOvertonandYuk-Sang e
Chan(DepartmentofGenetics,UniversityofCambridge)forflyinjections,Teresa c
EmmilieToudalKnudsenforhelpwithcloning,PaulFox(UniversityofCambridge) n
Larvalcrawlingassay e
Wanderingthird-instarlarvaewereplacedon2%agarplatesandallowed fmoremrebaedrinsgofthheismlaabnoursactorirpyt,foGruryeaTdainnegntthzeapmfa(UnunsivcerirpstitaynodfBgerintieshraCtinogluGmFbPia–)VaBnSd2, Sci
to acclimatise for 10s. 1min movies were captured at 30 frames per
andmembersoftheBrownlaboratoryforcomments. l
secondwithaJVCTKC1380cameramountedonaLeicaM8Dissecting el
microscope.Acustom-madeFijiscript(availablefromthecorresponding C
Competinginterests
author upon request) was used to track larvae and calculate average f
Theauthorsdeclarenocompetingorfinancialinterests. o
velocity.
l
a
Authorcontributions n
Generationoftaggedvinculinandtalinconstructs A.P.M.,J.W.,E.W.,B.K.andN.H.B.generatedconstructs;experimentswere ur
Generation of new Vinc alleles was achieved by imprecise P-element designedbyA.P.M.,J.W.andN.H.B.,andperformedbyA.P.M.,J.W.,E.W.and o
excision and is described in Klapholz et al. (2015) along with the H.G.;A.P.M.andN.H.B.wrotethepaper. J
4363