The structure and regulation ofvinculinWolfgang H. Ziegler1, Robert C. Liddington2 and David R. Critchley3

1 IZKF Leipzig, Faculty of Medicine, University of Leipzig, 04103 Leipzig, Germany2 Program on Cell Adhesion, The Burnham Institute, La Jolla, CA 92037, USA3 Department of Biochemistry, University of Leicester, Leicester, UK, LE2 7HD

Review TRENDS in Cell Biology Vol.16 No.9

Vinculin is a ubiquitously expressed actin-bindingprotein frequently used as a marker for both cell–celland cell–extracellular matrix (focal adhesion)adherens-type junctions, but its function has remainedelusive. Vinculin is made up of a globular head linked to atail domain by a short proline-rich sequence, and anintramolecular interaction between the head and tailmasks the numerous ligand-binding sites in the protein.Determination of the crystal structure of vinculin hasshed new light on the way that these ligand-bindingsites are regulated. The picture that emerges is one inwhich vinculin stabilizes focal adhesions and therebysuppresses cell migration, an effect that is relieved bytransient changes in the local concentrations of inositolphospholipids. However, the finding that vinculinmodulates the signalling pathways involved inapoptosis suggests that additional roles for vinculinremain to be discovered.

IntroductionVinculin is a 116 kDa actin-binding protein (1066amino acids) that is localized on the cytoplasmic face ofintegrin-mediated cell–extracellular matrix junctions(focal adhesions) and cadherin-mediated cell–celljunctions. Determination of the primary sequence ofvinculin, along with biochemical and electron microscope(EM) studies, show that vinculin is made up of a globularhead linked to a tail domain by a proline-rich region, andinteraction sites for numerous binding partners have beenmapped onto all three regions of the protein (Table 1) [1].Among the best characterized of these is talin, which isthought to couple the cytoplasmic domains of b-integrinsubunits to the actin cytoskeleton and is requiredfor integrin activation and focal adhesion assembly [2].Interestingly, all of the well characterized ligand-bindingsites in vinculin (including that for talin) are masked by anintramolecular interaction between the vinculin head andtail domains [3,4], and the molecule is thought to existin an equilibrium between active and inactivate states.Here, the activated state is used to describe a conformationin which the vinculin head–tail interaction has beendisengaged and all of the ligand-binding sites are exposed,although vinculin might exist in several differentconformational states.

Corresponding author: Critchley, D.R. (drc@le.ac.uk).Available online 8 August 2006.

www.sciencedirect.com 0962-8924/$ – see front matter � 2006 Elsevier Ltd. All rights reserve

Despite the extensive literature on vinculin, its preciserole in focal adhesions remains to be elucidated. Vinculinoverexpression reduces cell migration, whereas vinculindownregulation enhances cell motility. Moreover vinculin-null cells are less adherent, less well spread, are moremotile and have fewer and smaller focal adhesions thanwild-type cells, and signalling through both focal adhesionkinase (FAK) and paxillin are elevated [5,6], a featuretypical of motile cells. This might explain why vinculinbehaves as a tumour suppressor in model systems [7].

The recently determined crystal structures of full-length vinculin [8,9] now reveal how the intramolecularinteraction between the head domains (domains D1–D4)and tail domains (Vt, also called domain D5) inhibits theligand-binding sites in vinculin by steric and allostericmechanisms. Structures of the vinculin D1 domain boundto talin and a-actinin peptides show that these ligandsbind to a hydrophobic pocket in the D1 domain [10–12].Moreover, new data on talin show that it contains multiplevinculin-binding sites (VBSs), which are themselves buriedwithin the hydrophobic core of the helical bundles thatmake up the talin rod and therefore require activation [12–14]. The mechanism(s) underlying vinculin activation iscontroversial. As the head binds the tail with high affinity(Kd < 10�9 M) [8,15], vinculin might be activated by acombinatorial mechanism requiring at least two bindingpartners [8], but exposure of the high-affinity VBSs in talinor a-actinin might be sufficient [10,11,16] (see below).

Insights gained from the structure of vinculin are com-plemented by recent cellular and biochemical studies. Thefinding that the Arp2/3 complex, which drives the assem-bly of the dendritic actin networks in lamellipodia, tran-siently interacts with the proline-rich region in vinculinduring cell spreading offers a possible explanation for thespreading defects in vinculin-null cells [17]. Interestingly,vinexin-b, which also binds to the proline-rich region invinculin [18] and has been implicated in cell spreadingstimulated by epidermal growth factors (EGFs), is absentfrom the focal adhesions formed in vinculin-null cells [19].A role for tyrosine phosphorylation of vinculin in theregulation of cell spreading has also been suggested[20]. Vinculin has also been implicated in modulatingthe signalling pathways involved in apoptosis [6], andvinculin-null mouse F9 embryonal carcinoma cells areresistant to apoptosis (Box 1). Here, we focus on recentdata showing that (i) vinculin in focal adhesions doesindeed exist in an activated state, (ii) vinculin recruitment

d. doi:10.1016/j.tcb.2006.07.004

Table 1. Binding partners of vinculina

Ligand Vinculin

domain

Ligand site Regulation and function Methods Refs

Talin D1 Rod, up to 10 talin

VBSs [30]

Helix-bundle conversion, vinculin activation

[16]; FA strength, link to F-actin or

membrane

X-ray structure (D1/tVBS), Y2H,

PD, SPOT

[8,10,16,30,

54,72]

a-Actinin D1 Spectrin R4 [56]; CH

domain [58]

Helix-bundle conversion, vinculin activation

[16]; FA strength

X-ray structure (D1/aVBS),

cryoEM

[16,56,58,

73–75]

IpaA D1 n.d. Vinculin activation; bacterial entry, F-actin

depolymerization

OV, IP, ELISA [19,76,77]

a-Catenin D1–D3 aa 697–906 of a-

catenin; aa 326–509

of aE-catenin

Link to F-actin (?) [70], localization to cell–

cell junctions (adherens junctions);

PTEN stability (F9/g229 cells) [71]

OV, PD, SPR, ITC [8,69–71,

78–80]

b-Catenin D1–D3 n.d. Localization to cell–cell junctions, no

activation of F-actin binding

IP [70,78]

(intra.) D1 and D4 Vt (D5) Auto-inhibited cytoplasmic conformation,

release from FAs [35]

X-ray structure (vinculin), D5-

Ala-mutants, FRET, ITC, FRAP

[8,10,15,

19,35]

Vinexin PRR SH3–1/-2 Enhanced FA size and stress fibres,

promotes spreading; binds activated vinculin

Y2H, IP, SPR [18,19,81]

Ponsin PRR SH3–1/-2 IP, OV [25]

VASP PRR, FPPPP EVH1, EVH2 Link to F-actin IP, PD [23,24]

Arp 2/3 PRR,

P876/P878

n.d. Spreading, lamellipodial extension IP, PD [26]

Paxillin Vt (D5) LD1, LD2, LD4 Constitutive [82]; FAK/paxillin interaction

affects ERK activity, motility and apoptosis

(F9/g229 cells)

OV, PD [6,68,82,83]

F-actin Vt (D5) Two sites, separate

actin monomers

Actin filament bundling, vinculin

dimerization; link to actin cytoskeleton

Co-sedimentation,

D5-Ala-mutants, cryoEM

[15,27,28,31]

PKCa Vt (D5), C-

terminal arm

Regulatory domain D5 phosphorylation S1033/S1045;

PKCa recruitment

OV, IP; kinase assay [84,85]

Acidic

phospholipids

Vt (D5) n.d. Competition of F-actin binding;

FA turnover

PD, OV, bilayer insertion [24,34,62,86]

aAbbreviations: aa, amino acid; CH, calponin homology domain; cryoEM, cryo electron microscopy; ELISA, enzyme-linked immunosorbent assay; FA, focal adhesion;

FPPPP, PheProProProPro motif; FRET, Forster resonance energy transfer; IP, immunoprecipitation; ITC, isothermal titration calorimetry; LD, paxillin-LD motif;

n.d., not determined; OV, blot/gel overlay; PD, pull down; PRR, proline-rich region; SH3, Src homology domain 3; SPOT, SPOT-peptide assay;

SPR, surface plasmon resonance; Y2H, yeast-two-hybrid.

454 Review TRENDS in Cell Biology Vol.16 No.9

strengthens adhesions and (iii) vinculin suppresses focaladhesion turnover.

Vinculin structureThe crystal structure of full-length vinculin reveals afive-domain auto-inhibited conformation in which the tail(Vt) is grabbed in a pincer-like manner by domains D1–D3[8,9] (Figure 1a). The affinity between isolated D1 and Vt

Box 1. Vinculin modulates paxillin–FAK signalling and

apoptosis

Recent data suggest two new and unexpected roles for vinculin.

Vinculin-null mouse F9 embryonal carcinoma cells [47] are resistant

to caspase-3 activation triggered by serum withdrawal, treatment

with camptothecin (a cytotoxic anti-tumour drug that inhibits DNA-

topoisomerase I) or detachment from the extracellular matrix. The

cells can be resensitized by expressing full-length vinculin, but not

by vinculin with a Tyr822Phe mutation, although the significance of

phosphorylation of tyrosine 822 has not been established [6]. The

authors [6] noted that activity of the mitogen-activated protein

kinases ERK1 and ERK2 was significantly increased in the vinculin-

null cells, and this was correlated with suppression of the initiator

caspase-9. Inhibition of ERK resensitized the vinculin-null cells to

apoptosis. Interestingly, phosphorylation of FAK on Tyr397 and

paxillin on Tyr118 was increased in vinculin-null cells, and a vinculin

Vt polypeptide (residues 811–1066) suppressed both phosphoryla-

tion of FAK and paxillin and resensitized the vinculin-null cells to

apoptotic stimuli [6]. The vinculin and FAK binding sites in paxillin

partially overlap [68], and the authors [6] propose that Vt competes

with FAK for paxillin and that in the absence of vinculin, signalling of

FAK or paxillin through ERK is constitutive and sensitivity to

apoptosis is reduced [6].

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domains is relatively low (�10�6 M), but additionalcontacts between Vt and D4, together with the factthat the vinculin head is covalently linked to Vt by theproline-rich region, result in very tight head–tail binding(Kd < 10�9M) [8,15]. Studies with isolated domains haveidentified three distinct ligand-binding regions invinculin. Domain D1 contains binding sites for talin[21] and a-actinin [22]; so far, no specific ligand-bindingactivities have been shown for D2 or D3. The proline-rich region, which lies between D4 and Vt, binds toVASP [23,24], vinexin [18], ponsin [25] and the Arp2/3complex [26]. In the auto-inhibited conformation, thisregion is partly folded, and lies across the surface ofthe vinculin tail, obscuring binding sites within theproline-rich region as well as the binding sites forfilamentous (F)-actin [27,28] and phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) [8] on Vt (Figure 1b).

Recent studies have begun to reveal the nature ofvinculin regulation. Using crystal structures of vinculinD1 bound to peptide fragments of talin [10,29,30] and EMstructures of Vt bound to F-actin [31], together with bio-chemical and biophysical approaches, a model of vinculinactivation has been proposed [8,15,31]. Ligand binding isregulated both sterically and allosterically, and conforma-tional changes in the head, tail and proline-rich domainsare linked structurally and thermodynamically. The tightbinding between Vt and the head are thought to be toostrong for a single ligand to activate vinculin. This has ledto the proposal of a combinatorial pathway to activation inwhich vinculin is only activated at sites of cell adhesion

Figure 1. Structure of full-length vinculin and its complex with F-actin. (a) Crystal structure of vinculin in its auto-inhibited state, coloured by domain. Binding sites for

selected ligands are indicated in black. The vinculin tail (Vt) is held in place by contacts with domain D1 and domain D4. (b) The solvent accessible surface of Vt (grey) in the

context of the full-length molecule (magenta). Vt is surrounded on three sides by D1, D3 and D4. This organization inhibits the release of Vt from the vinculin head, sterically

blocking its interaction with F-actin. Contacts between Vt and D1 inhibit the conformational changes in D1 that would allow talin and a-actinin to bind (allosteric inhibition).

The ‘basic ladder’ and ‘basic collar’ are involved in binding PtdIns(4,5)P2. The ‘strap’ sterically hinders the exposure of the basic collar, and restrains the motion of the

proline-rich region, inhibiting binding of vinexin, ponsin and Arp2/3. The proline-rich region forms a loop connecting D4 and the strap. This loop is flexible and was

therefore not resolved in the crystal structure. (c) Solvent-accessible surface of two F-actin monomers within an F-actin filament, with two actin-binding sites, derived from

EM, highlighted in red. (d) Docked model of Vt (red) and two actin monomers (grey and green) making two contacts (‘top’ of Vt to ‘upper actin’ and ‘bottom’ of Vt to ‘lower

actin’. (e) Hypothetical structure of full-length vinculin bound to F-actin, showing the steric clashes between D1 and the upper site (blue); there are no clashes on the lower

site. This steric clash provides a structural basis for the low affinity of full-length vinculin for F-actin, whereas the accessibility of the lower site should allow full-length

vinculin to bind, at least weakly, to F-actin – this could be sufficient to cause the conformational change required for high-affinity binding. Reproduced with permission from

Refs. (a,b) [8] and (c–e) [31].

Box 2. An alternative concept of vinculin activation

Although the combinatorial model of vinculin activation [8] is

attractive, it has also been suggested that vinculin can be activated

by a single ligand, and the VBSs in talin and a-actinin (when

activated) have been proposed to be sufficient to drive vinculin

activation [10,11,16]. However, much of this original work was done

with isolated VBS peptides and fragments of vinculin head (D1) and

Vt, and the results can be explained by the fact that the D1–Vt

interaction between the fragments is �1000-fold weaker than that in

the full-length molecule [15], owing to additional contacts with D4

and the covalent linkage between the vinculin head and tail.

Bois et al. [16] also suggest that full-length vinculin binds to talin

VBS peptides with a high affinity. Using full-length vinculin

immobilized on a Biacore chip, the Kd of talin peptides were

calculated to be either 2 nM [22] or �70–80 nM [16]. Significantly,

the binding affinity between a VBS peptide (derived from a-actinin)

and the vinculin head was identical to that for binding to full-length

vinculin [22], which would appear to refute the existence of an

autoinhibited conformation. The concern here is that immobilization

of vinculin on or close to a solid substrate might act as a mild

denaturant and result in vinculin activation. Despite these reserva-

tions, the authors also demonstrated that both talin and a-actinin

VBS peptides stimulated the actin-binding activity of vinculin in

solution at molar ratios of >5:1 (VBS:vinculin) [16]. The results with

the talin VBS3 peptide are consistent with an earlier publication [54].

Further work will be required to establish the exact mechanisms of

vinculin activation.

Review TRENDS in Cell Biology Vol.16 No.9 455

when two or more of its binding partners are brought intoapposition. Although the combinatorial model of vinculinactivation is attractive, others have suggested that it canbe activated by a single ligand such as talin or a-actinin[10,11,16] (Box 2).

Given such a tight head–tail interaction, the sponta-neous off-rate of Vt from the head is likely to be very slowon the timescale of focal adhesion turnover, so a triggermust exist to open themolecule at least transiently to allowmultiple ligands to bind and achieve thermodynamic equi-librium. One possibility is suggested by the work of Jans-sen et al. [31], who recently showed that the actin-bindingsite on Vt comprises two distinct sub-sites, both of whichmust be engaged for high-affinity binding (Figure 1c,d).One binding site is exposed in the full-length auto-inhib-ited conformation, while the second is sterically occludedby the N-terminal domain of vinculin (Figure 1e). Thepartial accessibility of the binding site explains the struc-tural basis of affinity regulation for F-actin and also sug-gests how the binding of F-actin to the exposed site,although weak, can enhance the on-rate for the secondsub-site, providing a kinetic pathway towards achievingcombinatorial activation.

Binding of F-actin to the vinculin tail (once freed from itscontacts with the head) also promotes Vt dimerization andthereby actin cross-linking [32]. An atomic model for this

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456 Review TRENDS in Cell Biology Vol.16 No.9

interaction has been generated by fitting the crystalstructure onto difference maps derived from electrontomography [31]. The mode of dimerization is unexpectedbut results in a compelling model. Binding to F-actinactivates a cryptic dimerization potential by triggeringconformational changes within Vt [33], which seem toinvolve dislocation of the C-terminal arm from the baseof the Vt helical bundle [31]. This then allows dimerizationto occur and enables actin cross-linking.

Vinculin in focal adhesions is in an activatedconformationAlthough biochemical and now structural studies clearlyestablish that vinculin can be present in activated andauto-inhibited states, there has been no direct evidence toshow that these exist within the cell. Chen et al. [19] haverecently begun to investigate both when and where vincu-lin is activated using Forster resonance energy transfer(FRET). Positioning of the cyan and yellow fluorescentproteins (CFP and YFP) as a donor–acceptor pair at theN- and C-terminal ends of vinculin, which interact in theauto-inhibited conformation, did not generate a satisfac-tory FRET probe. The authors therefore evaluated a ‘tailprobe’ in which the YFP sequence was inserted within thevinculin molecule between the proline-rich region and Vt.Crucially, the introduction of CFP and YFP did not resultin activation of the tagged vinculin molecule, and the F-actin-binding sites remained in a low-affinity state. Onlyafter addition of the bacterial IpaA protein, which binds tothe vinculin D1 domain with high affinity [19], and relievesthe vinculin head–tail interaction, was F-actin bindingdetected. Concomitantly, a decrease in FRET ratio (1.48to 0.81) and efficiency (46% to 32%) of the probe wasobserved, establishing that the vinculin FRET ‘tail probe’reports on the conformational changes associated withactin binding [19]. When the probe was expressed in cells,the FRET signals observed provided the first direct evi-dence that vinculin in focal adhesions was in an activatedactin-bound conformation while the cytoplasmic pool wasin the auto-inhibited state [19]. The studies also showedthat activated vinculin was localized in those peripheraladhesions which turn over rapidly during cell spreading.Intriguingly, in gliding or dissolving focal adhesions, acti-vated vinculin was concentrated at the proximal edge ofthe adhesion, whereas, unexpectedly, only a fraction ofvinculin was seen in the activated state in stable or pro-truding adhesions [19]. This raises new questions about (i)how vinculin that is not in the active actin-bound confor-mation is localized to focal adhesions and (ii) the mechan-isms that activate and inactivate vinculin in focaladhesions.

Vinculin turnover is much more rapid than focaladhesion turnoverThe exchange rate of vinculin in stable focal adhesionshas been determined using green fluorescent protein(GFP)-tagged vinculin expressed in various cell lines,and fluorescence recovery after photo bleaching (FRAP).The measurements generally reflected conditions in whichthe release (koff) was time limiting and generated half-lifetimes (t1/2) around a minute [34,35] or 10 s [36,37]. This

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surprisingly fast turnover of vinculin is in line withthe kinetics observed for other cytoskeletal proteinsin adhesion sites, such as paxillin, zyxin and a-actinin[36–39]. The combinatorial model suggests that vinculinthat leaves the focal adhesion would revert to theauto-inhibited conformation and re-enter the solublecytoplasmic pool of inactive vinculin, although thepossibility that it might be targeted for destruction hasnot been explored. By contrast, focal adhesions turn overmuch more slowly, with life-times in the range of 10–20minutes. Local modulation of the exchange kinetics forfocal adhesion proteins might account for the glidingof adhesions observed in motile cells, with proteinincorporation and growth on one side and dissolution atthe opposite side.

A role for vinculin in strengthening cell–extracellularmatrix interactions?Cells respond to mechanical stress applied to ligand-boundintegrins either from outside the cell (e.g. stretching, pull-ing force or shear stress) or from inside the cell (e.g. RhoAstimulation of actomyosin contractility) [40–43] by enlar-ging their focal adhesions, and this is associated withincreased recruitment of vinculin (as well as talin andpaxillin) to focal adhesions [44]. In fibroblasts, mechanicalstretch induces an influx of Ca2+ at the leading edge of cells,and this regulates local traction forces [45]. Blocking theCa2+ signal with Gd3+ (gadolinium) correlated withreduced vinculin and phosphotyrosine at focal adhesionsas well as inhibition of cell migration [45]. By contrast, Racsignalling, which regulates membrane-protrusive activ-ities and cell spreading through Arp2/3-mediated actinpolymerization [43], was not affected by Gd3+ treatment[45], indicating that actomyosin contractility rather thanactin polymerization is required for vinculin recruitment.

Evidence that vinculin recruitment has a role instrengthening adhesions has been presented by Gallantet al. [46]. They measured the fluid shear force required todetach single cells from fibronectin-coated micropatternedislands of varying dimensions over time. To assess theeffect of focal adhesion assembly on adhesion strength,they used NIH 3T3 cells, which lack focal adhesions whencultured overnight in the absence of serum but rapidlyreassemble focal adhesions upon activation of RhoA byaddition of either serum or lysophosphatidic acid. Surpris-ingly, maturation of the initial adhesion sites into focaladhesions contributed only �20–30% of the adhesionstrength [46]. Adhesion strengthening was accompaniedby a 300% increase in vinculin and a 90% increase inrecruitment of talin to adhesion structures, whereas integ-rin levels were unchanged. These effects were completelyblocked by inhibition of actomyosin contractility (by bleb-bistatin) or Rho-kinase (by Y27632) [46]. Furthermore, invinculin-null F9 cells [47] or in NIH 3T3 cells in whichvinculin was downregulated by RNA interference, adhe-sion strength was reduced by 20–25% [46,48] and theserum-induced strengthening of adhesions was abolished[46]. The results suggest that much of the adhesion force isprovided by integrin binding to fibronectin and integrinclustering. However, adhesion strength is increased by afurther 20–30% bymechanisms that depend on actomyosin

Review TRENDS in Cell Biology Vol.16 No.9 457

contraction and the recruitment of vinculin to focaladhesions. How vinculin exerts these effects remain tobe elucidated, but recent studies on the interactionbetween vinculin and talin offer new insights.

The talin–vinculin interactionTalin is one of several proteins (including filamin,a-actinin, tensin and integrin-linked kinase) implicatedin coupling integrins to the actin cytoskeleton [1,49,50].The globular N-terminal talin FERM (band 4.1, ezrin,radixin and moesin) domain binds to the membraneproximal Asn-Pro-x-Tyr (NPxY) motif in b-integrincytoplasmic regions [51] and is linked to a flexible rodmadeup of a series of amphipathic helical bundles [12], themost C-terminal of which contains an actin-binding site[52,53]. Interestingly, the rod also containsmultiple VBSs[30], each of which is defined by hydrophobic residuesaligned down one face of a single amphipathic a-helix.Co-crystal andNMR structures [10,29] show that the talinhelix inserts into the vinculin D1 helical bundle andreplaces helix 1 of the D1 domain [10]. In full-lengthvinculin, helix 1 of D1 packs against the vinculin tail,and talin binding is inhibited (Figure 1a). Furtherevidence that a conformational change must occur in D1in order for talin to bind comes froma vinculinmutant thatstabilizes the D1 fold – this mutant does not affect tailbinding but reduces talin binding over 100-fold [8].

Interestingly, although the individual talin VBSpeptides bind vinculin D1 with relatively high affinity(Kd 15–40 nM) [11,54], talin purified from smooth musclebinds vinculin in solution with low affinity [13], suggesting

Figure 2. Vinculin dynamics in focal adhesions. Integrin heterodimers are shown bou

binding site in the talin rod. Talin dimers are not shown for simplicity. Actin filaments are

of talin. (a) Cytosolic vinculin in the auto-inhibited conformation is shown in steady-sta

simultaneously to talin (or other partners of D1) and other ligands. For example, Vt m

PtdIns(4,5)P2, PtdIns(3,4,5)P3 or phosphatidylserine) (1) or to F-actin (2). The proline-rich

vinexin-b or paxillin (Table 1) (3). (b) Mechanical load favours vinculin incorporation (so

perhaps by activating the VBSs in the talin rod and by bundling of actin filaments throu

generated or released locally (larger yellow shape) compete with F-actin binding to V

arrow) of vinculin from the focal adhesion (2) and destabilize the protein complex on th

Although talin and vinculin frequently co-localize in cells, it is interesting that vinculin is

synapse [67].

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that the activity of the VBSs in talin is also regulated. Thisis explained by the structures of the talin helical bundles,which show that the hydrophobic residues involved invinculin binding point towards the interior of the bundle[12,30]. Clearly, these bundles must unfold or otherwisereorganize to release the VBS helix to bind vinculin. Thisphenomenon has been observed directly using NMR andelectron paramagnetic resonance spectroscopy (EPR) tech-niques [12,14], and is inhibited by mutations that stabilizethe talin bundles [13]. The challenge now is to understandhow these VBSs become available in vivo, although at leastone VBS towards the N-terminal region of the talin rodappears to be constitutively active [13]. One attractivehypothesis is that force exerted by actomyosin contractionmight alter the conformation of the helical bundles in thetalin rod, leading to activation of the VBSs containedtherein. The response might depend on the stability ofindividual bundles, and be proportional to the force exertedupon them. The greater the force, the larger the number ofVBSs that would be activated, leading to a graded increasein vinculin recruitment and strengthening of adhesion(Figure 2a,b). A similar force-induced conformationalchange in the extracellular matrix protein fibronectinhas been shown to expose cryptic self association sitesand to drive fibronectin polymerization [55].

a-Actinin is also thought to couple integrins to F-actin,and an a-helical peptide derived from spectrin repeat 4 inthe a-actinin rod also binds to the vinculin D1 domain in atalin-like fashion [56]. However, the helix is buriedwithin an antiparallel coiled-coil dimer within full-lengtha-actinin [57], so that a major domain unfolding would

nd to talin either through the N-terminal FERM domain or an additional integrin-

shown bound to both the talin FERM domain and the C-terminal actin-binding site

te equilibrium (arrows) with activated vinculin in adhesion sites, where it is bound

ight bind to acidic phospholipids (yellow) in the lipid bilayer (phosphoinositides,

region and/or Vt might also recruit other binding partners (hexagon) such as VASP,

lid arrow) over release (dotted arrow), resulting in strengthening of adhesion sites,

gh vinculin Vt dimer formation [31]. (c) Phosphoinositides (or phosphatidylserine)

t (1). Ultimately this could favour release (solid arrow) over incorporation (dotted

e cytoplasmic face of focal adhesions, resulting in the dissolution of the adhesion.

apparently not recruited to the integrin–talin complex found at the immunological

Box 3. A function for vinculin in cell–cell junctions

Vinculin is localized in cell–cell junctions of epithelial sheets.

Experiments with vinculin-null F9 cells show that vinculin is not

required for the assembly of cadherin-mediated cell–cell junctions,

although a defect in formation of tight junctions was reported [69].

Rather, vinculin is believed to strengthen the mechanical links

between adhesion complexes (containing E-cadherin, b-catenin and

a-catenin) and the actin cytoskeleton. However, recent studies on

the protein dynamics in cell–cell junctions have called into question

the view [70]. Thus the dynamics of actin and actin-binding proteins

such as vinculin was not correlated with cadherin–catenin dy-

namics. The data suggest a somewhat loose association of

peripheral actin filaments in cell–cell junctions, leaving the role of

vinculin undefined.

Interestingly, vinculin-null F9 cells lack the lipid phosphatase

PTEN, although PTEN mRNA levels are comparable with wild-type

cells [71]. Levels of PTEN protein could be restored either by

expressing vinculin or inhibiting the proteasomal degradation

pathway with lactacystin, suggesting that vinculin protects PTEN

protein from destruction in some way. Subsequent experiments

indicate that vinculin is part of a protein complex on the cytoplasmic

face of E-cadherin, which includes b-catenin and its binding partners

MAGI-2 and a-catenin, and that this complex is required to stabilize

PTEN protein [71]. PTEN is among the most frequently mutated

tumour suppressor genes, but there are tumours in which no

mutations in the PTEN gene have been identified, even though

PTEN protein is not present. The authors suggest that loss of

expression of any of the components of the above complex could

explain the absence of PTEN [71].

458 Review TRENDS in Cell Biology Vol.16 No.9

have to occur (distinct from the local unfolding ofindividual bundles in talin) for this interaction to operatein vivo. Evidence that this occurs has yet to be presented.Moreover, recent EM diffraction studies have provided analternative model in which the vinculin head binds to theglobular ends of a-actinin without significant unfolding[58]. How recruitment of vinculin to focal adhesions mightstabilize these structures remains to be established.Vinculin has many binding partners (Table 1) and couldexert its effects through several distinct mechanisms. Forexample, vinculin might cross-link talin (or a-actinin) toF-actin (Figure 2a), but it has also been shown tomodulate FAK and paxillin signalling both of which areimportant in the regulation of cell motility [6].

Release of vinculin from focal adhesions andstimulation of cell motilityMutations that reduce the Kd of the vinculin head–tailinteraction from 1 nM to 100 nM are sufficient to enableconstitutive talin binding. Importantly, FRAP experi-ments show that expression of such vinculin mutants incells results in a significant reduction of turnover in bothvinculin and talin (but not paxillin) [35] and enlarged focaladhesions. These results are consistent with data showingthat vinculin negatively regulates focal adhesion dynamics[59] and suppresses cell motility [60]. The question thenarises how this suppressive effect of vinculin is relieved toenable focal adhesion turnover and cell migration.

Although relaxation of actomyosin contractility rapidlysignals the release of zyxin andVASP from focal adhesions,it does not signal the release of vinculin [61], despitesimilar turnover rates of these proteins in steady-stateadhesions. Acidic phospholipids, such as phosphoinositides(PtdIns(4,5)P2 or PtdIns(3,4,5)P3) or phosphatidylserine,bind to the lipid-interaction site in Vt and compete with F-actin binding to Vt in vitro [62]. This raises the possibilitythat transient increases in such lipids could enhancerelease of vinculin from adhesions. Indeed, PtdIns(3,4,5)P3

has been implicated in the PDGF-induced displacement ofvinculin (but not talin) from focal adhesions in mousefibroblasts [63]. In neuronal cells, increasing PtdIns(4,5)P2

levels by expressing PtdInsP 5-kinase a resulted in cellrounding, and vinculin was lost from focal adhesions fasterthan FAK and paxillin. Conversely, kinase-dead PtdInsP5-kinase a induced neurite outgrowth and retractionmediated by lysophosphatidic acid or semaphorin 3A (aneuronal guidance cue) was blocked, reflecting inhibition offocal adhesion turnover [64]. In B16-F1 cells, a GFP-vinculin mutant deficient in acidic phospholipid-binding(vinculin-LD) displayed release rates (koff) comparable towild-type protein in steady-state adhesions. However, thelifetime of focal adhesions was increased, while spreadingand migration of cells expressing vinculin-LD werestrongly inhibited [34]. Furthermore, overexpression ofPtdInsP 5-kinase a (but not the kinase-dead mutant)stimulated release of wild-type vinculin but not the LDvariant, and induced dissolution of adhesions [34].Similarly, a vinculin mutant lacking the C-terminal armof Vt and with reduced phosphoinositide-binding activityalso significantly inhibited focal adhesion turnover and cellspreading when expressed in vinculin-null fibroblasts [59].

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These results suggest a model in which focal adhesiondynamics is regulated by vinculin, and that transientincreases in local phosphoinositide levels, which inhibitthe vinculin–F-actin interaction, promote focal adhesionturnover and cell motility (Figure 2c). Interestingly, themuscle-specific splice variant of vinculin called metavin-culin (which contains a 68 amino acid insert in the Vtdomain), is localized in dense plaques and costameres,cell–extracellular matrix junctions that are much longerlived than focal adhesions. It could be significant that theVt/D5 domain of metavinculin interacts less strongly withacidic phospholipids than does the Vt/D5 domain of vincu-lin [65]. The association of metavinculin–vinculin hetero-dimers with F-actin might therefore be relatively resistantto phospholipid competition, resulting in more persistentadhesions.

ConclusionsThe past two years have seen major progress in our under-standing of the structure of vinculin, yet much remains tobe established about the role of the protein in a cellularcontext. Vinculin-null cells show a variety of phenotypesthat clearly indicate a role for vinculin in cell adhesion, cellspreading, focal adhesion stability and strengthening, cellmigration and resistance to apoptosis. Which of the multi-ple binding partners of the protein contribute to each ofthese characteristics, and how ligand binding is regulated,is still far from clear. There are also major gaps in ourknowledge about the role of vinculin in cell–cell junctions(Box 3). In mice, vinculin is required during embryonicdevelopment, and vinculin-null mice die at around embryo-nic day 10 from neural and cardiovascular defects [5].Elucidation of the role of vinculin in different tissues willclearly require a conditional knockout approach.

Review TRENDS in Cell Biology Vol.16 No.9 459

Interestingly, mice heterozygous for vinculin are pre-dis-posed to stress-induced cardiomyopathy [66]. Basal cardiacfunction and histology is normal, but the intercalated disksare abnormal and a-actinin-containing Z-lines in the mus-cles are misaligned. In conclusion, the new data reviewedhere offers the prospect of elucidating the function ofvinculin both at the cellular and organismal levels. How-ever, this will require a greater knowledge about the role ofthe many binding partners of vinculin. A more detailedcharacterization of the binding sites for these ligandsshould be facilitated by the availability of the recentlydetermined vinculin structure.

AcknowledgementsThis work was supported by grants from: the DeutscheForschungsgemeinschaft (ZI 545/2-4) and the Fonds der ChemischenIndustrie (W.H.Z.); National Institutes of Health (R01 GM059760 andU54 GM064346) (R.C.L.); and BBSRC, the Wellcome Trust and CancerResearch UK (D.R.C.).

References1 Critchley, D.R. (2000) Focal adhesions – the cytoskeletal connection.

Curr. Opin. Cell Biol. 12, 133–1392 Nayal, A. et al. (2004) Talin: an emerging focal point of adhesion

dynamics. Curr. Opin. Cell Biol. 16, 94–983 Johnson, R.P. and Craig, S.W. (1994) An intramolecular association

between the head and tail domains of vinculin modulates talin binding.J. Biol. Chem. 269, 12611–12619

4 Johnson, R.P. and Craig, S.W. (1995) F-actin binding site masked bythe intramolecular association of vinculin head and tail domains.Nature 373, 261–264

5 Xu,W. et al. (1998) Vinculin knockout results in heart and brain defectsduring embryonic development. Development 125, 327–337

6 Subauste, M.C. et al. (2004) Vinculin modulation of paxillin-FAKinteractions regulates ERK to control survival and motility. J. CellBiol. 165, 371–381

7 Rodriguez Fernandez, J.L. et al. (1992) Suppression of tumorigenicityin transformed cells after transfection with vinculin cDNA. J. Cell Biol.119, 427–438

8 Bakolitsa, C. et al. (2004) Structural basis for vinculin activation atsites of cell adhesion. Nature 430, 583–586

9 Borgon, R.A. et al. (2004) Crystal structure of human vinculin.Structure 12, 1189–1197

10 Izard, T. et al. (2004) Vinculin activation by talin through helicalbundle conversion. Nature 427, 171–175

11 Izard, T. and Vonrhein, C. (2004) Structural basis for amplifyingvinculin activation by talin. J. Biol. Chem. 279, 27667–27678

12 Papagrigoriou, E. et al. (2004) Activation of a vinculin-binding site inthe talin rod involves rearrangement of a five-helix bundle. EMBO J.23, 2942–2951

13 Patel, B. et al. (2006) The activity of the vinculin binding sites in talin isinfluenced by the stability of the helical bundles that make up the talinrod. J. Biol. Chem. 281, 7458–7467

14 Gingras, A.R. et al. (2006) Structural and dynamic characterizationof a vinculin binding site in the talin rod. Biochemistry 45, 1805–1817

15 Cohen, D.M. et al. (2005) Two distinct head-tail interfaces cooperate tosuppress activation of vinculin by talin.J.Biol. Chem. 280, 17109–17117

16 Bois, P.R. et al. (2006) The vinculin binding sites of talin and alpha-actinin are sufficient to activate vinculin. J. Biol. Chem. 281, 7228–7236

17 Demali, K.A. (2004) Vinculin–a dynamic regulator of cell adhesion.Trends Biochem. Sci. 29, 565–567

18 Kioka, N. et al. (1999) Vinexin: a novel vinculin-binding protein withmultiple SH3 domains enhances actin cytoskeletal organization. J.Cell Biol. 144, 59–69

19 Chen, H. et al. (2005) Spatial distribution and functional significance ofactivated vinculin in living cells. J. Cell Biol. 169, 459–470

20 Zhang, Z. et al. (2004) The phosphorylation of vinculin on tyrosineresidues 100 and 1065, mediated by Src kinases, affects cell spreading.Mol. Biol. Cell.

www.sciencedirect.com

21 Bass, M.D. et al. (1999) Talin contains three similar vinculin-bindingsites predicted to form an amphipathic helix. Biochem. J. 341, 257–263

22 Bois, P.R. et al. (2005) Structural dynamics of alpha-actinin-vinculininteractions. Mol. Cell. Biol. 25, 6112–6122

23 Brindle, N.P. et al. (1996) The focal-adhesion vasodilator-stimulatedphosphoprotein (VASP) binds to the proline-rich domain in vinculin.Biochem. J. 318, 753–757

24 Huttelmaier, S. et al. (1998) The interaction of the cell-contact proteinsVASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate. Curr. Biol. 8, 479–488

25 Mandai, K. et al. (1999) Ponsin/SH3P12: an l-afadin- and vinculin-binding protein localized at cell-cell and cell-matrix adherensjunctions. J. Cell Biol. 144, 1001–1017

26 DeMali, K.A. et al. (2002) Recruitment of the Arp2/3 complex tovinculin: coupling membrane protrusion to matrix adhesion. J. CellBiol. 159, 881–891

27 Menkel, A.R. et al. (1994) Characterization of an F-actin-bindingdomain in the cytoskeletal protein vinculin. J. Cell Biol. 126, 1231–1240

28 Huttelmaier, S. et al. (1997) Characterization of two F-actin-bindingand oligomerization sites in the cell-contact protein vinculin. Eur. J.Biochem. 247, 1136–1142

29 Fillingham, I. et al. (2005) A vinculin binding domain from the talin rodunfolds to form a complex with the vinculin head. Structure 13, 65–74

30 Gingras, A.R. et al. (2005) Mapping and consensus sequenceidentification for multiple vinculin binding sites within the talinrod. J. Biol. Chem. 280, 37217–37224

31 Janssen, M.E. et al. (2006) Three-dimensional structure of vinculinbound to actin filaments. Mol. Cell 21, 271–281

32 Johnson, R.P. and Craig, S.W. (2000) Actin activates a crypticdimerization potential of the vinculin tail domain. J. Biol. Chem.275, 95–105

33 Bakolitsa, C. et al. (1999) Crystal structure of the vinculin tail suggestsa pathway for activation. Cell 99, 603–613

34 Chandrasekar, I. et al. (2005) Vinculin acts as a sensor in lipidregulation of adhesion-site turnover. J. Cell Sci. 118, 1461–1472

35 Cohen, D.M. et al. (2006) A conformational switch in vinculin drivesformation and dynamics of a talin-vinculin complex at focal adhesions.J. Biol. Chem. 281, 16006–16015

36 Von Wichert, G. et al. (2003) Force-dependent integrin-cytoskeletonlinkage formation requires downregulation of focal complex dynamicsby Shp2. EMBO J. 22, 5023–5035

37 Lele, T.P. et al. (2006) Mechanical forces alter zyxin unbinding kineticswithin focal adhesions of living cells. J. Cell. Physiol. 207, 187–194

38 Webb, D.J. et al. (2004) FAK-Src signalling through paxillin, ERK andMLCK regulates adhesion disassembly. Nat. Cell Biol. 6, 154–161

39 Fraley, T.S. et al. (2005) Phosphoinositide binding regulates alpha-actinin dynamics: mechanism formodulating cytoskeletal remodelling.J. Biol. Chem. 280, 15479–15482

40 Giannone, G. et al. (2003) Talin1 is critical for force-dependentreinforcement of initial integrin-cytoskeleton bonds but not tyrosinekinase activation. J. Cell Biol. 163, 409–419

41 Balaban, N.Q. et al. (2001) Force and focal adhesion assembly: a closerelationship studied using elastic micropatterned substrates.Nat. CellBiol. 3, 466–472

42 Delanoe-Ayari, H. et al. (2004) Membrane and acto-myosin tensionpromote clustering of adhesion proteins. Proc. Natl. Acad. Sci. U. S. A.101, 2229–2234

43 Rottner, K. et al. (1999) Interplay between Rac and Rho in the control ofsubstrate contact dynamics. Curr. Biol. 9, 640–648

44 Zaidel-Bar, R. et al. (2003) Early molecular events in the assembly ofmatrix adhesions at the leading edge of migrating cells. J. Cell Sci. 116,4605–4613

45 Munevar, S. et al. (2004) Regulation of mechanical interactionsbetween fibroblasts and the substratum by stretch-activated Ca2+entry. J. Cell Sci. 117, 85–92

46 Gallant, N.D. et al. (2005) Cell adhesion strengthening: contributions ofadhesive area, integrin binding, and focal adhesion assembly. Mol.Biol. Cell 16, 4329–4340

47 Coll, J.L. et al. (1995) Targeted disruption of vinculin genes in F9 andembryonic stem cells changes cell morphology, adhesion, andlocomotion. Proc. Natl. Acad. Sci. U. S. A. 92, 9161–9165

460 Review TRENDS in Cell Biology Vol.16 No.9

48 Goldmann, W.H. et al. (1998) Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic forcemicroscopy. Exp. Cell Res. 239, 235–242

49 Calderwood, D.A. et al. (2003) Integrin beta cytoplasmic domaininteractions with phosphotyrosine-binding domains: a structuralprototype for diversity in integrin signalling. Proc. Natl. Acad. Sci.U. S. A. 100, 2272–2277

50 Brakebusch, C. and Fassler, R. (2003) The integrin-actin connection,an eternal love affair. EMBO J. 22, 2324–2333

51 Garcia-Alvarez, B. et al. (2003) Structural determinants of integrinrecognition by talin. Mol. Cell 11, 49–58

52 Hemmings, L. et al. (1996) Talin contains three actin-binding sites eachof which is adjacent to a vinculin-binding site. J. Cell Sci. 109, 2715–2726

53 Senetar, M.A. et al. (2004) Intrasteric inhibition mediates theinteraction of the I/LWEQ module proteins Talin1, Talin2, Hip1,and Hip12 with actin. Biochemistry 43, 15418–15428

54 Bass, M.D. et al. (2002) Further characterization of the interactionbetween the cytoskeletal proteins talin and vinculin. Biochem. J. 362,761–768

55 Wierzbicka-Patynowski, I. and Schwarzbauer, J.E. (2003) The ins andouts of fibronectin matrix assembly. J. Cell Sci. 116, 3269–3276

56 Bois, P.R. et al. (2005) Structural dynamics of alpha-actinin-vinculininteractions. Mol. Cell. Biol. 25, 6112–6122

57 Ylanne, J. et al. (2001) Crystal structure of the alpha-actinin rodreveals an extensive torsional twist. Structure 9, 597–604

58 Kelly, D.F. et al. (2006) Structure of the alpha-actinin-vinculin headdomain complex determined by cryo-electron microscopy. J. Mol. Biol.357, 562–573

59 Saunders, R.M. et al. (2006) Role of vinculin in regulating focaladhesion turnover. Eur. J. Cell Biol. 85, 487–500

60 Xu, W. et al. (1998) Rescue of the mutant phenotype by reexpression offull-length vinculin in null F9 cells; effects on cell locomotion by domaindeleted vinculin. J. Cell Sci. 111, 1535–1544

61 Lele, T.P. et al. (2006) Mechanical forces alter zyxin unbinding kineticswithin focal adhesions of living cells. J. Cell. Physiol. 207, 187–194

62 Steimle, P.A. et al. (1999) Polyphosphoinositides inhibit the interactionof vinculin with actin filaments. J. Biol. Chem. 274, 18414–18420

63 Greenwood, J.A. et al. (2000) Restructuring of focal adhesion plaquesby PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin. J. Cell Biol. 150, 627–642

64 van Horck, F.P. et al. (2002) Essential role of type I(alpha)phosphatidylinositol 4-phosphate 5-kinase in neurite remodelling.Curr. Biol. 12, 241–245

65 Witt, S. et al. (2004) Comparative biochemical analysis suggests thatvinculin and metavinculin cooperate in muscular adhesion sites. J.Biol. Chem. 279, 31533–31543

66 Zemljic-Harpf, A.E. et al. (2004) Heterozygous inactivation of thevinculin gene predisposes to stress-induced cardiomyopathy. Am. J.Pathol. 165, 1033–1044

67 Monks, C.R.F. et al. (1998) Three-dimensional segregtion ofsupramolecular activation clusters in T cells. Nature 395, 82–86

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68 Turner, C.E. (2000) Paxillin interactions. J. Cell Sci. 113, 4139–414069 Watabe-Uchida, M. et al. (1998) alpha-Catenin-vinculin interaction

functions to organize the apical junctional complex in epithelial cells. J.Cell Biol. 142, 847–857

70 Yamada, S. et al. (2005) Deconstructing the cadherin-catenin-actincomplex. Cell 123, 889–901

71 Subauste, M.C. et al. (2005) Vinculin controls PTEN protein level bymaintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2. J. Biol. Chem. 280,5676–5681

72 Burridge, K. and Mangeat, P. (1984) An interaction between vinculinand talin. Nature 308, 744–746

73 Wachsstock, D.H. et al. (1987) Specific interaction of vinculin withalpha-actinin. Biochem. Biophys. Res. Commun. 146, 554–560

74 McGregor, A. et al. (1994) Identification of the vinculin-bindingsite in the cytoskeletal protein alpha-actinin. Biochem. J. 301, 225–233

75 Kroemker, M. et al. (1994) Intramolecular interactions in vinculincontrol alpha-actinin binding to the vinculin head. FEBS Lett. 355,259–262

76 Bourdet-Sicard, R. et al. (1999) Binding of the Shigella proteinIpaA to vinculin induces F-actin depolymerization. EMBO J. 18,5853–5862

77 Tran Van Nhieu, G. et al. (1997) Modulation of bacterial entry intoepithelial cells by association between vinculin and the Shigella IpaAinvasin. EMBO J. 16, 2717–2729

78 Hazan, R.B. et al. (1997) Vinculin is associated with the E-cadherinadhesion complex. J. Biol. Chem. 272, 32448–32453

79 Weiss, E.E. et al. (1998) Vinculin is part of the cadherin-cateninjunctional complex: complex formation between alpha-catenin andvinculin. J. Cell Biol. 141, 755–764

80 Imamura, Y. et al. (1999) Functional domains of alpha-catenin requiredfor the strong state of cadherin-based cell adhesion. J. Cell Biol. 144,1311–1322

81 Takahashi, H. et al. (2005) Role of interaction with vinculin inrecruitment of vinexins to focal adhesions. Biochem. Biophys. Res.Commun. 336, 239–246

82 Gilmore, A.P. and Burridge, K. (1996) Regulation of vinculin binding totalin and actin by phosphatidyl-inositol-4-5-bisphosphate. Nature 381,531–535

83 Wood, C.K. et al. (1994) Characterisation of the paxillin-binding siteand the C-terminal focal adhesion targeting sequence in vinculin. J.Cell Sci. 107, 709–717

84 Weekes, J. et al. (1996) Acidic phospholipids inhibit the intramolecularassociation between the N- and C-terminal regions of vinculin,exposing actin-binding and protein kinase C phosphorylation sites.Biochem. J. 314, 827–832

85 Ziegler, W.H. et al. (2002) A lipid-regulated docking site on vinculin forprotein kinase C. J. Biol. Chem. 277, 7396–7404

86 Johnson, R.P. et al. (1998) A conserved motif in the tail domain ofvinculin mediates association with and insertion into acidicphospholipid bilayers. Biochemistry 37, 10211–10222

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