4319Research Article

IntroductionAfadin is a protein that binds nectin and actin filaments (F-actin)and thus connects nectins to the actin cytoskeleton (Mandai et al.,1997; Takahashi et al., 1999). Afadin comprises multiple domains:two Ras-binding (RA) domains, a dilute (DIL) domain, a PDZ (postsynaptic density protein, Drosophila disc large tumor suppressor,and zonula occludens-1 protein) domain, three proline-rich domains,and an F-actin-binding (FAB) domain in this order from the N-terminus (Takai et al., 2003). Nectins are Ca2+-independentimmunoglobulin-like cell-cell adhesion molecules and consist offour members: nectin-1, nectin-2, nectin-3 and nectin-4 (Ogita andTakai, 2008; Takai and Nakanishi, 2003). Our previous studies haverevealed the roles and modes of actions of nectins and afadin inthe formation of adherens junctions (AJs) (Ogita and Takai, 2008;Sakisaka et al., 2007; Takai et al., 2008a). The formation of AJsbegins with trans-interaction of nectins between neighboring cells.This trans-interaction induces the activation of the small G proteinsRap1, Cdc42 and Rac through the activation of c-Src. ActivatedCdc42 and Rac bind to the GTPase activator protein IQGAP1 (alsoknown as p195) and these molecules cooperatively reorganize theactin cytoskeleton to recruit the E-cadherin–catenin complex tonectin-based cell-cell contact sites in epithelial cells. At this stage,E-cadherin molecules have only weak adhesion activity and do nottrans-interact with each other. However, afadin binds activated Rap1and then associates with p120ctn, which prevents the endocytosis

of non-trans-interacting E-cadherin and enhances the adhesionactivity of E-cadherin to facilitate its trans-interaction, eventuallyestablishing AJs. In addition, afadin functions as a connectorbetween the cadherin-catenin and nectin-afadin systems at AJs byinteracting with ponsin, LIM domain only 7 (LMO7), and afadinDIL domain-interacting protein (ADIP), or by directly binding to-catenin (Asada et al., 2003; Mandai et al., 1999; Ooshio et al.,2004; Pokutta et al., 2002; Tachibana et al., 2000).

In addition to its role in the formation of AJs, afadin is involvedin cell polarization (together with Par complex proteins) and theformation of tight junctions (TJs) by assembly of TJ components,such as claudin, at the apical site of AJs (Komura et al., 2008; Ooshioet al., 2007; Yamada et al., 2006). The nectin-afadin complex alsoplays a key role in cell survival by supporting the platelet-derivedgrowth factor (PDGF)-induced activation of the phosphatidylinositol3-kinase (PI3K)-Akt pathway (Kanzaki et al., 2008). Moreover,afadin is indispensable for morphogenesis in embryonicdevelopment because the lack of afadin in mice causes embryoniclethality owing to disorganized cell-cell junctions, improperdifferentiation of cells and impaired cell migration duringgastrulation (Ikeda et al., 1999; Zhadanov et al., 1999). Therefore,afadin has multiple important functions in the formation of cell-cell junctions, as shown by in vitro and in vivo experiments.

Afadin has been shown to be present in the cytosolic fraction aswell as at cell-cell adhesion sites (Mandai et al., 1997). We recently

Afadin is an actin-filament-binding protein that binds to nectin,an immunoglobulin-like cell-cell adhesion molecule, and playsan important role in the formation of adherens junctions. Here,we show that afadin, which did not bind to nectin and waslocalized at the leading edge of moving cells, has another role:enhancement of the directional, but not random, cell movement.When NIH3T3 cells were stimulated with platelet-derivedgrowth factor (PDGF), afadin colocalized with PDGF receptor,v3 integrin and nectin-like molecule-5 at the leading edgeand facilitated the formation of leading-edge structures anddirectional cell movement in the direction of PDGF stimulation.However, these phenotypes were markedly perturbed byknockdown of afadin, and were dependent on the binding ofafadin to active Rap1. Binding of Rap1 to afadin was necessary

for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightlyregulate the activation of PDGF receptor and its downstreamsignaling pathway for the formation of the leading edge. Theseresults indicate that afadin has a novel role in PDGF-induceddirectional cell movement, presumably in cooperation withactive Rap1 and SHP-2.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/23/4319/DC1

Key words: Growth factor receptor, Integrin, Nectin-like molecule,SHP-2

Summary

Localization of nectin-free afadin at the leading edgeand its involvement in directional cell movementinduced by platelet-derived growth factorMuneaki Miyata1, Hisakazu Ogita1, Hitomi Komura1, Shinsuke Nakata2, Ryoko Okamoto2, Misa Ozaki1,Takashi Majima2, Naomi Matsuzawa1, Satoshi Kawano1, Akihiro Minami1, Masumi Waseda1, Naoyuki Fujita2,Kiyohito Mizutani1, Yoshiyuki Rikitake1 and Yoshimi Takai1,*1Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine,Kobe 650-0017, Japan2Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0871 Japan*Author for correspondence (ytakai@med.kobe-u.ac.jp)

Accepted 21 September 2009Journal of Cell Science 122, 4319-4329 Published by The Company of Biologists 2009doi:10.1242/jcs.048439

Jour

nal o

f Cel

l Sci

ence

4320

demonstrated that, in sparsely cultured cells that do not contact withother cells, afadin prevents PDGF-induced hyperactivation ofPDGF receptor and its downstream signaling molecules Ras andERK by increasing the phosphatase activity of SHP-2 (Nakata etal., 2007). The Ras-ERK signaling pathway is involved in theregulation of cytoskeletal dynamics and directional cell movement(Chernyavsky et al., 2005; Ho et al., 2001; Huang et al., 2004;Matsubayashi et al., 2004). However, the role of afadin in thecytosolic fraction is not fully understood. Thus, in this study, weinvestigated the role of afadin in moving cells and found that afadinwas localized at the leading edge of moving cells and was involvedin directional cell movement. To promote directional cell movement,nectin-like molecule-5 (Necl-5; also known as poliovirus receptor),a cell-adhesion molecule structurally similar to nectins, forms acomplex with v3 integrin and PDGF receptor, and enhances thesignaling pathways needed for the formation of leading-edgestructures (Amano et al., 2008; Minami et al., 2007; Nagamatsu et

al., 2008; Takahashi et al., 2008). On the basis of these results, wealso examined the association of afadin with these molecules toelucidate the molecular mechanism by which afadin is involved inPDGF-induced directional cell movement.

ResultsInvolvement of afadin in directional cell movementWe first examined the effect of afadin on cell movement in responseto PDGF stimulation using wild-type and afadin-knockdownNIH3T3 cells. The expression level of afadin in both cell types isshown in Fig. 1A. Wild-type and afadin-knockdown NIH3T3 cellswere sparsely plated on -Slide VI Flow dishes pre-coated withvitronectin, an extracellular matrix protein that binds to v3integrin (Schvartz et al., 1999), and were directionally stimulatedwith PDGF. Wild-type NIH3T3 cells became polarized with thewell-spreading leading edge toward the higher concentration ofPDGF, whereas afadin-knockdown NIH3T3 cells showed elongated

Journal of Cell Science 122 (23)

Fig. 1. Impaired formation of the leading edge and reduction of directional cell movement. (A)Expression level of afadin in wild-type and afadin-knockdownNIH3T3 cells. Lysates from wild-type and afadin-knockdown NIH3T3 cells were separated by SDS-PAGE, followed by western blotting with the anti-afadin andanti-N-cadherin mAbs. (B)Morphology of wild-type and afadin-knockdown NIH3T3 cells in response to PDGF stimulation. After wild-type and afadin-knockdown, NIH3T3 cells were seeded on the-Slide VI Flow, cells were directionally stimulated with 30 ng/ml PDGF for 30 minutes from the bottom. Cellswere then fixed and stained with the anti-actin mAb. Scale bars: 50m. (C)Directional cell movement estimated by the wound-healing assay. Confluent cellmonolayers of wild-type and afadin-knockdown NIH3T3 cells were manually scratched and cultured for 8 hours in the presence of 30 ng/ml PDGF. Cells werestained with the anti-afadin pAb (green) and the anti-actin mAb (red). Nuclei were counterstained with DAPI (blue). Percentage wound closure was calculated asdescribed in the Materials and Methods. *P<0.05 vs. wild-type (WT). Scale bars: 50m. (D)Reorientation of the Golgi complex during the wound-healing assay.Cells were stained with the anti-GM130 mAb (red) and nuclei were counterstained with DAPI (blue). The dotted line indicates the edge of the wound. Thepercentage of Golgi complexes facing the wound was calculated as described in the Materials and Methods. *P<0.05 vs. WT. Scale bars: 10m. (E)Directionalcell movement estimated by the Boyden chamber assay. Wild-type and afadin-knockdown NIH3T3 cells were incubated on cell-culture inserts coated withvitronectin in the presence or absence of 30 ng/ml PDGF in the upper or lower well for 12 hours. To count the number of cells that had migrated into the bottomwell, cells were fixed and stained with DAPI. *P<0.05 vs. WT. Scale bars: 50m. (F)Mobility of cells estimated by the phagokinetic track motility assay. Wild-type and afadin-knockdown NIH3T3 cells were seeded on colloidal gold-coated coverslips and incubated for 18 hours before fixation. Phase-contrast images weretaken to measure the area free of gold particles around a single cell. Results are representative of three independent experiments. Scale bars: 10m.

Jour

nal o

f Cel

l Sci

ence

4321Novel role of afadin in cell movement

shapes and had a small leading edge that was randomly directedand was independent of the direction of the higher concentrationof PDGF (Fig. 1B).

These results led us to assume that afadin is involved indirectional cell movement. To examine this assumption, weperformed a wound-healing assay by scratching the confluentmonolayer of wild-type and afadin-knockdown NIH3T3 cells inthe presence or absence of PDGF. In the absence of PDGF, thewound was still open at 8 hours after scratching in both wild-typeand afadin-knockdown NIH3T3 cells, and the degree of the woundclosure was similar between both types of cells (supplementarymaterial Fig. S1A). In the presence of PDGF, however, the woundmade by the scratch gradually closed in wild-type NIH3T3 cells(Fig. 1C). In this process, cells efficiently formed protrusions, andthe immunofluorescence signal for afadin was observed at theleading edge of these protrusions as well as at the cell-cell adhesionsites. By contrast, the wound did not close in the afadin-knockdowncell monolayer. Formation of the leading edge in afadin-knockdowncells was less obvious than that in wild-type cells and theorganization of actin stress fibers appeared more prominently inafadin-knockdown than wild-type cells due to the increasedactivation of RhoA in afadin-knockdown cells (Miyata et al., 2009).The velocity of cell proliferation and the degree of apoptosis inwild-type and afadin-knockdown cells during the experimentalperiod (8 hours) were not significantly different, as shown in ourprevious report (Nakata et al., 2007) and in our unpublished results.We further analyzed the alignment of the Golgi complex in cellsat the wound edge because the reorientation of the Golgi complexin moving cells was reported to correlate with directional cellmovement (Kupfer et al., 1982; Nobes and Hall, 1999). The ratioof the Golgi complex facing the wound was lower in afadin-knockdown than wild-type cells (Fig. 1D). In addition, the Boydenchamber assay showed that afadin-knockdown NIH3T3 cells wereless responsive to PDGF than wild-type cells when PDGF was addedinto only the bottom chamber (Fig. 1E). However, the number ofwild-type NIH3T3 cells crossing the membrane was similar to thatof afadin-knockdown cells when PDGF was added into both upperand lower sides of chambers, suggesting that afadin might notcontribute to random chemokinesis. Furthermore, the area of cellmovement of afadin-knockdown NIH3T3 cells was similar to thatof wild-type cells, as assessed by the phagokinetic track motilityassay that visualizes the tracks of moving cells (Fig. 1F), assumingthat cell movement is not impaired by knockdown of afadin. Takentogether, these results indicate that afadin regulates the directionalityof cell movement in moving cells in response to PDGF.

This hypothesis was further confirmed by time-lapse microscopy.Wild-type NIH3T3 cells formed a large leading edge in the directionof the higher concentration of PDGF and this leading-edge formationwas persistent, keeping the direction of cell movement constant(supplementary material Movie 1). By contrast, formation of theleading edge in afadin-knockdown NIH3T3 cells was less stableand was unrelated to the direction of PDGF stimulation, resultingin impaired directional cell movement (supplementary materialMovie 2). Tracking of cell movement also revealed that the PDGF-induced directional cell movement was markedly perturbed byknockdown of afadin in NIH3T3 cells (Fig. 2).

To prove that the impairment of leading-edge formation anddirectional cell movement in afadin-knockdown NIH3T3 cells isdependent on the reduction of afadin itself, we used afadin-knockdown cells stably re-expressing green fluorescent protein(GFP)-tagged full-length afadin (GFP-afadin), which was resistant

to RNAi against afadin. The expression level of GFP-afadin wassimilar to that of endogenous afadin in wild-type NIH3T3 cells (Fig.3A). Formation of the leading edge induced by stimulation withPDGF was rescued by re-expression of GFP-afadin in afadin-knockdown NIH3T3 cells (Fig. 3B). Similarly to endogenous afadin,GFP-afadin was recruited to the leading edge. The accumulationof PDGF receptor at the leading edge was also recovered by re-expression of GFP-afadin in afadin-knockdown NIH3T3 cells (Fig.3C). Consistent with these results, directional cell movementassessed by the wound-healing assay, the reorientation of the Golgicomplex and the Boyden chamber assay were restored by re-expression of GFP-afadin in afadin-knockdown NIH3T3 cells (Fig.3D-F).

Necessity of afadin for the recruitment of Necl-5, v3 integrin,and PDGF receptor to the leading edgeWe have reported that Necl-5, v3 integrin, and PDGF receptorform a complex at the leading edge in moving cells, and that thiscomplex is important for cell movement and proliferation (Amanoet al., 2008; Ikeda et al., 2004; Kakunaga et al., 2004; Minami etal., 2007; Nagamatsu et al., 2008; Takahashi et al., 2008). Basedon this observation, we examined the association of these moleculeswith afadin in NIH3T3 cells stimulated with PDGF. Theimmunofluorescence signals for v3 integrin, PDGF receptor, andNecl-5 were concentrated at the leading edge formed in the directionof the higher concentration of PDGF, as previously described, andcolocalized with that for afadin (Fig. 4A). In GFP-expressingNIH3T3 cells, the signal for N-cadherin was detected on the entireplasma membrane, but was not highly concentrated at the leadingedge, as previously reported (Fujito et al., 2005). The ectopicallyexpressed GFP signal was not observed on the cell periphery,providing the notion that the accumulation and colocalization ofv3 integrin, PDGF receptor and Necl-5 with afadin at the leadingedge are not merely dependent on the thickening of the cell front.This colocalization was further confirmed by the bead-cell contactassay. When microbeads coated with vitronectin were incubatedwith NIH3T3 cells, the signal for v3 integrin was preferentiallyobserved at the bead-cell contact sites and the signal for afadin also

Fig. 2. Cell movement tracking of PDGF-stimulated NIH3T3 cells. Wild-typeand afadin-knockdown NIH3T3 cells seeded on the-Slide VI Flow weredirectionally stimulated with 30 ng/ml PDGF for 2 hours from the bottom.Cell movement of randomly selected 13 wild-type and afadin-knockdownNIH3T3 cells was manually traced and analyzed as described in the Materialsand Methods. The mean value for the directionality of cell movement in wild-type NIH3T3 cells was significantly higher than that in afadin-knockdownNIH3T3 cells (P<0.01). The small blue cross in each panel indicates the centerof mass of all endpoints.

Jour

nal o

f Cel

l Sci

ence

4322

accumulated there, although these signals were not observed at thecontact sites between cells and the control Concanavalin A (ConA)-coated beads (Fig. 4B). Furthermore, the signals for PDGF receptorand Necl-5 were also accumulated at the contact sites between cellsand vitronectin-coated beads; however, the signal for nectin-1 wasnot accumulated there (Fig. 4C). This suggests that afadin, whichlocalizes at the bead-cell contact sites, associates with v3integrin, PDGF receptor and Necl-5, but not with nectin. Theseresults are also consistent with our previous report that thelocalization and function of nectins and Necls are different, eventhough their molecular structures are similar (Takai et al., 2008b).The wound-healing assay further confirmed that afadin at the leadingedge was free from nectin. Just after scratching the wild-typeNIH3T3 cell monolayer, the leading edge was not formed and afadinand nectin-3 colocalized at the cell-cell adhesion sites, whereas at2 hours after scratching, afadin, but not nectin-3, was accumulatedat the leading edge (supplementary material Fig. S1B). At this time,the immunofluorescence signals for afadin and nectin-3 becameweaker at the cell-cell adhesion sites behind the wound because of

the loose cell-cell adhesion, but their colocalization was stillobserved at several cell-cell adhesion sites. These results indicatethe different intracellular behavior between afadin and nectin.

We then examined whether afadin is necessary for theaccumulation of v3 integrin, PDGF receptor and Necl-5 at theleading edge. When afadin was knocked down in NIH3T3 cells,the signal for PDGF receptor was hardly detected at the leadingedge in response to PDGF (Fig. 4D). Similar results were obtainedfor the signals for v3 integrin and Necl-5 (data not shown). Theseresults indicate that afadin plays a role in recruiting and tetheringv3 integrin, PDGF receptor and Necl-5 to the leading edge ofmoving cells to promote the PDGF-induced directional cellmovement.

Role of binding of Rap1 to afadin in directional cell movementOur recent study showed that Rap1, which is activated in responseto PDGF, is recruited to the leading edge and is crucial for PDGF-induced formation of leading-edge structures (Takahashi et al.,2008). We, and another research group, have also reported that

Journal of Cell Science 122 (23)

Fig. 3. Restoration of directional cell movement by re-expression of full-length afadin, but not afadinRA, inafadin-knockdown NIH3T3 cells. (A)Expression levels ofafadin in each cell line. Lysates from the cells were separatedby SDS-PAGE, followed by western blotting with the anti-afadin mAb and the anti-GAPDH mAb as a loading control.WT, wild-type; KD, afadin-knockdown. (B)Morphology ofdifferent types of NIH3T3 cells in response to PDGF. Afterthe indicated types of NIH3T3 cells were seeded on the-Slide VI Flow and were directionally stimulated with 30ng/ml PDGF for 30 minutes from the bottom, cells werefixed and stained with the anti-actin mAb (red). Arrowheadindicates the accumulation of GFP-afadin at the leading edge.Scale bars: 10m. (C)Localization of PDGF receptor inafadin-knockdown NIH3T3 cells re-expressing GFP-afadinor GFP-afadinRA. After the indicated types of NIH3T3cells were treated as described in B, cells were fixed andstained with the anti-PDGF receptor pAb (red) and the anti-actin mAb (blue). Scale bars: 10m. (D)Directional cellmovement estimated by the wound-healing assay. Confluentcell monolayers of each type of NIH3T3 cell were manuallyscratched and cultured for 8 hours in the presence of 30ng/ml PDGF. Nuclei were counterstained with DAPI (blue).Arrowheads indicate the accumulation of GFP-afadin at theleading edge. The percentage wound closure was calculatedas described in the Materials and Methods. *P<0.05 vs. WT+ GFP. Scale bars: 100m. (E)Reorientation of the Golgicomplex during the wound-healing assay. Cells were stainedwith the anti-GM130 mAb (red) and nuclei werecounterstained with DAPI (blue). The dotted line indicatesthe edge of wound. The percentage of the Golgi complexfacing the wound was calculated as described in the Materialsand Methods. *P<0.05 vs. WT + GFP. Scale bars: 20m.(F)Directional cell movement estimated by the Boydenchamber assay. Types of NIH3T3 cells were incubated oncell-culture inserts coated with vitronectin in the presence of30 ng/ml PDGF in the bottom well for 12 hours. The numberof cells that migrated into the bottom well was counted.*P<0.05 vs. WT + GFP. Results are representative of threeindependent experiments.

Jour

nal o

f Cel

l Sci

ence

4323Novel role of afadin in cell movement

activated Rap1 binds afadin (Hoshino et al., 2005; Linnemann etal., 1999). To confirm this, we conducted immunoprecipitation andin vitro binding assays. The association of afadin with Rap1 wasdependent on the activation of Rap1 because a constitutively activemutant of Rap1 (V12Rap1), but not Rap1 inactivated by Rap1GAP,could be co-immunoprecipitated with afadin (Fig. 5A). Rap1GAPis a GTPase-activating protein (GAP) for Rap1. In the in vitrobinding assay, we used pure recombinant proteins of afadin fusedto maltose-binding protein (MBP-afadin) and V12Rap1 tagged withsix repeats of His (His-V12Rap1). When His-V12Rap1 wasincubated with MBP-afadin immobilized on amylose resin beads,the direct binding of His-V12Rap1 to MBP-afadin was dose-dependent (Fig. 5B). As negative controls, His-V12Rap1 did notbind to MBP, nor did His-wild-type Rap1 (Rap1WT), on whichGTPS was not loaded, bind to MBP-afadin. In addition, the Rasassociation (RA) domain of afadin as well as full-length afadin wereco-immunoprecipitated with V12Rap1, but afadinRA, whichlacks the RA domain, was incapable of being co-immunoprecipitatedwith V12Rap1 (Fig. 5C). This indicates that the binding of afadinto the active form of Rap1 is mediated by the RA domain of afadin.

On the basis of these results, we examined whether the bindingof Rap1 to afadin is required for its localization at the leading edge,the formation of the leading edge, and directional cell movementin response to PDGF. When GFP-afadinRA was re-expressed in

afadin-knockdown NIH3T3 cells, this re-expression failed to rescuethe formation of the leading edge that was observed in wild-typeNIH3T3 cells expressing GFP and in afadin-knockdown NIH3T3cells re-expressing GFP-afadin (Fig. 3B). GFP-afadinRA did notaccumulate at the cell edge, indicating a role of Rap1 in therecruitment of afadin to the leading edge. Similarly, PDGF receptordid not accumulate at the cell edge (Fig. 3C). As expected, re-expression of GFP-afadinRA in afadin-knockdown NIH3T3 cellsdid not restore directional cell movement (Fig. 3D-F). These resultssuggest that afadin promotes the formation of the leading edge andPDGF-induced directional cell movement by binding to Rap1.

The activation of Rap1 has been reported to be mediated by anadaptor protein Crk, which directly interacts with phosphorylatedPDGF receptor, and by the Rap1 guanine exchange factor C3G,which is activated downstream of Crk (Ichiba et al., 1997;Matsumoto et al., 2000). Therefore, we hypothesized thataccumulation of the Rap1-afadin complex is involved in theassociation of Rap1 with PDGF receptor through Crk and C3G,and performed the immunoprecipitation assay to test this. V12Rap1was co-immunoprecipitated with PDGF receptor in the presence ofCrk and C3G (Fig. 5D). However, this co-immunoprecipitation wasperturbed by the dominant-negative mutant of Crk. This perturbationwas partial but statistically significant. Furthermore, such co-immunoprecipitation was observed at the endogenous protein level,

Fig. 4. Localization of afadin in movingNIH3T3 cells. (A)Colocalization of afadinwith v3 integrin, PDGF receptor andNecl-5 at the leading edge. After wild-typeNIH3T3 cells seeded on the-Slide VIFlow were directionally stimulated with 30ng/ml PDGF for 30 minutes from thebottom, cells were fixed and stained withthe indicated Abs. Arrowheads indicate thecolocalization of immunofluorescencesignals. (B,C)The bead-cell contact assay.After NIH3T3 cells were cultured with thevitronectin- or ConA-coated beads for 1hour, cells were fixed and stained with theindicated Abs. Insets show the highermagnification of the bead-cell contact sites.DIC, differential interference contrast;asterisks, beads. (D)Inhibition of PDGF-induced accumulation of PDGF receptor atthe leading edge by knockdown of afadin.After wild-type and afadin-knockdownNIH3T3 cells were treated as described inA, cells were fixed and stained with theanti-PDGF receptor pAb and the anti-actinmAb. Results are representative of threeindependent experiments. Scale bars:10m.

Jour

nal o

f Cel

l Sci

ence

4324

and was inhibited by Rap1GAP (Fig. 5E). The results from theseimmunoprecipitation assays and the immunofluorescencemicroscopy shown in Fig. 3 indicate that afadin associates withPDGF receptor through active Rap1 for its accumulation at theleading edge.

Inhibition of directional cell movement by blockade of theafadin-Rap1 interactionAs described above, the RA domain of afadin is essential for thebinding of afadin to Rap1. Therefore, we examined whether theRA domain itself inhibits the interaction between afadin and Rap1.

When GFP-afadin and GFP-afadin-RA were simultaneouslytransfected with FLAG-V12Rap1 in HEK293 cells, GFP-afadin-RA was preferentially co-immunoprecipitated with FLAG-V12Rap1and inhibited the interaction between GFP-afadin and FLAG-V12Rap1 (Fig. 6A), indicating an inhibitory effect of the RA domainof afadin on the afadin-Rap1 interaction. We next examined whetherblockade of the afadin-Rap1 interaction by overexpression of theRA domain affects the formation of the leading edge and directionalcell movement. After stimulation with PDGF, NIH3T3 cellsexpressing GFP formed the leading edge, whereas NIH3T3 cellsexpressing GFP-afadin-RA hardly formed the leading edge, similarto the situation observed in afadin-knockdown NIH3T3 cells (Fig.6B). Consistent with this, the PDGF-induced directional cellmovement determined by the Boyden chamber assay was impairedin NIH3T3 cells expressing GFP-afadin-RA compared with cellsexpressing GFP (Fig. 6C). These results suggest that the interactionbetween afadin and activated Rap1 is involved in formation of theleading edge, which is important for directional cell movement.

Involvement of afadin, Rap1 and SHP-2 in the stable formationof the leading edgeIn the last set of experiments, we investigated how the Rap1-afadincomplex regulates directional cell movement. We recentlydemonstrated that, in response to PDGF, SHP-2 binds to thetyrosine-phosphorylated form of afadin, and that formation of theSHP-2–afadin complex is necessary for the interaction betweenSHP-2 and the tyrosine-phosphorylated form of PDGF receptor andfor the proper regulation of the Ras-MEK-ERK pathway (Nakataet al., 2007). Appropriate activation of this signaling pathwaycontrols the formation of leading-edge structures throughreorganization of the actin cytoskeleton, resulting in enhanceddirectional cell movement (Chernyavsky et al., 2005; Ho et al., 2001;Huang et al., 2004). However, it remains elusive how the SHP-2–afadin complex, which crucially regulates the PDGF-inducedRas-ERK signaling, is recruited to PDGF receptor and how thiscomplex is associated with Rap1.

To explore these issues, we examined the intracellular localizationof afadin, SHP-2 and PDGF receptor in NIH3T3 cells in responseto PDGF. As expected, SHP-2 was recruited to the leading edgeand colocalized with PDGF receptor and afadin in wild-typeNIH3T3 cells, whereas this colocalization was absent in afadin-knockdown NIH3T3 cells (Fig. 7A). When GFP-afadin was re-expressed in afadin-knockdown NIH3T3 cells, the cells formed theleading edge, and the colocalization of SHP-2 with PDGF receptorand afadin at the leading edge was rescued (Fig. 7B). By contrast,when GFP-afadinRA was re-expressed instead of GFP-afadin, thecells failed to form the leading edge and SHP-2 did not colocalizewith PDGF receptor. These results suggest that the interaction ofafadin with Rap1 is necessary for the recruitment of SHP-2 to PDGFreceptor and that the associations between SHP-2, afadin and Rap1are crucial for PDGF-induced formation of the leading edge.

We further examined whether the associations between SHP-2,afadin and Rap1 are involved in the proper regulation of ERKactivation. As previously reported, ERK is hyperactivated in afadin-knockdown cells compared with wild-type NIH3T3 cells (Nakataet al., 2007). This hyperactivation was canceled by re-expressionof GFP-afadin in afadin-knockdown NIH3T3 cells, but not by re-expression of GFP-afadinRA (Fig. 7C). In addition, the associationbetween PDGF receptor and Necl-5, which plays a crucial role inthe formation of the leading edge, was reduced by expression ofGFP-afadinRA compared with that of GFP-afadin in HEK293 cells

Journal of Cell Science 122 (23)

Fig. 5. Association of afadin with Rap1 and PDGF receptor. (A)Co-immunoprecipitation of afadin with active Rap1. Lysates of HEK293 cellstransiently expressing the indicated molecules were immunoprecipitated withthe anti-FLAG mAb. The immunoprecipitates were subjected to westernblotting with the anti-GFP, anti-FLAG and anti-Myc mAbs. (B)Direct bindingof afadin to active Rap1. His-V12Rap1 bound to MBP-afadin was analyzed byprotein staining with Coomassie brilliant blue. As negative controls, MBP andHis-Rap1WT (GTPS-unloaded) were used. (C)Co-immunoprecipitation ofafadin mutants with active Rap1. The immunoprecipitation was performed asdescribed in A. The immunoprecipitates were subjected to western blottingwith the anti-GFP pAb and anti-FLAG mAb. (D)Co-immunoprecipitation ofPDGF receptor with active Rap1. The immunoprecipitation was performed asdescribed in A with the anti-HA mAb. The immunoprecipitates were subjectedto western blotting with the anti-GFP pAb and the anti-HA, anti-FLAG,andanti-Myc mAbs. WT, wild-type; DN, dominant-negative mutant. Arrowheadindicates the IgG heavy chain. Histogram shows the relative band intensity ofco-immunoprecipitated GFP-V12Rap1 in the right two lanes. The value of thesecond lane from the right is expressed as 1. *P<0.05. (E)Co-immunoprecipitation of endogenous PDGF receptor and afadin. Lysates ofNIH3T3 cells transiently expressing Myc-Rap1GAP or not, which werestimulated with 30 ng/ml PDGF for 10 minutes after serum starvation, wereimmunoprecipitated with the anti-PDGF receptor pAb or pre-immune IgG as acontrol. The immunoprecipitates were subjected to western blotting with theindicated Abs. Results are representative of three independent experiments.

Jour

nal o

f Cel

l Sci

ence

4325Novel role of afadin in cell movement

(Fig. 7D). Taken together, these results indicate that afadin, whichis recruited to the leading edge by binding to Rap1, supports theassociation of PDGF receptor with Necl-5 at the leading edge andthat afadin, together with Rap1, SHP-2 and ERK, positivelyregulates the formation of the leading edge, thus facilitating PDGF-induced directional cell movement.

DiscussionMoving cells form protrusions, such as lamellipodia and filopodia,ruffles, focal complexes and focal adhesions, by reorganization ofthe actin cytoskeleton at the leading edge. This reorganization ofthe actin cytoskeleton is performed by several F-actin-bindingproteins, including N-WASP, WAVEs, IQGAP1 and cortactin(Briggs and Sacks, 2003; Daly, 2004; Takai et al., 2001; Takenawaand Miki, 2001). In addition, many of these F-actin-binding proteinsfunction downstream of Cdc42 and Rac, the activation of which isinduced by growth factors in an integrin-dependent manner (Arthuret al., 2004; DeMali et al., 2003; Ridley, 2001). We recentlydemonstrated that PDGF-activated Rap1 subsequently induces theactivation of Rac and its downstream molecules and is crucial forthe formation of the leading edge and for cell movement (Takahashi

et al., 2008). A pool of afadin that localizes in the cytosol, but notat the cell-cell adhesion, is also shown to be involved in PDGF-induced formation of the leading edge by regulating the activationof ERK through SHP-2 (Nakata et al., 2007). However, the role ofafadin in cell movement was unclear: does afadin affect themobility of cells itself or does afadin only affect the directionalityof cell movement? It was also unclear how afadin, Rap1 and SHP-2 are involved in cell movement. Here, we show that afadin localizesat the leading edge of moving NIH3T3 cells where Necl-5, v3integrin and PDGF receptor (all of which are important for cellmovement) also localize, and that afadin preferentially controls thedirectionality of cell movement but does not affect the mobility ofcells.

We also revealed the molecular mechanisms involved in PDGF-induced and afadin-dependent formation of the leading edge. It hasbeen shown that Necl-5 physically interacts with v3 integrin andinduces the clustering of v3 integrin and PDGF receptor for theformation of leading edge structures including lamellipodia,peripheral ruffles, focal complexes and focal adhesions, eventuallyenhancing cell movement (Ikeda et al., 2004; Minami et al., 2007).These actions of Necl-5 are dependent on PDGF-induced activationof Rac and the binding of v3 integrin to its specific extracellularmatrix proteins, such as vitronectin and fibronectin (Minami et al.,2007). The extracellular region of Necl-5 directly interacts with theextracellular region of v3 integrin and is necessary for directionalcell movement, but not for random cell movement, whereas thecytoplasmic region of Necl-5 is necessary for both directional andrandom cell movement (Ikeda et al., 2004). In addition, previousstudies have clarified that PDGF receptor physically interacts withv3 integrin (Schneller et al., 1997; Woodard et al., 1998),suggesting the formation of a ternary complex with PDGF receptor,v3 integrin and Necl-5. This ternary complex is formed inresponse to PDGF at the leading edge, where Rap1 and Rac areactivated (Takahashi et al., 2008). Considering our recent andpresent results, on one hand, activated Rap1 causes reorganizationof the actin cytoskeleton together with Rac and other F-actin-bindingproteins (Miyata et al., 2009). On the other hand, activated Rap1recruits the afadin–SHP-2 complex to PDGF receptor and thiscomplex regulates PDGF-induced activation of ERK and formationof the leading edge (Fig. 8). These signaling networks in turnenhance the clustering of the Necl-5–v3-integrin–PDGF receptorcomplex in a feedback amplification manner and stabilize theformation of the well-spreading leading edge, resulting in thefacilitation of directional cell movement. Although there is no directevidence that ERK promotes the formation of the ternary complexwith Necl-5, v3 integrin and PDGF receptor at the leading edge,ERK participates in reorganization of the actin cytoskeleton byregulating the activity of myosin light chain (MLC) kinase and MLCphosphorylation (Klemke et al., 1997), and contributes to integrinactivation and focal adhesion turnover through the phosphorylationof focal adhesion kinase and paxillin (Chou et al., 2003; Hunger-Glaser et al., 2003; Liu et al., 2002). Thus, ERK might indirectlysupport and stabilize the formation of the ternary complex, but itsprecise role and mechanism of action remain to be clarified.

Our previous study demonstrated that PDGF-induced and PDGF-receptor-mediated activation of Rap1 through the Crk-C3G complexand the subsequent activation of Rac are essential for the formationof the leading edge and for directional cell movement (Takahashiet al., 2008). In addition to these observations, this present studyhas revealed that afadin, SHP-2 and ERK are also necessary forformation of the leading edge and for directional cell movement.

Fig. 6. Inhibitory effect of GFP-afadin-RA on PDGF-induced directional cellmovement. (A)Inhibition of the association of afadin with active Rap1 by theexpression of the RA domain of afadin. Lysates of HEK293 cells transientlyexpressing the indicated molecules were immunoprecipitated with the anti-FLAG mAb. The immunoprecipitates were subjected to western blotting withthe anti-GFP and anti-FLAG mAbs. (B)Morphology of NIH3T3 cellsexpressing GFP-afadin-RA in response to PDGF. After types of NIH3T3 cellswere seeded on the-Slide VI Flow, the cells were directionally stimulatedwith 30 ng/ml PDGF for 30 minutes from the bottom and were fixed andstained with the anti-afadin pAb (red). WT, wild-type. Scale bars: 10m.(C)Directional cell movement estimated by the Boyden chamber assay. Eachtype of NIH3T3 cells were incubated on cell-culture inserts coated withvitronectin in the presence of 30 ng/ml PDGF in the bottom well for 12 hours.The percentage of migrated cells was calculated as the number of migratedcells expressing GFP into the bottom well divided by the total number of cellsexpressing GFP. *P<0.05. Results are representative of three independentexperiments. Scale bars: 50m.

Jour

nal o

f Cel

l Sci

ence

4326

Considering this and previous studies, it is clear that several typesof molecules participate in and regulate directional cell movement.Although we have extensively investigated the mechanism ofdirectional cell movement, the underlying mechanism seems to bequite complicated and is not yet fully understood. Therefore, furtherstudies are required to better understand the underlying mechanisms.

In contrast to the results of our present study, one study has shownthat knockdown of afadin enhances cell movement and accelerateswound healing in epithelial MCF10A cells (Lorger and Moelling,2006). In that report, when afadin is knocked down, the intercellularadhesion mediated by E-cadherin is impaired because the associationof E-cadherin with p120ctn and F-actin becomes weaker. Thisimpaired intercellular adhesion loosens the cell-cell connection andincreases the directionality of cell movement in MCF10A cells.Similar to the results of Lorger and Moelling, the signal for N-cadherin at the cell-cell contact sites is reduced in afadin-knockdownNIH3T3 cells (data not shown). However, the directionality of cellmovement is attenuated in these NIH3T3 cells. Although the celltypes used in the experiments differ, the reason for this discrepancyremains to be elucidated, and further studies are therefore necessary.

There is a report that p120ctn regulates RhoA activation throughp190RhoGAP (Wildenberg et al., 2006). Both afadin and p120ctn

are peripheral membrane proteins and directly interact with nectinsand cadherins, respectively, and they can also interact with each

other (Hoshino et al., 2005). The knockdown of p120ctn in NIH3T3cells induces the enhancement of RhoA activation and formationof actin stress fibers and suppresses the velocity of the woundclosure: these phenotypes are similar to those observed in afadin-knockdown NIH3T3 cells, as shown in this work and in our recentstudy (Miyata et al., 2009). Wildenberg and colleagues propose thatp120ctn regulates RhoA activation through p190RhoGAP, whichinteracts with p120ctn (Wildenberg et al., 2006), whereas we haverecently demonstrated that afadin regulates the RhoA activationthrough Rap1 and ARAP1 (Miyata et al., 2009). ARAP1 is a Rap1-dependent RhoGAP. Afadin prevents the inactivation of Rap1,which is activated by PDGF at the leading edge, and promotes thebinding of activated Rap1 to ARAP1. This results in enhancementof the ARAP1 function in modulating the activation state of RhoA.Although the detailed molecular mechanisms are different, it mightbe of interest that afadin and p120ctn, which are both classified asperipheral membrane proteins, have similar effects on cellularphenotypes.

Afadin located at the leading edge is unlikely to bind to nectin,because nectin was not concentrated at the leading edge or at thecontact sites between the vitronectin-coated beads and the cellswhere afadin was concentrated (Fig. 4C). However, afadin isconcentrated and binds nectin at AJs after the initial formation ofcell-cell contact and the bound afadin supports the nectin-induced

Journal of Cell Science 122 (23)

Fig. 7. Involvement of Rap1 in the recruitment of the afadin–SHP-2complex to the leading edge and in stabilization of the formation ofthe leading edge by supporting the association of PDGF receptorwith Necl-5. (A)Colocalization of PDGF receptor with SHP-2 inresponse to PDGF. After wild-type (WT) or afadin-knockdown(KD) NIH3T3 cells were seeded on the-Slide VI Flow and weredirectionally stimulated with 30 ng/ml PDGF for 30 minutes fromthe bottom, the cells were fixed and stained with the indicated Abs.Scale bars: 10m. (B)Colocalization of PDGF receptor, SHP-2 andafadin in a Rap1-dependent manner. After afadin-knockdown cellsexpressing GFP-afadin or GFP-afadinRA were treated asdescribed in A, cells were fixed and stained with the indicated Abs.Arrowheads indicate the colocalization of immunofluorescencesignals. Scale bars: 10m. (C)Blockade of the hyperactivation ofERK by re-expression of GFP-afadin, but not GFP-afadinRA, inafadin-knockdown NIH3T3 cells. At 10 minutes after 15 ng/mlPDGF stimulation, lysates of types of NIH3T3 cells were subjectedto western blotting with the anti-phospho-ERK and anti-ERKmAbs. Histogram shows the relative band intensities ofphosphorylated ERK1 normalized to the total amount of ERK1 ascompared with the value from WT, which is expressed as 1. Errorbars indicate s.e.m. *P<0.05 vs. WT. (D)Association betweenPDGF receptor and Necl-5 in a Rap1-dependent manner. At 10minutes after 15 ng/ml PDGF stimulation, lysates of HEK293 cellstransfected with the indicated plasmids were immunoprecipitatedwith the anti-FLAG mAb. The immunoprecipitates were subjectedto western blotting with the anti-FLAG and anti-HA mAbs. Resultsare representative of three independent experiments.

Jour

nal o

f Cel

l Sci

ence

4327Novel role of afadin in cell movement

formation of AJs (Asakura et al., 1999; Takahashi et al., 1999). Onthe basis of these results, we conclude that afadin is localized in atleast two pools in two different states: free afadin at the leadingedge and nectin-bound afadin at the cell-cell contact site. Thus,afadin plays key roles, not only in the formation of cell-cell junctionsbetween contacting cells, but also in directional cell movement inmoving cells.

When normal cells become confluent, they cease movement andproliferation, and start to form cell-cell junctions (Abercrombie,1970; Zegers et al., 2003). This phenomenon is referred to as‘contact inhibition’ of cell movement and proliferation. Contactinhibition is also important for mesenchymal-epithelial transition(MET), which is observed during organogenesis in embryonicdevelopment. The mechanism underlying contact inhibition iscomplicated, but Necl-5 and nectin are at least involved in thismechanism (Fujito et al., 2005). In moving cells, Necl-5predominantly localizes at the leading edge and enhances cellmovement (Ikeda et al., 2004). However, Necl-5 is downregulatedfrom the cell surface by its trans-interaction with nectin-3 at thecell-cell contact sites, resulting in the reduction in cell movementand proliferation by inhibiting the signaling mediated by PDGFreceptor and v3 integrin (Fujito et al., 2005). We have shownhere that afadin at the leading edge of moving cells regulates cellmovement in addition to its important role in nectin-inducedformation of cell-cell junctions. During the formation of cell-celljunctions, afadin might also help Necl-5 to recruit nectin-3 as wellas help nectin-3 to recruit nectin-1 for their trans-interaction at thecell-cell adhesion sites. Therefore, afadin might play a crucial rolein MET through these functions.

Materials and MethodsCell culture and knockdown experimentsNIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% calf serum. Afadin-knockdown NIH3T3 cells were generatedas previously described (Kanzaki et al., 2008). Briefly, pEB-H1-afadin vectorcontaining a short hairpin RNA (shRNA) sequence against afadin was transfectedinto NIH3T3 cells, followed by selection with 500 g/ml G418 (Nacalai Tesque).

Afadin-knockdown NIH3T3 cells stably expressing GFP were generated byadditionally transfecting pCAGpuro-EGFP-N3 into afadin-knockdown NIH3T3 cells,followed by selection with both 500 g/ml G418 and 10 g/ml puromycin (Sigma-Aldrich). NIH3T3 cells stably expressing GFP were generated by transfectingpCAGpuro-EGFP-N3 into wild-type NIH3T3 cells, followed by selection with 10g/ml puromycin. HEK293 cells were cultured in DMEM supplemented with 10%fetal calf serum. For DNA transfection, Lipofectamine 2000 (Invitrogen) and anAmaxa Nucleofector kit (Amaxa) were used.

For rescue experiments, expression vectors for RNAi-resistant GFP-tagged rat full-length afadin (amino acids 1-1829; pMSCVpuro-GFP-afadin*) and GFP-afadinRA(amino acids 351-1829; pMSCVpuro-GFP-afadinRA) were created. Afadin-knockdown NIH3T3 cells stably expressing GFP-afadin and GFP-afadinRA weregenerated by retrovirus-mediated introduction and selection with both 500 g/ml G418and 10 g/ml puromycin, as previously described (Kakunaga et al., 2004; Kanzakiet al., 2008). AfadinRA cDNA did not contain the target sequence for afadin shRNA.

Plasmid constructionsV12Rap1B, in which a glycine residue at amino acid 12 of bovine Rap1B was replacedby a valine residue (V), was a constitutively active mutant of Rap1 and preparedusing the QuickChange site-directed mutagenesis kit (Stratagene). The cDNA ofRap1GAP was a gift from Patrick Casey (Duke University, Durham, NC). Expressionvectors for FLAG-Rap1WT, FLAG-V12Rap1, GFP-V12Rap1, Myc-Rap1GAP,FLAG-CrkI and Myc-C3G were prepared as previously described (Fukuyama et al.,2005). An expression vector for a FLAG-tagged dominant-negative mutant of CrkI(pIRM21-FLAG-CrkI-W169L) was kindly provided by Michiyuki Matsuda (KyotoUniversity, Kyoto, Japan). Expression vectors for GFP-afadin and GFP-afadinRAwere prepared as described (Nakata et al., 2007). Expression vectors for FLAG–Necl-5 and HA-PDGF receptor were prepared as described (Amano et al., 2008). Toconstruct an expression vector for GFP-afadin-RA, the rat afadin cDNA fragmentthat corresponds to amino acids 1-350 was subcloned into pEGFP-C1 (Clontech).

Antibodies and reagentsA rabbit anti-afadin polyclonal antibody (pAb) and a mouse anti-afadin monoclonalAb (mAb) were prepared as described (Sakisaka et al., 1999). A rat mAb against theextracellular region of Necl-5 (mAb-i, 1A8-8) was prepared as previously described(Ikeda et al., 2003). Hybridoma cells expressing a mouse anti-Myc mAb (9E10) wereobtained from American Type Culture Collection and prepared as described (Kodamaet al., 2000). Hamster anti-integrin v and 3 mAbs (H9.2B8 and 2C9.G2,respectively) were purchased from BD Biosciences. The following mouse mAbs werepurchased from commercial sources: anti-GM130 (Pharmingen), anti-N-cadherin(Pharmingen), anti-SHP-2 (Pharmingen), anti-actin (Chemicon), anti-phospho-ERK1/2 (Cell Signaling Technology), anti-FLAG (Sigma-Aldrich), anti-HA (Babco)and anti-GFP (Clontech). Rabbit anti-PDGF receptor (Y92) and anti-GAPDH(14C10) mAbs were purchased from Abcam and Cell Signaling Technology,respectively. The following rabbit pAbs were purchased from commercial sources:anti-PDGF receptor (Santa Cruz Biotechnology and Upstate), anti-Rap1GAP (SantaCruz Biotechnology), anti-ERK1/2 (Cell Signaling Technology), and anti-GFP(MBL). Fatty-acid-free BSA, trypsin inhibitor and 4,6-diamidino-2-phenylindole(DAPI) were purchased from Sigma-Aldrich. Horseradish-peroxidase-conjugated andfluorophore-conjugated secondary Abs were purchased from GE Healthcare andChemicon, respectively. Pre-immune IgG was purchased from JacksonImmunoResearch Laboratories. Vitronectin was purified from human plasma(Kohjinbio) as previously described (Yatohgo et al., 1988).

Directional stimulation by PDGFTo generate a concentration gradient of PDGF, a -Slide VI Flow (uncoated; Ibidi)was used as previously described (Minami et al., 2007). Briefly, cells were seededat a density of 5�103 cells/cm2 on the vitronectin-coated -Slide VI Flow, culturedfor 18 hours, and serum-starved with DMEM containing 0.5% BSA for 1 hour. Theconcentration gradient of 30 ng/ml PDGF was made according to the manufacturer’sprotocol. After 30 minutes, cells were fixed with acetone-methanol (1:1), incubatedwith 1% BSA in PBS, and then incubated with 20% BlockAce in PBS, followed byimmunofluorescence microscopy. Tracking of cell movement was analyzed by theChemotaxis and Migration Tool (version 1) (Ibidi), which was plugged into the ImageJsoftware (NIH, Bethesda, MD). The directionality of cell movement was defined asthe endpoint y-value divided by the accumulated distance. Data are expressed as means± s.e.m. of 13 randomly selected wild-type or afadin-knockdown NIH3T3 cells.

Assay for polarization of the Golgi complexPolarization of the Golgi complex in the direction of movement was assessed aspreviously described (Nobes and Hall, 1999). Briefly, correctly polarized cells weredefined as those that could reorient their Golgi complex to the 120° sector of thecells facing the wound front. Random polarization would mean that 33% of the cellshad a Golgi complex in any sector. Wider wounds (~200 m wide) were made inmonolayers of cells, and the cells fixed for 2 hours after wounding. In each experiment,at least 100 cells within the row of cells adjacent to the wound were examined. Dataare expressed as means ± s.e.m. of three independent experiments. Paired Student’st-test was performed for statistical analysis.

Fig. 8. Schematic model for the afadin-mediated stabilization of the leadingedge and directional cell movement. Details are given in the Discussion.

Jour

nal o

f Cel

l Sci

ence

4328

Boyden chamber assayThe Boyden chamber assay was performed as previously described (Fujito et al.,2005). Falcon cell-culture inserts (8.0-m pores; Becton Dickinson) were coated with3 g/ml vitronectin at 37°C for 1 hour. The inserts were then blocked with 1% BSAat 37°C for 30 minutes. The cells, which had been serum-starved in DMEMsupplemented with 0.5% fatty-acid-free BSA for 1 hour, were detached with 0.05%trypsin and 0.53 mM EDTA and then treated with a trypsin inhibitor. Cells were re-suspended in DMEM supplemented with 0.5% fatty-acid-free BSA and seeded at adensity of 2.5�104 cells per insert. The cells were incubated at 37°C for 12 hoursin the presence or absence of 30 ng/ml PDGF. After incubation, the inserts werewashed with PBS and the cells fixed using 3.7% formaldehyde and subsequentlystained with DAPI (Sigma-Aldrich). After removing the cells on the upper part ofthe filter using cotton sticks, the number of stained cells that crossed the filter wascounted in five randomly chosen fields per filter using a microscope. Data areexpressed as means ± s.e.m. of three independent experiments.

Western blotting and immunoprecipitationAfter being washed with ice-cold PBS, cells were harvested using pre-warmedLaemmli buffer containing 1 mM Na3VO4, 10 mM NaF and a phosphatase inhibitorcocktail (Sigma-Aldrich), boiled for 5 minutes, and sonicated three times for 10seconds with 20-second cooling periods. The protein concentrations of the sampleswere determined using an RC DC protein assay kit (Bio-Rad) with BSA as a referenceprotein. The samples were separated by SDS-PAGE, and this was followed by westernblotting with the indicated Abs. The band intensity measured by densitometry wasanalyzed using ImageJ software. For the immunoprecipitation assay, cells expressingvarious combinations of the indicated molecules were lysed with Buffer A (20 mMTris-HCl at pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% NP-40, 1 mMNa3VO4, 1 mM APMSF, 3 g/ml leupeptin and 5 g/ml aprotinin). The cell lysateswere centrifuged at 20,000 g for 15 minutes, and the supernatant was then incubatedwith the anti-FLAG mAb at 4°C for 2 hours, followed by incubation with protein-G-Sepharose beads at 4°C for 2 hours. After the beads had been extensively washedwith Buffer A, bound proteins were eluted from the beads by boiling in SDS samplebuffer for 5 minutes and were subjected to SDS-PAGE, followed by western blottingwith the indicated Abs.

Direct binding of afadin to Rap1Recombinant maltose-binding protein (MBP) and MBP-fused full-length afadin(MBP-afadin) were prepared as described (Hoshino et al., 2005; Nakata et al., 2007;Sakisaka et al., 2008). cDNA of wild-type bovine Rap1B or V12Rap1B was insertedinto the pQE-30 vector to express His-tagged Rap1WT (His-Rap1WT) or V12Rap1(His-V12Rap1) in E. coli. Purified His-V12Rap1 was preloaded with GTPS aspreviously described (Yamada et al., 2005). To examine the affinities of Rap1WT(GTPS-unloaded) and V12Rap1 (GTPS-loaded) for afadin, Rap1WT or His-V12Rap1-GTPS were incubated with MBP (20 pmol) or MBP-afadin (20 pmol)immobilized on 20 l of amylose resin beads in 400 l of Buffer B (25 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 M GTPS, 48 mM MgCl2, 8 mM EDTA, 1 mMDTT and 0.08% CHAPS) at 4°C for 2 hours. After extensively washing the beadswith Buffer B, the bound proteins were eluted by boiling the beads in the SDS samplebuffer. The samples were then subjected to SDS-PAGE and stained with Coomassiebrilliant blue.

Other proceduresAssays for phagokinetic track motility, wound healing and bead-cell contact wereperformed as previously described (Ikeda et al., 2004). In the wound-healing assay,the width of the wound space at 0, 2 and 8 hours after scratching the confluent cellmonolayer was measured at at least five different points in each experiment to quantifythe extent of wound closure (Goldfinger et al., 1999). Data are expressed as means± s.e.m. of three independent experiments.

We are grateful to Patrick Casey (Duke University), MichiyukiMatsuda (Kyoto University), Hitoshi Shibuya (Tokyo Medical andDental University), and Yoshihiro Miwa (University of Tsukuba) forthe generous gifts of reagents. This study was supported by grants-in-aid for Scientific Research and for Cancer Research and by TargetedProteins Research Program (TPRP) from the Ministry of Education,Culture, Sports, Science, and Technology (MEXT), Japan (2008), OnoMedical Research Foundation, Kanae Foundation for the Promotion ofMedical Science, and Hyogo Science and Technology Association.

ReferencesAbercrombie, M. (1970). Contact inhibition in tissue culture. In Vitro 6, 128-142.Amano, H., Ikeda, W., Kawano, S., Kajita, M., Tamaru, Y., Inoue, N., Minami, Y.,

Yamada, A. and Takai, Y. (2008). Interaction and localization of Necl-5 and PDGFreceptor at the leading edges of moving NIH3T3 cells: implications for directional cellmovement. Genes Cells 13, 269-284.

Arthur, W. T., Quilliam, L. A. and Cooper, J. A. (2004). Rap1 promotes cell spreadingby localizing Rac guanine nucleotide exchange factors. J. Cell Biol. 167, 111-122.

Asada, M., Irie, K., Morimoto, K., Yamada, A., Ikeda, W., Takeuchi, M. and Takai,Y. (2003). ADIP, a novel afadin- and -actinin-binding protein localized at cell-celladherens junctions. J. Biol. Chem. 278, 4103-4111.

Asakura, T., Nakanishi, H., Sakisaka, T., Takahashi, K., Mandai, K., Nishimura, M.,Sasaki, T. and Takai, Y. (1999). Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of thepolarized junctional alignment in epithelial cells. Genes Cells 4, 573-581.

Briggs, M. W. and Sacks, D. B. (2003). IQGAP proteins are integral components ofcytoskeletal regulation. EMBO Rep. 4, 571-574.

Chernyavsky, A. I., Arredondo, J., Karlsson, E., Wessler, I. and Grando, S. A. (2005).The Ras/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediatescholinergic regulation of keratinocyte directional migration. J. Biol. Chem. 280, 39220-39228.

Chou, F. L., Hill, J. M., Hsieh, J. C., Pouyssegur, J., Brunet, A., Glading, A., Uberall,F., Ramos, J. W., Werner, M. H. and Ginsberg, M. H. (2003). PEA-15 binding toERK1/2 MAPKs is required for its modulation of integrin activation. J. Biol. Chem.278, 52587-52597.

Daly, R. J. (2004). Cortactin signalling and dynamic actin networks. Biochem. J. 382, 13-25.

DeMali, K. A., Wennerberg, K. and Burridge, K. (2003). Integrin signaling to the actincytoskeleton. Curr. Opin. Cell Biol. 15, 572-582.

Fujito, T., Ikeda, W., Kakunaga, S., Minami, Y., Kajita, M., Sakamoto, Y., Monden,M. and Takai, Y. (2005). Inhibition of cell movement and proliferation by cell-cellcontact-induced interaction of Necl-5 with nectin-3. J. Cell Biol. 171, 165-173.

Fukuyama, T., Ogita, H., Kawakatsu, T., Fukuhara, T., Yamada, T., Sato, T., Shimizu,K., Nakamura, T., Matsuda, M. and Takai, Y. (2005). Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherensjunctions. J. Biol. Chem. 280, 815-825.

Goldfinger, L. E., Hopkinson, S. B., deHart, G. W., Collawn, S., Couchman, J. R. andJones, J. C. (1999). The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrincoordinately regulate wound healing in cultured epithelial cells and in the skin. J. CellSci. 112, 2615-2629.

Ho, W., Uniyal, S., Meakin, S. O., Morris, V. L. and Chan, B. M. (2001). A differentialrole of extracellular signal-regulated kinase in stimulated PC12 pheochromocytoma cellmovement. Exp. Cell Res. 263, 254-264.

Hoshino, T., Sakisaka, T., Baba, T., Yamada, T., Kimura, T. and Takai, Y. (2005).Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn. J.Biol. Chem. 280, 24095-24103.

Huang, C., Jacobson, K. and Schaller, M. D. (2004). MAP kinases and cell migration.J. Cell Sci. 117, 4619-4628.

Hunger-Glaser, I., Salazar, E. P., Sinnett-Smith, J. and Rozengurt, E. (2003). Bombesin,lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesionkinase phosphorylation at Ser-910: requirement for ERK activation. J. Biol. Chem. 278,22631-22643.

Ichiba, T., Kuraishi, Y., Sakai, O., Nagata, S., Groffen, J., Kurata, T., Hattori, S. andMatsuda, M. (1997). Enhancement of guanine-nucleotide exchange activity of C3G forRap1 by the expression of Crk, CrkL, and Grb2. J. Biol. Chem. 272, 22215-22220.

Ikeda, W., Nakanishi, H., Miyoshi, J., Mandai, K., Ishizaki, H., Tanaka, M., Togawa,A., Takahashi, K., Nishioka, H., Yoshida, H. et al. (1999). Afadin: A key moleculeessential for structural organization of cell-cell junctions of polarized epithelia duringembryogenesis. J. Cell Biol. 146, 1117-1132.

Ikeda, W., Kakunaga, S., Itoh, S., Shingai, T., Takekuni, K., Satoh, K., Inoue, Y.,Hamaguchi, A., Morimoto, K., Takeuchi, M. et al. (2003). Tage4/Nectin-like molecule-5 heterophilically trans-interacts with cell adhesion molecule nectin-3 and enhances cellmigration. J. Biol. Chem. 278, 28167-28172.

Ikeda, W., Kakunaga, S., Takekuni, K., Shingai, T., Satoh, K., Morimoto, K., Takeuchi,M., Imai, T. and Takai, Y. (2004). Nectin-like molecule-5/Tage4 enhances cellmigration in an integrin-dependent, nectin-3-independent manner. J. Biol. Chem. 279,18015-18025.

Kakunaga, S., Ikeda, W., Shingai, T., Fujito, T., Yamada, A., Minami, Y., Imai, T. andTakai, Y. (2004). Enhancement of serum- and platelet-derived growth factor-inducedcell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling. J. Biol. Chem. 279, 36419-36425.

Kanzaki, N., Ogita, H., Komura, H., Ozaki, M., Sakamoto, Y., Majima, T., Ijuin, T.,Takenawa, T. and Takai, Y. (2008). Involvement of the nectin-afadin complex in PDGF-induced cell survival. J. Cell Sci. 121, 2008-2017.

Klemke, R. L., Cai, S., Giannini, A. L., Gallagher, P. J., de Lanerolle, P. and Cheresh,D. A. (1997). Regulation of cell motility by mitogen-activated protein kinase. J. CellBiol. 137, 481-492.

Kodama, A., Matozaki, T., Fukuhara, A., Kikyo, M., Ichihashi, M. and Takai, Y. (2000).Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growthfactor/scatter factor-induced cell scattering. Mol. Biol. Cell 11, 2565-2575.

Komura, H., Ogita, H., Ikeda, W., Mizoguchi, A., Miyoshi, J. and Takai, Y. (2008).Establishment of cell polarity by afadin during the formation of embryoid bodies. GenesCells 13, 79-90.

Kupfer, A., Louvard, D. and Singer, S. J. (1982). Polarization of the Golgi apparatus andthe microtubule-organizing center in cultured fibroblasts at the edge of an experimentalwound. Proc. Natl. Acad. Sci. USA 79, 2603-2607.

Linnemann, T., Geyer, M., Jaitner, B. K., Block, C., Kalbitzer, H. R., Wittinghofer,A. and Herrmann, C. (1999). Thermodynamic and kinetic characterization of theinteraction between the Ras binding domain of AF6 and members of the Ras subfamily.J. Biol. Chem. 274, 13556-13562.

Journal of Cell Science 122 (23)

Jour

nal o

f Cel

l Sci

ence

4329Novel role of afadin in cell movement

Liu, Z. X., Yu, C. F., Nickel, C., Thomas, S. and Cantley, L. G. (2002). Hepatocytegrowth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association. J. Biol. Chem. 277, 10452-10458.

Lorger, M. and Moelling, K. (2006). Regulation of epithelial wound closure andintercellular adhesion by interaction of AF6 with actin cytoskeleton. J. Cell Sci. 119,3385-3398.

Mandai, K., Nakanishi, H., Satoh, A., Obaishi, H., Wada, M., Nishioka, H., Itoh, M.,Mizoguchi, A., Aoki, T., Fujimoto, T. et al. (1997). Afadin: a novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherensjunction. J. Cell Biol. 139, 517-528.

Mandai, K., Nakanishi, H., Satoh, A., Takahashi, K., Satoh, K., Nishioka, H.,Mizoguchi, A. and Takai, Y. (1999). Ponsin/SH3P12: an l-afadin- and vinculin-bindingprotein localized at cell-cell and cell-matrix adherens junctions. J. Cell Biol. 144, 1001-1017.

Matsubayashi, Y., Ebisuya, M., Honjoh, S. and Nishida, E. (2004). ERK activationpropagates in epithelial cell sheets and regulates their migration during wound healing.Curr. Biol. 14, 731-735.

Matsumoto, T., Yokote, K., Take, A., Takemoto, M., Asaumi, S., Hashimoto, Y.,Matsuda, M., Saito, Y. and Mori, S. (2000). Differential interaction of CrkII adaptorprotein with platelet-derived growth factor alpha- and beta-receptors is determined byits internal tyrosine phosphorylation. Biochem. Biophys. Res. Commun. 270, 28-33.

Minami, Y., Ikeda, W., Kajita, M., Fujito, T., Amano, H., Tamaru, Y., Kuramitsu, K.,Sakamoto, Y., Monden, M. and Takai, Y. (2007). Necl-5/poliovirus receptor cis-interacts with integrin v3 and enhances its clustering. J. Biol. Chem. 282, 18481-18496.

Miyata, M., Rikitake, Y., Takahashi, M., Nagamatsu, Y., Yamauchi, Y., Ogita, H.,Hirata, K. I. and Takai, Y. (2009). Regulation by Afadin of cyclical activation andinactivation of Rap1, Rac1, and RhoA Small G proteins at leading edges of movingNIH3T3 cells. J. Biol. Chem. 284, 24595-24609.

Nagamatsu, Y., Rikitake, Y., Takahashi, M., Deki, Y., Ikeda, W., Hirata, K.-I. andTakai, Y. (2008). Roles of Necl-5/poliovirus receptor and Rho-associated kinase(ROCK) in the regulation of transformation of integrin v3-based focal complexes intofocal adhesions. J. Biol. Chem. 283, 14532-14541.

Nakata, S., Fujita, N., Kitagawa, Y., Okamoto, R., Ogita, H. and Takai, Y. (2007).Regulation of platelet-derived growth factor receptor activation by afadin through SHP-2: implications for cellular morphology. J. Biol. Chem. 282, 37815-37825.

Nobes, C. D. and Hall, A. (1999). Rho GTPases control polarity, protrusion, and adhesionduring cell movement. J. Cell Biol. 144, 1235-1244.

Ogita, H. and Takai, Y. (2008). Cross-talk among integrin, cadherin, and growth factorreceptor: roles of nectin and nectin-like molecule. Int. Rev. Cytol. 265, 1-54.

Ooshio, T., Irie, K., Morimoto, K., Fukuhara, A., Imai, T. and Takai, Y. (2004).Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectinand E-cadherin, through afadin and alpha-actinin in epithelial cells. J. Biol. Chem. 279,31365-31373.

Ooshio, T., Fujita, N., Yamada, A., Sato, T., Kitagawa, Y., Okamoto, R., Nakata, S.,Miki, A., Irie, K. and Takai, Y. (2007). Cooperative roles of Par-3 and afadin in theformation of adherens and tight junctions. J. Cell Sci. 120, 2352-2365.

Pokutta, S., Drees, F., Takai, Y., Nelson, W. J. and Weis, W. I. (2002). Biochemical andstructural definition of the l-afadin- and actin-binding sites of alpha-catenin. J. Biol.Chem. 277, 18868-18874.

Ridley, A. J. (2001). Rho GTPases and cell migration. J. Cell Sci. 114, 2713-2722.Sakisaka, T., Nakanishi, H., Takahashi, K., Mandai, K., Miyahara, M., Satoh, A.,

Takaishi, K. and Takai, Y. (1999). Different behavior of l-afadin and neurabin-II duringthe formation and destruction of cell-cell adherens junction. Oncogene 18, 1609-1617.

Sakisaka, T., Ikeda, W., Ogita, H., Fujita, N. and Takai, Y. (2007). The roles of nectinsin cell adhesions: cooperation with other cell adhesion molecules and growth factorreceptors. Curr. Opin. Cell Biol. 19, 593-602.

Sakisaka, T., Yamamoto, Y., Mochida, S., Nakamura, M., Nishikawa, K., Ishizaki, H.,Okamoto-Tanaka, M., Miyoshi, J., Fujiyoshi, Y., Manabe, T. et al. (2008). Dual

inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitterrelease. J. Cell Biol. 183, 323-337.

Schneller, M., Vuori, K. and Ruoslahti, E. (1997). v3 integrin associates with activatedinsulin and PDGF receptors and potentiates the biological activity of PDGF. EMBOJ. 16, 5600-5607.

Schvartz, I., Seger, D. and Shaltiel, S. (1999). Vitronectin. Int. J. Biochem. Cell Biol. 31,539-544.

Tachibana, K., Nakanishi, H., Mandai, K., Ozaki, K., Ikeda, W., Yamamoto, Y.,Nagafuchi, A., Tsukita, S. and Takai, Y. (2000). Two cell adhesion molecules, nectinand cadherin, interact through their cytoplasmic domain-associated proteins. J. Cell Biol.150, 1161-1176.

Takahashi, K., Nakanishi, H., Miyahara, M., Mandai, K., Satoh, K., Satoh, A., Nishioka,H., Aoki, J., Nomoto, A., Mizoguchi, A. et al. (1999). Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions throughinteraction with Afadin, a PDZ domain-containing protein. J. Cell Biol. 145, 539-549.

Takahashi, M., Rikitake, Y., Nagamatsu, Y., Hara, T., Ikeda, W., Hirata, K.-i. andTakai, Y. (2008). Sequential activation of Rap1 and Rac1 small G proteins by PDGFlocally at leading edges of NIH3T3 cells. Genes Cells 13, 549-569.

Takai, Y. and Nakanishi, H. (2003). Nectin and afadin: novel organizers of intercellularjunctions. J. Cell Sci. 116, 17-27.

Takai, Y., Sasaki, T. and Matozaki, T. (2001). Small GTP-binding proteins. Physiol. Rev.81, 153-208.

Takai, Y., Irie, K., Shimizu, K., Sakisaka, T. and Ikeda, W. (2003). Nectins and nectin-like molecules: roles in cell adhesion, migration, and polarization. Cancer Sci. 94, 655-667.

Takai, Y., Ikeda, W., Ogita, H. and Rikitake, Y. (2008a). The immunoglobulin-like celladhesion molecule nectin and its associated protein afadin. Annu. Rev. Cell Dev. Biol.24, 309-342.

Takai, Y., Miyoshi, J., Ikeda, W. and Ogita, H. (2008b). Nectins and nectin-like molecules:roles in contact inhibition of cell movement and proliferation. Nat. Rev. Mol. Cell. Biol.9, 603-615.

Takenawa, T. and Miki, H. (2001). WASP and WAVE family proteins: key molecules forrapid rearrangement of cortical actin filaments and cell movement. J. Cell Sci. 114, 1801-1809.

Wildenberg, G. A., Dohn, M. R., Carnahan, R. H., Davis, M. A., Lobdell, N. A.,Settleman, J. and Reynolds, A. B. (2006). p120-catenin and p190RhoGAP regulatecell-cell adhesion by coordinating antagonism between Rac and Rho. Cell 127, 1027-1039.

Woodard, A. S., Garcia-Cardena, G., Leong, M., Madri, J. A., Sessa, W. C. andLanguino, L. R. (1998). The synergistic activity of v3 integrin and PDGF receptorincreases cell migration. J. Cell Sci. 111, 469-478.

Yamada, A., Fujita, N., Sato, T., Okamoto, R., Ooshio, T., Hirota, T., Morimoto, K.,Irie, K. and Takai, Y. (2006). Requirement of nectin, but not cadherin, for formationof claudin-based tight junctions in annexin II-knockdown MDCK cells. Oncogene 25,5085-5102.

Yamada, T., Sakisaka, T., Hisata, S., Baba, T. and Takai, Y. (2005). RA-RhoGAP, Rap-activated Rho GTPase-activating protein implicated in neurite outgrowth through Rho.J. Biol. Chem. 280, 33026-33034.

Yatohgo, T., Izumi, M., Kashiwagi, H. and Hayashi, M. (1988). Novel purification ofvitronectin from human plasma by heparin affinity chromatography. Cell Struct. Funct.13, 281-292.

Zegers, M. M., Forget, M. A., Chernoff, J., Mostov, K. E., ter Beest, M. B. and Hansen,S. H. (2003). Pak1 and PIX regulate contact inhibition during epithelial wound healing.EMBO J. 22, 4155-4165.

Zhadanov, A. B., Provance, D. W., Jr, Speer, C. A., Coffin, J. D., Goss, D., Blixt, J.A., Reichert, C. M. and Mercer, J. A. (1999). Absence of the tight junctional proteinAF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.Curr. Biol. 9, 880-888.

Jour

nal o

f Cel

l Sci

ence