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Binding of APC and dishevelled mediates Wnt5a-regulated focal adhesion dynamics in migrating cells

2010; Springer Nature; Volume: 29; Issue: 7 Linguagem: Inglês

10.1038/emboj.2010.26

1460-2075

Shinji Matsumoto, Katsumi Fumoto, Tetsuji Okamoto, Kozo Kaibuchi, Akira Kikuchi,

Cellular Mechanics and Interactions

Article11 March 2010free access Binding of APC and dishevelled mediates Wnt5a-regulated focal adhesion dynamics in migrating cells Shinji Matsumoto Shinji Matsumoto Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan Search for more papers by this author Katsumi Fumoto Katsumi Fumoto Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, Netherlands Search for more papers by this author Tetsuji Okamoto Tetsuji Okamoto Department of Molecular Oral Medicine and Maxillofacial Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Kozo Kaibuchi Kozo Kaibuchi Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Akira Kikuchi Corresponding Author Akira Kikuchi Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan Search for more papers by this author Shinji Matsumoto Shinji Matsumoto Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan Search for more papers by this author Katsumi Fumoto Katsumi Fumoto Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, Netherlands Search for more papers by this author Tetsuji Okamoto Tetsuji Okamoto Department of Molecular Oral Medicine and Maxillofacial Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Kozo Kaibuchi Kozo Kaibuchi Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Akira Kikuchi Corresponding Author Akira Kikuchi Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan Search for more papers by this author Author Information Shinji Matsumoto1, Katsumi Fumoto2, Tetsuji Okamoto3, Kozo Kaibuchi4 and Akira Kikuchi 1 1Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Japan 2Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, Netherlands 3Department of Molecular Oral Medicine and Maxillofacial Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan 4Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan *Corresponding author. Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 3410; Fax: +81 6 6879 3419; E-mail: [email protected] The EMBO Journal (2010)29:1192-1204https://doi.org/10.1038/emboj.2010.26 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Wnt5a is a representative ligand that activates the Wnt/β-catenin-independent pathway, resulting in the regulation of cell adhesion, migration, and polarity, but its molecular mechanism is poorly understood. This report shows that Dishevelled (Dvl) binds to adenomatous polyposis coli (APC) gene product, and this binding is enhanced by Wnt5a. Dvl was involved in the stabilization of the plus end dynamics of microtubules as well as APC. Frizzled2 (Fz2) was present with Wnt5a at the leading edge of migrating cells and formed a complex with APC through Dvl. Fz2 also interacted with integrins at the leading edge, and Dvl and APC associated with and activated focal adhesion kinase and paxillin. The binding of APC to Dvl enhanced the localization of paxillin to the leading edge and was involved in Wnt5a-dependent focal adhesion turnover. Furthermore, this new Wnt5a signalling pathway was important for the epithelial morphogenesis in the three-dimensional culture. These results suggest that the functional and physical interaction of Dvl and APC is involved in Wnt5a/Fz2-dependent focal adhesion dynamics during cell migration and epithelial morphogenesis. Introduction Wnts are secretory proteins that are essential for animal development (Logan and Nusse, 2004). A subset of Wnts activates the β-catenin-independent pathways (also referred to as non-canonical Wnt pathways) that primarily modulate cell movement, as initially observed during embryogenesis (Veeman et al, 2003; Kikuchi and Yamamoto, 2008). This activation occurs through multiple mechanisms, which overlap with other signalling pathways, including Rac, Rho, Jun N-terminal kinase (JNK), Rho-associated kinase, and protein kinase C (PKC). Wnt5a, which is a representative ligand that activates the β-catenin-independent pathway, is known to regulate cell adhesion, migration, and polarity (Weeraratna et al, 2002; Veeman et al, 2003; Kikuchi and Yamamoto, 2008). Although its molecular mechanism is not well understood, possible mechanisms have been demonstrated. For instance, Wnt5a activates JNK in convergent extension of Xenopus embryos (Yamanaka et al, 2002) or recruits actin, myosin IIB, and melanoma cell adhesion molecule into a polarized structure in migrating melanoma cells (Witze et al, 2008). It has also been shown that Wnt5a activates Rac and focal adhesion kinase (FAK) and that it is required for the turnover of focal adhesions in migrating cells (Kurayoshi et al, 2006, 2007). Dishevelled (Dvl) is a critical component of Wnt signalling and mediates both the β-catenin-dependent and -independent pathways (Wharton, 2003). It has been shown that Dvl is observed as punctate cytoplasmic structures (Kishida et al, 1999; Capelluto et al, 2002), which correlates to the protein assembling, and translocated to the cell-surface membrane in a Wnt-dependent manner (Schwarz-Romond et al, 2007). It has also been reported that Dvl localizes to focal adhesions in mammalian cultured cells (Torres and Nelson, 2000) and that its translocation to the cell-surface membrane is dependent on fibronectin in Xenopus embryos (Marsden and DeSimone, 2001). In addition, Dvl is known to stabilize microtubules by binding directly to them (Krylova et al, 2000; Ciani et al, 2004). These results suggest that Dvl localizes to specific sites where it regulates the cytoskeleton and cell-substrate adhesion, but the involvement of Dvl in Wnt5a-dependent cell-substrate adhesion and cell migration has not yet been clarified. The adhesion of a cell to a substrate (cell-substrate adhesion) is necessary for the cell to spread and migrate (Small and Kaverina, 2003). Small focal adhesions, referred to as focal complexes, at the cell periphery of spreading and migrating cells are regulated by small GTP-binding proteins (G proteins) Rac and Cdc42 (Etienne-Manneville and Hall, 2002). On the generation of tension, these small adhesions can mature into large, more organized adhesions, such as focal adhesions, which are found both at the cell periphery and more centrally. Spreading and migrating cells continuously form and disassemble their adhesions at the leading edge by the activation of small G proteins and FAK (Mitra et al, 2005). Recent imaging studies have implicated microtubule targeting to focal adhesions in focal adhesion disassembly, although the molecular mechanism is not fully understood (Small and Kaverina, 2003). Adenomatous polyposis coli (APC) protein was originally identified as a tumour suppressor of human colon cancer and subsequently found to be involved in the stability of β-catenin in Wnt signalling (Polakis, 2000; Aoki and Taketo, 2007). Evidence has shown that APC regulates cell–cell adhesion, cell polarization, and migration through the stabilization of microtubules by binding to their plus ends (Mimori-Kiyosue et al, 2000; Dikovskaya et al, 2001). APC is transported along microtubules by kinesin motor protein complexes containing KAP3 and accumulates at microtubule plus ends specifically at the protrusion of the migrating cells (Akiyama and Kawasaki, 2006; Kroboth et al, 2007). As Wnt5a and Dvl are involved in the accumulation of APC to the plus end of microtubules (Schlessinger et al, 2007), it has been suggested that Wnt5a, Dvl, and APC interact functionally to regulate cell-substrate adhesions and migration. However, how Wnt5a signalling links to the regulation of focal adhesions remains to be clarified. Here, it is reported that Dvl bound to APC directly and that the complex played a role in the Wnt5a-dependent formation of cell-substrate adhesions and polarized cell migration. It was also shown that Frizzled2 (Fz2), a Wnt5a receptor, is concentrated to the leading edge of migrating cells where it forms a complex with APC through Dvl. Furthermore, Fz2 interacted with integrins at the leading edge, and Dvl and APC associated and colocalized with FAK and paxillin. These results show that Dvl and APC mediate a Wnt5a-Fz2 signal to focal adhesions to regulate cell-substrate adhesions and migration. Results Roles of Wnt5a, Dvl, and APC in cell-substrate adhesion and migration To investigate whether Dvl mediates the ability of Wnt5a to regulate cell-substrate adhesion and migration, the phenotypes by Dvl knockdown cell lines were analysed. The cells in which all Dvls (Dvl1, Dvl2, and Dvl3) were knocked down were used (Supplementary Figure S1A). Adhesion of HeLaS3 (human cervical cancer) cells to collagen or fibronectin was suppressed by the knockdown of Dvls (Figure 1A). In Dvl knockdown cells, the levels of α2, α3, α5, αv, β1, and β3 integrins were unchanged (Supplementary Figure S1B), suggesting that Dvl is necessary for cell-substrate signalling after integrin engagement with extracellular matrix proteins. When cells were seeded into a collagen-coated dish from a suspension culture, FAK was activated as assessed by the phosphorylation of Tyr397 (pY397), and paxillin was phosphorylated at Tyr118 (pY118) (Figure 1B). The cell adhesion-dependent activation of FAK and phosphorylation of paxillin were dramatically reduced in Dvl knockdown cells (Figure 1B). Immunocytochemical analyses also showed that depletion of Dvl inhibits the activation (FAK pY397 staining) and formation (FAK and cortical actin staining) of cell-substrate adhesion at the cell periphery in the initiation of cell spreading (Supplementary Figure S2A). Knockdown of Dvl in Vero (monkey kidney epithelium) cells also suppressed cell-substrate adhesion and FAK activation (Supplementary Figure S3), suggesting that these functions of Dvl are conserved in at least HeLaS3 and Vero cells. Figure 1.Dvl is required for cell-substrate adhesion. (A) HeLaS3 cells transfected with Dvl siRNA were plated on collagen (Col) or fibronectin (FN) for 15 min and subjected to the adhesion assay. The results are expressed as the ratio of adhesion activity of cells treated with control siRNA and indicate means±s.e. from three independent experiments. (B) After HeLaS3 cells transfected with Dvl siRNA were suspended in serum-free medium, they were kept in suspension or plated onto collagen-coated dishes for 1 h. Lysates were probed with anti-pY397-FAK and anti-pY118-paxillin antibodies. FAK and paxillin were used as loading controls. (C) HeLaS3 cells transfected with Dvl or Wnt5a siRNA were stained with anti-paxillin antibody. The individual areas of paxillin staining were quantified in five different focal adhesions per cell (counted cells: 20 per siRNA), and the results were shown in the lower right panel. The region in the white box is shown enlarged. (D) The dynamics of GFP-paxillin in HeLaS3 cells transfected with Dvl siRNA were visualized. The percentages of adhesions turning over within 45 min were calculated for 10 different focal adhesions per cell (counted cells: 20 per siRNA), and the results were shown in the bottom panel. The region in the white box is shown enlarged. (E) HeLaS3 cells transfected with Dvl siRNA were stained with anti-paxillin and anti-tubulin antibodies. The numbers of microtubules in a 1 μm × 10 μm area (white box) from the cell edge were counted (counted cells: 30 per siRNA). Scale bars in (C) and (D), 10 μm; in (E), 2 μm. *P<0.01. Download figure Download PowerPoint Knockdown of Dvl induced enlargement of the sizes of focal adhesions, which indicated the inhibition of the focal adhesion turnover, and expression of FLAG-Dvl2 rescued this phenotype (Figure 1C; Supplementary Figures S1C and S2B). When the processes of focal adhesion formation were visualized in a time-lapse imaging study, dynamic remodelling of focal adhesions was less dynamic in Dvl knockdown cells (Figure 1D). These results suggested that Dvl regulates the dynamics of focal adhesions. It has been shown that microtubule growth promotes the disassembly of focal adhesions (Small and Kaverina, 2003). Although microtubules extended towards the cell edges and got over focal adhesions in control cells, knockdown of Dvl created a microtubule-free border behind the adhesion sites (Figure 1E). The numbers of microtubule filaments were also decreased more at the periphery of Dvl knockdown cells when cells were treated with a low dose of nocodazole, which affects the dynamics of the plus ends of microtubules (Supplementary Figure S2C). In addition, cell adhesion-dependent extension of microtubules to the cell periphery was reduced in Dvl knockdown cells (Supplementary Figure S2D). Consistent with these observations, knockdown of Dvl in HeLaS3 and NIH3T3 cells reduced cell migration in transwell migration and wound-healing assays (Supplementary Figure S4). Taken together, these results showed that Dvl is involved in the regulation of the dynamics of cell-substrate adhesion and migration probably through the stabilization of microtubules at the cell periphery. It has already been shown that overexpression of Wnt5a enhances cell-substrate adhesion and adhesion-dependent FAK activation (Kurayoshi et al, 2006, 2007). Consistent with the results, knockdown of Wnt5a decreased these cellular responses (Supplementary Figure S5A–C). In addition, knockdown of Wnt5a induced enlargement of the sizes of focal adhesions and enhanced nocodazole sensitivity of microtubules predominantly at the cell periphery as well as knockdown of Dvl (Figure 1C; Supplementary Figure S5D). Although it is known that APC regulates cell–cell adhesion, cell migration, and microtubule dynamics, the involvement of APC in cell-substrate adhesion is not clear. Knockdown of APC decreased cell-substrate adhesion activity and suppressed adhesion-dependent FAK activation and paxillin phosphorylation as well as knockdown of Dvl (Supplementary Figure S6A–C). Furthermore, extension of microtubules to the cell periphery in initial cell spreading was reduced in APC knockdown cells (Supplementary Figure S6D). Thus, the knockdown of Wnt5a, Dvl, and APC showed similar phenotypes, suggesting that there are functional relationships between Wnt5a, Dvl, and APC in cell-substrate adhesion and cell migration. Interaction of Dvl with APC To address this possibility, the physiological interaction between Dvl and APC was examined. Dvl2 and APC formed a complex at endogenous levels (Figure 2A and B). Deletion mutant analyses showed that the C-terminal region of Dvl1 (Dvl1(DEP+)) interacts with APC in COS7 cells and that the armadillo domain of APC (APC(Arm+)) formed a complex with Dvl2 (Figure 2C–E). In vitro binding studies using recombinant proteins revealed that GST-APC(Arm+) binds to MBP-Dvl1 directly in a dose-dependent manner (Figure 2F). Furthermore, MBP-APC(Arm+) bound to GST-Dvl1(395–670) directly (Supplementary Figure S7). Figure 2.Dvl interacts with APC. (A) Lysates of HeLaS3 cells were immunoprecipitated with anti-Dvl (DIX) antibody and the immunoprecipitates were probed with the indicated antibodies. (B) Lysates of MDCK cells were immunoprecipitated with anti-APC antibody and the immunoprecipitates were probed with the indicated antibodies. (C) Regions of Dvl and APC required for the interaction are shown. Amino acid numbers are indicated. (D) Lysates of COS7 cells expressing HA-Dvl1 deletion mutants were immunoprecipitated with anti-APC antibody. The immunoprecipitates were probed with anti-HA and anti-APC antibodies. Arrowheads indicate HA-Dvl1(WT) and HA-Dvl1(DEP+) coimmunoprecipitated with endogenous APC. (E) Lysates of COS7 cells expressing EGFP-APC deletion mutants were immunoprecipitated with anti-GFP antibody. The immunoprecipitates were probed with anti-Dvl2 and anti-GFP antibodies. An arrowhead indicates endogenous Dvl2 coimmunoprecipitated with EGFP-APC(Arm+). (F) The indicated concentrations of GST-APC(Arm+) were incubated with MBP-Dvl1. Band intensities of GST-APC(Arm+) precipitated with MBP-Dvl1 were quantified in the bottom panel. Download figure Download PowerPoint FLAG-Dvl2 was localized to the leading edge where F-actin was accumulated and microtubules were extended in polarized Vero cells (Figure 3A). Endogenous Dvl2, Dvl3, and APC were detected at the leading edge to where the ends of microtubules were extended, and localization of APC was more restrictive than that of Dvl (Figure 3B). Knockdown of Dvl or APC reduced their staining at the leading edge (Supplementary Figure S8A and B), indicating that these findings are not simply because of non-specific signals by antibodies. It was also observed that ectopically expressed FLAG-Dvl2 is colocalized with APC-GFP to the cell edge in liver progenitor HPPL cells (Supplementary Figure S8C). Consistent with the previous observations using embryonic fibroblasts (Schlessinger et al, 2007), knockdown of Dvl in Vero and HPPL cells decreased the localization of APC to the leading edge (Supplementary Figure S9A and B), whereas knockdown of APC did not affect the localization of Dvl (Supplementary Figure S9C). Localization of Dvl and APC at the leading edge was decreased significantly by a high dose of nocodazole to disrupt microtubules (Supplementary Figure S9D). These results suggested that Dvl is localized to the leading edge independently of APC, whereas Dvl is required for the localization of APC to the leading edge of polarized cells. In addition, their membrane localization would depend on microtubules. Figure 3.APC localizes to the cell periphery with Dvl in response to Wnt5a. (A) Vero cells expressing FLAG-Dvl2 were plated onto collagen-coated dishes for 1 h, followed by the staining with anti-FLAG antibody and phalloidin or anti-tubulin antibody. Cortical localization of FLAG-Dvl2 was observed in 42% of 30 cells expressing FLAG-Dvl2. (B) Vero cells were fixed with methanol and stained with anti-β-tubulin, anti-Dvl2+3 (a mixture of anti-Dvl2 and anti-Dvl3 antibodies), and anti-APC antibodies. Polarized Dvl at the cell cortex was observed in 42% of 96 cells and colocalization of Dvl with APC was observed in 27% of 41 cells with cortical Dvl staining. (C) After NIH3T3 cells were treated with Wnt5a or Wnt3a for 30 min, lysates were immunoprecipitated with anti-Dvl (DIX) antibody. The immunoprecipitates were probed with anti-APC and anti-Dvl2 antibodies, and band intensities of precipitated APC were quantified in the right-hand panel. (D) After HeLaS3 cells were serum-starved for 36 h and stimulated with or without 400 ng/ml purified Wnt5a for 60 min, the cells were stained with anti-APC antibody. Cells with a high intensity of APC at the cell periphery that was 1.5–2 times stronger than at the cell centre were defined as (+), and 2 times or more stronger were defined as (++). Cells with accumulated APC (−, +, ++) at the cell periphery was counted in at least 50 cells per each treatment. Scale bars in (A), (B), and (D), 10 μm. *P<0.01. Download figure Download PowerPoint To clarify the role of interaction of Dvl and APC at the cell periphery, the effect of Wnts on the complex formation was examined. Wnt5a enhanced the formation of a complex between Dvl and APC in intact cells but Wnt3a did not (Figure 3C). Consistent with these results, Wnt5a induced the accumulation of APC to the cell periphery (Figure 3D). Furthermore, Wnt5a-dependent localization of APC to the cell periphery was reduced in Dvl knockdown cells (Supplementary Figure S10). Taken together, these results suggest that the binding of Dvl and APC is involved in Wnt5a-dependent recruitment of APC to the cell periphery. Wnt5a and Fz2 are colocalized with Dvl and APC to the leading edge FLAG-Fz2, a Wnt5a receptor, was concentrated to the leading edge (Figure 4A) and was present as punctate structures at the dorsal cell surface (Figure 4B) in polarized Vero cells. The findings were also observed in the front cells of directionally migrating cells (Supplementary Figure S11A). When Wnt5a was expressed alone, it could not be detected at the cell surface (data not shown). When Wnt5a and FLAG-Fz2 were expressed simultaneously, both proteins were colocalized to the leading edge and also to the boundary of cell-to-cell adhesions (Figure 4C). To show the colocalization of Wnt5a and Fz2 more clearly, their expression at the dorsal cell surface was examined. FLAG-Fz2 was indeed colocalized with Wnt5a as punctate structures (Figure 4C). FLAG-Fz2 expressed on the cell surface formed a weakly visible complex with endogenous APC, and expression of HA-Dvl2 enhanced the complex formation in HEK293T cells (Figure 4D). When FLAG-Fz2, HA-Dvl2, or APC-GFP was expressed alone, they revealed different subcellular localizations (Supplementary Figure S11B–D). It has been shown that Dvl binds to the Wnt receptor Fz (Schulte and Bryja, 2007). Consistent with these observations, when FLAG-Fz2 and HA-Dvl2 were coexpressed, HA-Dvl2 was localized to the cell-surface membrane with FLAG-Fz2 (Supplementary Figure S11E). Coexpression of HA-Dvl2 and APC-GFP induced their colocalization as cytoplasmic punctate structures (Supplementary Figure S11F). APC-GFP was distributed in the cytoplasm along microtubules without the expression of HA-Dvl2 even though FLAG-Fz2 was expressed (Figure 4E, top panels). However, APC-GFP was colocalized with FLAG-Fz2 at the cell-surface membrane when HA-Dvl2 was expressed (Figure 4E, bottom panels). Taken together, these results suggest that Dvl and APC are localized to the leading edge of polarized cells with the Wnt5a and Fz2 complex. Figure 4.Fz2 forms a complex with APC and Dvl. (A, B) Vero cells expressing FLAG-Fz2 were fixed and incubated with polyclonal anti-FLAG antibody for 1 h without permeabilization. After the removal of free antibody, cells were probed with Alexa 546-conjugated anti-rabbit Ig antibodies. Polarized localization of FLAG-Fz2 was observed in 32% of 76 cells expressing FLAG-Fz2 (A). Punctate structures of FLAG-Fz2 were observed at the dorsal cell surface (B). (C) Vero cells expressing FLAG-Fz2 and Wnt5a were wounded. After 4 h, the cells were incubated with anti-FLAG and anti-Wnt5a antibodies for 1 h before fixation. White arrows indicate migration direction. Dashed lines indicate the front line of scratched cells. Bottom panels, colocalization of FLAG-Fz2 with Wnt5a was observed in 65% of 150 FLAG-Fz2 puncta at the dorsal cell surface. (D) HEK293T cells expressing FLAG-Fz2 and HA-Dvl2 as indicated were incubated with anti-FLAG antibody for 1 h before lysis. Lysates were incubated with protein sepharose-A beads, and the immunoprecipitates were probed with the indicated antibodies. (E) HEK293T cells expressing FLAG-Fz2 and APC-GFP with (bottom panels) or without (top panels) HA-Dvl2 were fixed and stained with polyclonal anti-FLAG antibody before permeabilization. Thereafter, Dvl and APC were stained with anti-HA and anti-GFP antibodies. Cells with membrane localization of APC were counted in at least 50 cells per condition, and the results are shown in the right-hand panel. *P<0.01. Scale bars in (A), (B) (left panel), (C) (top panels), and (E), 10 μm; in (B) (right panel) and (C) (bottom panels), 2 μm. Download figure Download PowerPoint Axin has an essential role in the regulation of β-catenin stability by associating with GSK-3β, APC, and Dvl (Kikuchi et al, 2009). HA-Axin was observed as cytoplasmic punctate structures in Vero cells (Supplementary Figure S12A). When HA-Axin and FLAG-Dvl2 were coexpressed, HA-Axin was colocalized with cytoplasmic FLAG-Dvl2 but not with cell-surface FLAG-Dvl2 (Supplementary Figure S12B). Overexpression of FLAG-Fz2 did not affect the distribution of HA-Axin, either (Supplementary Figure S12C). Therefore, it is conceivable that Axin is not recruited with APC to the Fz2-associated Dvl. Dvl and APC bind to components of the focal adhesion complex Integrins are essential molecules to regulate cell-substrate adhesion and migration (Legate et al, 2009). Endogenous cell-surface β1 integrin was accumulated at the leading edge where FLAG-Fz2 was also accumulated in polarized Vero cells (Figure 5A, top three panels). When localization of β1 integrin and FLAG-Fz2 at the ventral (substrate facing) cell surface was examined, β1 integrin puncta localized adjacent to puncta of FLAG-Fz2 (Figure 5A, bottom panels). Furthermore, complex formation between FLAG-Fz2 and α2 integrin in HEK293T cells was observed using cross-linker methods (Figure 5B). These findings raised the possibility that there is a connection between Wnt/Fz and integrin signalling. Figure 5.Fz2, Dvl, and APC associate with focal adhesions. (A) Vero cells expressing FLAG-Fz2 were fixed and stained with anti-β1 integrin and anti-FLAG polyclonal antibodies without permeabilization (top three panels). Right panel, the distribution of fluorescence intensity was measured along the dotted line. Bottom panels, localization of FLAG-Fz2 adjacent to β1 integrin was observed in 45% of 118 FLAG-Fz2 puncta at the ventral (substrate facing) cell surface. (B) HEK293T cells expressing FLAG-Fz2 were treated with or without 2 mM Dithiobis (succinimidyl propionate) (DSP) for 1 h at 4°C. The lysates were immunoprecipitated with polyclonal anti-FLAG antibody, and the immunoprecipitates were probed with anti-α2 integrin and anti-FLAG antibodies. The samples for immunoblotting were treated with or without 2-mercaptoethanol (2ME). Upper and lower arrows indicate cross-linked and monomeric α2 integrin, respectively. (C) Vero cells expressing FLAG-Dvl2 were plated on collagen for 1 h and stained with anti-FLAG and anti-paxillin antibodies. The regions in the white boxes (c1 and c2) are shown enlarged in bottom panels. Localization of FLAG-Dvl2 at the tip of paxillin staining was observed in 83% of 60 cortical Dvl staining. (D) Wounded HPPL cells were stained with anti-APC and anti-paxillin antibodies. The regions in the white boxes (d1 and d2) are shown enlarged in the right-hand panels. Localization of APC at the tip of paxillin was observed in 61% of 96 cortical APC staining. (E, F) Lysates of HeLaS3 cells were immunoprecipitated with anti-Dvl(DIX) (E) or anti-APC (F) antibody, and the immunoprecipitates were probed with indicated antibodies. (G) Lysates of COS7 cells expressing HA-Dvl1 deletion mutants were immunoprecipitated with anti-FAK antibody and the immunoprecipitates were probed with anti-HA and anti-FAK antibodies. Arrowheads indicate HA-Dvl1(WT) and HA-Dvl1(DEP+) coimmunoprecipitated with endogenous FAK. (H) Lysates of COS7 cells expressing EGFP-APC deletion mutants were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were probed with anti-paxillin and anti-GFP antibodies. An arrowhead indicates endogenous paxillin coimmunoprecipitated with EGFP-APC(Arm+). Scale bars in (A) (top three panels), (C), and (D), 10 μm; in (A) (bottom panels), 1 μm. Download figure Download PowerPoint In the initiation of spreading of Vero cells, paxillin was observed throughout the cell periphery and FLAG-Dvl2 was found to be localized at the tip of the paxillin-positive region (Figure 5C). APC was also localized at the tip of the paxillin-positive region in the focal complex of migrating and spreading HPPL cells (Figure 5D; Supplementary Figure S13). These results suggested that Dvl and APC are colocalized to focal adhesions. To clarify the roles of Dvl and APC in focal complex formation, the interaction of Dvl and APC with the components of cell-substrate adhesion was examined. Dvl formed a complex with FAK at endogenous levels in HeLaS3 cells, but it did not interact with paxillin (Figure 5E). In contrast, APC associated with paxillin at endogenous levels but not with FAK (Figure 5F). Dvl and APC did not form a complex with talin, vinculin, or β1 integrin (Figure 5E and F). Deletion mutant analyses showed that Dvl1(DEP+) forms a complex with FAK and that APC(Arm+) associates with paxillin (Figure 5G and H). The binding activity of GFP-Dvl2(ΔDEP) or GFP-Dvl2(1-509) to APC was similar to that of GFP-Dvl2, but GFP-Dvl2(506–736) did not form a complex with APC (Supplementary Figure S14). Therefore, either the DEP domain or the C-terminal region after the DEP domain is necessary for the binding of Dvl to APC, but the C-terminal region alone is not sufficient for it. GFP-Dvl2(ΔDEP) and GFP-Dvl2(506-736) associated with FAK as well as GFP-Dvl2, but GFP-Dvl2(1-509) did not (Supplementary Figure S14). Therefore, the DEP domain is not necessary for the binding of Dvl to FAK. The C-terminal region after the DEP domain of Dvl is necessary and sufficient for the binding of Dvl to FAK. These results suggest that Dvl forms a complex with APC and FAK at different sites although they might be overlapped partially. Paxillin formed a complex with EGFP-APC(Arm) with the simil