Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling
2018; Springer Nature; Volume: 19; Issue: 7 Linguagem: Inglês
10.15252/embr.201745471
ISSN1469-3178
AutoresKoji Kikuchi, Akira Nakamura, Masaki Arata, Dongbo Shi, Mami Nakagawa, Tsubasa Tanaka, Tadashi Uemura, Toshihiko Fujimori, Akira Kikuchi, Akiyoshi Uezu, Yasuhisa Sakamoto, Hiroyuki Nakanishi,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle7 June 2018free access Source DataTransparent process Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling Koji Kikuchi Corresponding Author [email protected] orcid.org/0000-0002-7616-8200 Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Akira Nakamura Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Masaki Arata Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Dongbo Shi orcid.org/0000-0002-4408-3042 Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Mami Nakagawa Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Tsubasa Tanaka Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Tadashi Uemura Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshihiko Fujimori Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Akira Kikuchi Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Akiyoshi Uezu Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Yasuhisa Sakamoto Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Hiroyuki Nakanishi Corresponding Author [email protected] orcid.org/0000-0002-9765-0266 Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Koji Kikuchi Corresponding Author [email protected] orcid.org/0000-0002-7616-8200 Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Akira Nakamura Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Masaki Arata Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Dongbo Shi orcid.org/0000-0002-4408-3042 Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Mami Nakagawa Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Tsubasa Tanaka Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Tadashi Uemura Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshihiko Fujimori Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan Search for more papers by this author Akira Kikuchi Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Search for more papers by this author Akiyoshi Uezu Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Yasuhisa Sakamoto Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Hiroyuki Nakanishi Corresponding Author [email protected] orcid.org/0000-0002-9765-0266 Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan Search for more papers by this author Author Information Koji Kikuchi *,1, Akira Nakamura2,3, Masaki Arata4, Dongbo Shi5, Mami Nakagawa5, Tsubasa Tanaka2,3, Tadashi Uemura4, Toshihiko Fujimori5, Akira Kikuchi6, Akiyoshi Uezu1, Yasuhisa Sakamoto1 and Hiroyuki Nakanishi *,1 1Department of Molecular Pharmacology, Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan 2Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-ku, Kumamoto, Japan 3Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto, Japan 4Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan 5Division of Embryology, National Institute for Basic Biology, Okazaki, Aichi, Japan 6Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan *Corresponding author (Lead contact): +Tel: +81 96 373 5076; E-mail: [email protected] *Corresponding author. +Tel: +81 96 373 5074; E-mail: [email protected] EMBO Rep (2018)19:e45471https://doi.org/10.15252/embr.201745471 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 Abstract The Wnt signaling pathway can be grouped into two classes, the β-catenin-dependent and β-catenin-independent pathways. Wnt5a signaling through a β-catenin-independent pathway promotes microtubule (MT) remodeling during cell-substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT-associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus-ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus-ends, and depletion of the Kinesin-1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar-polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved. Synopsis Microtubule-associated proteins Map7/7D1/Ens and Dishevelled form an evolutionarily conserved axis in β-catenin-independent Wnt signaling and/or PCP formation. Map7/7D1 cooperate with a Kinesin-1 Kif5b to coordinate Dishevelled dynamics and microtubule remodeling in the Wnt5a signaling pathway. Map7/7D1 and their fly homolog Ens show planar-polarized distribution in both mouse and fly epithelia. Ens is required for polarized localization of Dishevelled during PCP formation in pupal wing epithelia. Map7/7D1/Ens play an evolutionarily conserved role in Dishevelled localization. Introduction Wnt signaling participates in various cellular functions 1. The Wnt signaling pathway can be divided into two groups, the β-catenin-dependent and β-catenin-independent pathways. β-catenin-independent Wnt signaling plays important roles in cell-substrate adhesion, cell migration, and planar cell polarity (PCP) formation, all of which are essential features of tissue morphogenesis 2, 3. β-catenin-independent Wnt signaling is transduced to its effectors through Disheveled (Dvl), whose intracellular localization is important for actin remodeling 4. A formin family actin nucleator, Daam1, is an effector of this process. Microtubule (MT) remodeling is also promoted by β-catenin-independent Wnt signaling 5-8. However, the mechanism by which β-catenin-independent Wnt signaling regulates MT remodeling remains elusive. Notably, MT remodeling and β-catenin-independent Wnt signaling appear to be regulated interdependently. For example, during front-rear polarity formation in migrating cells, MTs are polarized by elongating toward the leading edge. This MT attachment to the leading edge depends on β-catenin-independent Wnt signaling 6, 7. Conversely, treating cells with nocodazole, which induces MT depolymerization, disturbs the proper localization of Dvl and adenomatous polyposis coli (APC), both of which are critical component of β-catenin-independent Wnt signaling 6, suggesting that the β-catenin-independent Wnt signal transduction requires proper MT alignment. Similarly, during PCP formation in the multiciliated cells of the mouse airway, β-catenin-independent Wnt signaling promotes apical MT arrays to be organized in a planar-polarized manner along the lung-oral axis, and this MT organization is required for the proper localization of several proteins involved in PCP formation 8. These observations indicate that the interdependent regulation of MT remodeling and β-catenin-independent Wnt signaling establishes robust cell polarity. However, the factor that mutually connects them is completely unknown. Microtubule remodeling is usually regulated by the coordinated actions of various MT-binding proteins, such as MT-associated proteins (MAPs) 9, 10. Thus, we hypothesized that MT-binding protein(s) may be responsible for the interdependent regulation. Since one of β-catenin-independent pathways, the Wnt5a signaling pathway regulates cell-substrate adhesion and cell migration in HeLa cells 6, 11, 12, as well as PCP formation during mammalian development 13, we used HeLa cells to screen uncharacterized MT-binding proteins for their involvement in Wnt5a signaling. Here, we report that Map7 and its paralog, Map7D1 (Map7/7D1) participate in a feedback loop between MT remodeling and Wnt5a signaling through a direct interaction with Dvl. We also showed that the Kinesin-1 member Kif5b, which is known as the binding partner of Map7 14, is required for both Map7/7D1 dynamics and Dvl localization. Furthermore, we found that Map7/7D1 and its Drosophila ortholog, Ensconsin (Ens) show planar-polarized distribution in epithelial cells of mouse oviducts and fly pupal wings, respectively, and that Ens is required for proper localization of Drosophila Disheveled (Dsh). These results suggest that Map7/7D1 cooperate with Kif5b to coordinate a feedback loop between Dvl dynamics and MT remodeling in the Wnt5a signaling pathway, and that the role of Map7/7D1 family proteins in Dvl/Dsh localization is evolutionarily conserved. Results Paralogous MT-associated proteins Map7 and Map7D1 are required for cell adhesion and migration in HeLa cells To identify MT-binding proteins that are potentially involved in the β-catenin-independent Wnt5a signaling pathway, we performed a siRNA-based screen in HeLa cells for previously identified MT co-sedimented proteins 15 (Fig 1A; Appendix Fig S1; see Materials and Methods for details). In HeLa cells, cell-substrate adhesion and directional cell migration (hereafter, cell adhesion and migration, respectively) is regulated by endogenously expressing Wnt5a. By observing the effects of their knockdown on cell adhesion and migration, two genes, encoding Map7 and Map7D1, were identified as candidates that regulate cell adhesion and migration in response to endogenous Wnt5a (Fig 1A–C; Appendix Figs S2 and S3). Map7 and Map7D1 are members of the MAP7 family, which also includes Map7D2 and Map7D3 (Appendix Fig S3A). RT–qPCR analysis revealed that MAP7, MAP7D1, and MAP7D3, but not MAP7D2, were detectably expressed in HeLa cells (Appendix Fig S3A). Unlike Map7 and Map7D1, however, Map7D3 depletion neither triggered blebbing nor slowed migration (Fig 1B and C; Appendix Fig S3C). In contrast, depletion of both Map7 and Map7D1 caused much severer defects in cell adhesion and migration than that of each single gene product (Fig 1B and C). Thus, Map7 and Map7D1, but not Map7D3, have overlapping functions in the adhesion and migration of HeLa cells. Figure 1. Paralogous MT-associated proteins Map7 and Map7D1 are required for cell-substrate adhesion and migration in HeLa cells Experimental flow chart for the siRNA-based screen in HeLa cells to discover MT-binding protein(s) potentially involved in β-catenin-independent Wnt5a signaling. For details, see Materials and Methods. Images of the indicated cells at various times after being replated on a fibronectin-coated glass-bottom dish (top). Lamellipodial extension was observed during cell-substrate adhesion in control cells. Defective cell adhesion caused membrane blebbing instead of lamellipodial extension. Graph shows the percentage of blebbing cells 60 min after replating. Images of the indicated cells after inducing cell migration (top). Dotted and solid lines show the wounded edge 0 or 6 h after wounding, respectively. Average distance moved by the wounded edge 6 h after wounding (lower left). HeLa cells were transfected with validated CDS-targeting siRNAs. Map7D3 was depleted with a mixture of three validated siRNAs (see Appendix Fig S3C). Distance moved by the wounded edge was measured over time after wounding in the indicated cells (lower right). Map7 or Map7D1 was depleted with a mixture of three or two validated siRNAs, respectively. For the double depletion, cells were treated with combined siRNAs against Map7 and Map7D1. Depletion efficiency of Map7 in wild-type or Map7-EGFPKI cells transfected with the indicated siRNAs (top). At 72 h post-transfection, the protein level of Map7 was analyzed with an anti-Map7 antibody. Graph shows the average distance moved by the wounded edge 6 h after wounding. siMAP7 CDS, Map7 was depleted with a mixture of three validated siRNAs targeting the CDS. siMAP7 3′ UTR, Map7 was depleted with a mixture of two validated siRNAs targeting the 3′ UTR. Data information: Scale bars, 10 μm in (B) and 50 μm in (C). Data shown in (B–D) are from three or four independent experiments, and represent the average ± SD. *P < 0.002; **P < 0.005: ***P < 0.015 (the Student's t-test). Source data are available online for this figure. Source Data for Figure 1 [embr201745471-sup-0016-SDataFig1.zip] Download figure Download PowerPoint The ectopic expression of Map7 or Map7D1 induced aberrant MT bundling 16 and therefore did not rescue the defects of Map7- or Map7D1-depleted cells, respectively. To confirm the specificity of the siRNAs for depleting Map7 or Map7D1, by applying CRISPR-Cas9 technique 17, 18, we generated Map7-EGFP knocked-in (Map7-EGFPKI) HeLa cell lines, in which the endogenous 3′ untranslated region (UTR) was replaced by the SV40 polyadenylation signal (Fig 1D, top panel; Appendix Fig S4; Movie EV1). The siRNAs targeting the 3′ UTR or the coding sequence (CDS) of MAP7 caused slower migration in unmodified HeLa cells (Fig 1D, bottom). In contrast, siRNAs targeting the CDS but not the 3′ UTR decreased the migration rate of Map7-EGFPKI cells (Fig 1D, bottom). These results indicate that the siRNAs used in our assay specifically deplete the target genes as designed, and that Map7 and Map7D1 play overlapping functions in cell adhesion and migration. Because of their functional overlap (Fig 1B and C), Map7 and Map7D1 (Map7/7D1) were simultaneously depleted in the following experiments. Map7/7D1 are critical for the cortical targeting of MT plus-ends As Map7/7D1 depletion caused slower cell migration, it may affect MT stability. To test this possibility, we measured the levels of acetylated and detyrosinated tubulins, which are enriched in stable MTs. Map7/7D1 depletion did not affect the bulk levels of these modified tubulins (Fig 2A). We also evaluated the stability of MTs in migrating cells. Since MT turnover is fast at the leading edge during cell migration, the signal for acetylated tubulin was obvious only in the inner region, and not at the cell periphery, in control cells. These staining patterns were unaffected by Map7/7D1 depletion (Appendix Fig S5A). Thus, it is unlikely that Map7/7D1 regulate MT stability. Figure 2. Map7/7D1 are critical for the cortical targeting of MT plus-ends Immunoblot analysis for detyrosinated (Detyr-) and acetylated (Ace-) tubulins in the indicated cells. β-Tubulin (loading control) and Map7/7D1 were also analyzed. Asterisk shows unspecific band. Images of peripheral MT arrays at the leading edge in the indicated cells 1 h after wounding (left). Images were focused on the Paxillin signal, a marker of focal adhesions (FAs), because the polarized MT arrays were observed close to the adherent side of the cells. Graph shows the percentage of cells with polarized MT arrays (from three independent experiments). Dynamics of EB1-GFP comets observed by live-cell imaging 1 h after wounding (left). Time-lapse images were taken at 5-s intervals for 2 min (see Movies EV2, EV3, EV11 and EV12). Comet trails were measured at three time points (red: 0 s, green: 5 s, blue: 10 s) in the indicated cells (arrowheads). The measurement of the vector angle of EB1-GFP comets with respect to the leading edge (top). The vector angle of 0° indicates direction toward the leading edge. Arrow indicates the direction of migrating cells. Rose diagram shows the vector angle of EB1-GFP comets in the indicated cells (control, n = 328 comets; siMAP7/7D1, n = 277 comets; siWNT5A, n = 318 comets; siDVLs, n = 320 comets from three independent experiments). Graph shows the velocity of EB1-GFP comets in the indicated cells (control, n = 270 comets; siMAP7/7D1, n = 270 comets; siWNT5A, n = 450 comets; siDVLs, n = 300 comets from three independent experiments). Images of filopodia at the leading edge in the indicated cells 1 h after wounding (left). Graph shows the number of filopodia at the leading edge of the front cells divided by the length of the leading edge. Twenty images (1,024 × 1,024 pixels, one image included about 4–5 front cells) from four independent experiments were analyzed for each cell type. FAs at the leading edge in the indicated cells 1 h after wounding (left). FAs were visualized by staining for focal adhesion kinase (FAK). Graph shows the FA area at the leading edge of the front cells measured 1 h after wounding (control, n = 114 cells; siMAP7/7D1, n = 81 cells from four independent experiments). Data information: Panels in (B–D, F, and G) are arranged to show that cells are migrating in an upward direction. Scale bars, 10 μm in (B, C, F, and G). Data shown in (B and F) are from three or four independent experiments, and represent the average ± SD. The bars of box-and-whisker plots show the 5 and 95 percentiles, and the box limits are the 1st and 3rd quartile in (E and G). Statistical significance was tested with the Student's t-test in (B and E–G) or the Mardia-Watson-Wheeler test in (D). Source data are available online for this figure. Source Data for Figure 2 [embr201745471-sup-0017-SDataFig2.xlsx] Download figure Download PowerPoint We next examined the proportion of polarized MTs elongating toward the leading edge (hereafter, the polarized MT arrays) by antibody staining for MTs and found that Map7D/7D1 depletion caused a decrease in the polarized MT arrays at the leading edge (Fig 2B). Since the polarized MT arrays are established by the cortical targeting of MT plus-ends 9, we examined the dynamics of the MT plus-ends by live imaging using the MT plus-end marker, EB1-GFP 19. In control cells, EB1-GFP comets moved toward the leading edge during cell migration (Fig 2C and D; Movie EV2). However, the proportion of EB1-GFP comets moving toward the leading edge was severely decreased in Map7/7D1-depleted cells (Fig 2C and D; Movie EV3). Notably, the gross velocity of EB1-GFP movement was unaffected by Map7/7D1 depletion (Fig 2E). These results suggest that Map7/7D1 are required for the cortical targeting of MT plus-ends without affecting the growth of EB1-decorated MTs. Map7/7D1 coordinate MT and actin dynamics The cortical targeting of MT plus-ends regulates the actin dynamics, which plays essential roles in cell adhesion and migration 20, 21. Since Map7/7D1 depletion compromised the cortical targeting of MT plus-ends, we wondered if it would also affect the filopodia organization. In control migrating cells, extended filopodia with thick F-actin bundles were observed at the leading edge (Fig 2F). Map7/7D1 depletion caused significant decreases in the number of filopodia and thickness of F-actin bundles (Fig 2F). Filopodia formation is required for the proper assembly and disassembly of focal adhesions (FAs), which is called FA turnover 22. In control migrating cells, small FAs, visualized by staining for focal adhesion kinase (FAK), formed at the leading edge, due to the increased turnover of FA 23 (Fig 2G). In contrast, larger FAs were observed at the leading edge in Map7/7D1-depleted cells (Fig 2G), suggesting that the FA turnover rate was reduced. Similar phenotypes were observed during cell adhesion (Appendix Fig S5B and C). These results indicate that Map7/7D1 depletion causes a failure in the cortical targeting of MT plus-ends, which in turn leads to reduced actin and FA dynamics, and consequently to cell migration and adhesion defects. Map7/7D1 bind to the key mediator of Wnt5a signaling, Dvl Map7/7D1 depletion shows similar phenotypes as when Wnt5a signaling is disrupted, including multinucleated cells 5, 6 (Appendix Fig S5D). To further examine whether Map7/7D1 are involved in the β-catenin-dependent pathway, we analyzed the effects of Map7/7D1 depletion on the β-catenin-dependent induction of AXIN2 expression by RT–qPCR 24. By the Wnt3a administration, AXIN2 mRNA was increased in both control and Map7/7D1-depleted cells (Appendix Fig S5E), suggesting that Map7/7D1 are dispensable for the β-catenin-dependent pathway in response to Wnt3a. Because the key mediator in Wnt5a signaling, Dvl is known to participate in the cortical targeting of MT plus-ends 6, we examined whether Map7/7D1 form a complex with Dvl. Endogenous Dvl2 was co-precipitated with hMap7-V5His6 (Fig 3A). Furthermore, an anti-Dvl2 antibody co-precipitated endogenous Map7 and Map7D1 (Fig 3B). In contrast, a trace amount of overexpressed Map7D2 was co-immunoprecipitated with Dvl2 (Appendix Fig S6A and B). These results indicate that Map7 and Map7D1 form a complex with Dvl2 under physiological conditions. Figure 3. Map7/7D1 bind to Dvl, a key mediator of Wnt5a signaling Lysates from HeLa cells transfected with control vector or pcDNA3.1-hMAP7-V5His6 were immunoprecipitated with an anti-V5 antibody, and the immunoprecipitates were probed with anti-Dvl2 and anti-V5 antibodies. Lysates from HeLa cells were subjected to immunoprecipitation with control IgG or an anti-Dvl2 antibody and analyzed by immunoblotting with an anti-Map7/7D1 or anti-Dvl2 antibody. Lysates from HeLa cells co-expressing various deletion derivatives of hMap7-V5His6 with EGFP-mDvl2 were immunoprecipitated with an anti-GFP antibody, and the immunoprecipitates were probed with anti-V5 and anti-GFP antibodies. Lysates from HeLa cells co-expressing various derivatives of EGFP-mDvl2 with hMap7-V5His6 were immunoprecipitated with an anti-V5 antibody, and the immunoprecipitates were probed with anti-GFP and anti-V5 antibodies. MBP-hMap71-265ΔCC1 (30 pmol) conjugated to amylose resin was incubated with purified GST, GST-hDvl1DIX, or GST-hDvl1DEP (30 pmol of each), and the bound proteins were analyzed by immunoblotting with anti-GST and anti-MBP antibodies. The positions of GST, GST-hDvl1DIX, and GST-hDvl1DEP were revealed by loading 1.5 pmol (5%) of each purified protein. Depletion efficiency of siWNT5A or siDVLs. Lysates derived from the indicated cells were probed with anti-Dvl2 and anti-Wnt5a antibodies. The blot was reprobed for γ-tubulin as a loading control (left). Effects of siWNT5A or siDVLs on the protein levels of Map7 and Map7D1 were also analyzed (right). The blot was reprobed for γ-tubulin as a loading control. Graph shows the relative mRNA levels of MAP7 and MAP7D1 in the indicated cells 72 h after siRNA transfection. Expression levels of MAP7 and MAP7D1 transcripts were quantified by normalization to the GAPDH expression. Data are from three independent experiments and represent average ± SD. Source data are available online for this figure. Source Data for Figure 3 [embr201745471-sup-0018-SDataFig3.zip] Download figure Download PowerPoint To determine which domains of Map7 interact with Dvl2, we expressed various deletion derivatives of Map7 together with Dvl2 in HeLa cells. Map7 lacking amino acids (aa) 159–246 interacted poorly with Dvl2. Further deletion (aa 89–246), which includes the coiled-coil domain 1 (CC1), abolished the Map7–Dvl2 interaction, whereas deletion of the CC1 domain alone (aa 89–152) did not affect the interaction (Fig 3C). We next examined which region of Dvl2 is required for the association with Map7, and found that Dvl2 lacking the DIX or DEP domain failed to associate with Map7 (Fig 3D). Although it is well known that overexpressed Dvl2 is highly phosphorylated in HeLa cells, this phosphorylation was abolished in the truncated Dvl2 that lacked the DIX domain. In contrast, another truncated Dvl2 with the DEP deletion remained phosphorylated (Appendix Fig S6C), suggesting that the conformational change of Dvl2 arising from DIX deletion may affect the Map7–Dvl2 interaction. We further performed in vitro biding assay using purified GST-hDvl1DIX and GST-hDvl1DEP. Purified GST-hDvl1DEP, but not GST-hDvl1DIX, was pulled down with MBP-hMap71-265ΔCC1 in vitro (Fig 3E; Appendix Fig S6D). Taken together, these results indicate that the DEP domain of Dvl is sufficient for, and the aa 159–246 region of Map7 is required for their interaction. The DEP domain in Dvls is critical for both β-catenin-dependent and β-catenin-independent Wnt signaling 25-28. Because Map7/7D1 are not involved in β-catenin-dependent Wnt3a signaling (Appendix Fig S5E), these results further support the idea that Map7/7D1 play a role in the Wnt5a signaling pathway through interaction with Dvl. We also noticed that depleting all three human Dvl proteins (Dvls) reduced the steady state of Map7/7D1 protein levels without affecting the MAP7/7D1 mRNA levels (Fig 3F and G). In contrast, Wnt5a depletion did not affect the Map7/7D1 levels. These results indicate that Dvl is required for Map7/7D1's stability, and that this regulation is independent of Wnt5a signaling. Map7/7D1 direct the Dvl localization to the cell cortex We next examined whether Map7/7D1 regulate Dvl's localization in the Wnt5a signaling pathway. Dvl can bind to actin 29 and is preferentially accumulated at lamellipodia of the cell periphery in response to Wnt5a 6, 30. To observe Dvl's localization to the cell periphery in living cells, we generated Dvl2-EGFP knock-in (Dvl2-EGFPKI) HeLa cell lines (Appendix Fig S7). Consistent with previous immunostaining results 6, 30, Dvl2-EGFPKI accumulated at the cell cortex during cell migration in control cells (Fig 4A; Movie EV4). In contrast, this Dvl2-EGFPKI accumulation was compromised in Map7/7D1-depleted cells (Fig 4A; Movie EV5). Importantly, Map7/7D1 depletion did not affect the expression or secretion of Wnt5a, which provides the autocrine signal that is essential for the cortical accumulation of Dvl in HeLa cells (Fig 4B). The expression and localization of adenomatous polyposis coli (APC), which binds to Dvl and maintains the polarized MT arrays 6, were also unaffected by Map7/7D1 depletion (Fig 4C and D). Furthermore, upon Wnt5a administration into the medium, a robust cortical accumulation of Dvl2-EGFPKI was induced in the control cells, but not in Map7/7D1-depleted cells (Fig 4E). In contrast, the Wnt5a-induced Dvl2 phosphorylation was intact in Map7/7D1-depleted cells (Fig 4C). Thus, Map7/7D1 depletion disrupted the cortical accumulation of Dvl2, even though the Wnt5a signaling-mediated Dvl phosphorylation was intact. Figure 4. Map7/7D1 direct Dvl localization to the cell cortex Live-cell imaging of Dvl2-EGFPKI cells 1 h after wounding. Movies were taken at 10-s intervals for 3 min (see Movies EV4 and EV5). Graph shows the percentage of cells with accumulated Dvl2-EGFPKI at the cell periphery. Data are from four independent experiments and represent average ± SD. Statistical significance was tested with the Student's t-test. Effects of Map7/7D1 depletion on the secretion and expression levels of Wnt5a. Conditioned medium and lysates derived from the indicated cells were separated on SDS–PAGE and were immunoblotted with an anti-Wnt5a antibody. To assess the levels of Map7/7D1 depletion and loading control, the blot was reprobed for Map7/7D1 and γ-tubulin, respectively. Asterisk shows unspecific band. Effects of Map7/7D1 d
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