Abstract: Article14 September 2018free access Source DataTransparent process CDK1-mediated BCL9 phosphorylation inhibits clathrin to promote mitotic Wnt signalling Jianxiang Chen Corresponding Author [email protected]net orcid.org/0000-0002-8193-191X Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Muthukumar Rajasekaran Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hongping Xia Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Shik Nie Kong Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Amudha Deivasigamani Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Karthik Sekar Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hengjun Gao Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hannah LF Swa Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Jayantha Gunaratne Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author London Lucien Ooi Division of Surgery, Singapore General Hospital, Singapore City, Singapore Search for more papers by this author Tian Xie Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Search for more papers by this author Wanjin Hong Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Kam Man Hui Corresponding Author [email protected] orcid.org/0000-0003-1820-1399 Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore City, Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Search for more papers by this author Jianxiang Chen Corresponding Author [email protected] orcid.org/0000-0002-8193-191X Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Muthukumar Rajasekaran Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hongping Xia Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Shik Nie Kong Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Amudha Deivasigamani Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Karthik Sekar Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hengjun Gao Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Search for more papers by this author Hannah LF Swa Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Jayantha Gunaratne Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author London Lucien Ooi Division of Surgery, Singapore General Hospital, Singapore City, Singapore Search for more papers by this author Tian Xie Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Search for more papers by this author Wanjin Hong Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Search for more papers by this author Kam Man Hui Corresponding Author [email protected] orcid.org/0000-0003-1820-1399 Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore City, Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Search for more papers by this author Author Information Jianxiang Chen *,1,2,3, Muthukumar Rajasekaran2, Hongping Xia2, Shik Nie Kong2, Amudha Deivasigamani2, Karthik Sekar2, Hengjun Gao2, Hannah LF Swa3, Jayantha Gunaratne3, London Lucien Ooi4, Tian Xie1, Wanjin Hong3 and Kam Man Hui *,1,2,3,5,6 1Key Laboratory of Elemene Class Anti-Cancer Chinese Medicine of Zhejiang Province, Engineering Laboratory of Development and Application of Traditional Chinese Medicine from Zhejiang Province, School of Medicine, Holistic Integrative Pharmacy Institutes (HIPI), Hangzhou Normal University, Hangzhou, China 2Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore City, Singapore 3Institute of Molecular and Cell Biology, A*STAR, Proteos, Singapore 4Division of Surgery, Singapore General Hospital, Singapore City, Singapore 5Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore City, Singapore 6Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore *Corresponding author. Tel: +65 64368760; E-mail: [email protected] *Corresponding author. Tel: +65 64368338; E-mail: [email protected] EMBO J (2018)37:e99395https://doi.org/10.15252/embj.201899395 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 Uncontrolled cell division is a hallmark of cancer. Deregulation of Wnt components has been linked to aberrant cell division by multiple mechanisms, including Wnt-mediated stabilisation of proteins signalling, which was notably observed in mitosis. Analysis of Wnt components revealed an unexpected role of B-cell CLL/lymphoma 9 (BCL9) in maintaining mitotic Wnt signalling to promote precise cell division and growth of cancer cell. Mitotic interactome analysis revealed a mechanistic role of BCL9 in inhibiting clathrin-mediated degradation of LRP6 signalosome components by interacting with clathrin and the components in Wnt destruction complex; this function was further controlled by CDK1-driven phosphorylation of BCL9 N-terminal, especially T172. Interestingly, T172 phosphorylation was correlated with cancer patient prognosis and enriched in tumours. Thus, our results revealed a novel role of BCL9 in controlling mitotic Wnt signalling to promote cell division and growth. Synopsis A novel role of B-cell CLL/lymphoma 9 (BCL9) in maintaining mitotic Wnt signalling extends beyond its conventional transcriptional roles in interphase, illustrating a potential new model of how mitotic BCL9 phosphorylation can promote mitotic Wnt signaling and thereby promote tumorigenesis. BCL9 acts as a positive regulator in mitotic Wnt signaling. BCL9 localizes to mitotic spindles and regulates precise cell division by maintaining basal mitotic Wnt signaling activity. BCL9 regulates the mitotic β-catenin destruction complex by competing with clathrin binding to LRP6/Axin1. BCL9 stabilizes the mitotic LRP6-signalosome by inhibiting clathrin-mediated-endocytosis of signalosome components. CDK1-mediated BCL9 phosphorylation on T172 inhibits its binding to clathrin complexes and stabilizes mitotic LRP6-signalosomes. Introduction One of the notable properties of cancer is uncontrolled and/or improper chromosome segregation during mitosis (Jallepalli & Lengauer, 2001; Funk et al, 2016), which is primarily due to the deregulation of cell division regulators and/or mitotic signalling pathways (Johnson & Dent, 2013; Dominguez-Brauer et al, 2015). As the nuclear envelope breaks down at the beginning of mitosis, some mitotic-associated complexes are reassembled by the mixture of nuclear and cytoplasmic factors, which probably directly regulate mitotic events to promote normal cell division and sustain proliferation. Elucidation of the cancer-associated mitotic signalling network and corresponding regulators would provide valuable therapeutic targets for cancer diagnosis or drug discovery. Canonical Wnt signalling plays a crucial and complex role in cancers (Polakis, 2012; Zhan et al, 2017). Wnt signalling is activated in a spatiotemporal manner in current models (Clevers & Nusse, 2012; Nusse & Clevers, 2017). Wnt ligand binding to two co-receptors, Frizzled (Fzd) and LRP5 or LRP6, promotes the phosphorylation of the cytoplasmic tail of LRP6 by casein kinase 1 (CK1) and later GSK3, which are recruited together with axis inhibition protein (Axin) from a cytosolic “destruction” complex (consisting of APC, Axin, CK1 and GSK3, normally to degrade β-catenin). This further triggers polymerisation of dishevelled (DVL) and LRP6 on the plasma membrane and subsequent endocytosis of the Wnt receptor complex to form the LRP6 signalosome, which is crucial for Wnt signalling activation (Bilic et al, 2007; Gammons et al, 2016). In contrast, Dickkopf 1 (DKK1), which inhibits Wnt-dependent stabilisation of β-catenin, induces the internalisation of LRP6 via clathrin-mediated endocytosis (CME) to promote LRP6 receptor complex degradation. These changes result in failure to induce signalosome formation and β-catenin stabilisation, leading to Wnt signalling inhibition (Semenov et al, 2008; Yamamoto et al, 2008). Wnt signalling peaks in the G2/M phase due to priming of low-density lipoprotein receptor-related 6 (LRP6) phosphorylation by cyclin Y and CDK14 (Davidson et al, 2009; Davidson & Niehrs, 2010) indicating a potential mitotic role of Wnt signalling. In cancer cells, mitotic Wnt signalling has been shown to be involved in the stabilisation of proteins (STOP; Acebron et al, 2014), including well-known oncogenic drivers such as c-Myc and β-catenin. Basal mitotic Wnt signalling has been further implicated in ensuring spindle assembly and precise chromosome segregation to promote embryogenesis and cancer cell proliferation (Huang et al, 2015; Stolz et al, 2015). Moreover, Wnt signalling components have been detected in mitotic spindle, centrosome, kinetochore and microtubule ends to modulate the spindle orientation (Schlesinger et al, 1999; Kikuchi et al, 2010; Segalen et al, 2010; Morin & Bellaiche, 2011), chromosome alignment and precise cell division (Kaplan et al, 2001; Hadjihannas et al, 2006; Kikuchi et al, 2010; Ong et al, 2010; Niehrs & Acebron, 2012; Poulton et al, 2013; Stolz et al, 2015), eventually promoting efficient cancer cell growth. However, the intrinsic mechanism and potential regulators underlying mitotic Wnt signalling are still poorly understood. BCL9/legless (LGS, a Drosophila homologue of human BCL9) has been identified as an essential component in canonical Wnt signalling at the level of nuclear β-catenin to promote Wnt target transcription (Kramps et al, 2002; Sampietro et al, 2006). BCL9 is highly amplified and expressed in many types of cancers described in The Cancer Genome Atlas, TCGA (TCGA cbioportal, http://www.cbioportal.org/). BCL9 has been implicated in several cancers primarily via its HD2 domain-mediated transcription (de la Roche et al, 2012; Takada et al, 2012). Beyond this function, the mechanical role of BCL9 in human cancers is poorly understood. Here, BCL9 was shown to have an unexpected role in sustaining mitotic Wnt signalling to stabilise proteins by modulating the interaction with LRP6/Axin1 and the clathrin complex, which is further modulated by CDK1-driven N-terminal phosphorylation on BCL9. Thus, BCL9 would further ensure the high efficiency of cancer cell division and promote tumorigenesis. Results Identification of BCL9 in mitotic Wnt signalling to promote tumorigenesis Deregulation of mitosis and Wnt signalling are both frequently observed in human cancer (Polakis, 2012; Dominguez-Brauer et al, 2015; Funk et al, 2016; Zhan et al, 2017). Recently, a new role of Wnt signalling in mitosis has been reported and plays a crucial role in cancer cells (Acebron et al, 2014; Zhan et al, 2017); however, its regulatory mechanism and potential regulators are largely unknown. To identify mitotic Wnt signalling regulators, we employed a mini-screen involving siRNAs targeting 47 reported Wnt signalling regulators expressed in HeLa cells (MacDonald et al, 2009; Davidson, 2010; Clevers, 2012; Fig EV1A and B, and Tables EV1 and EV2), a cell line confirmed with active mitotic Wnt signalling (Acebron et al, 2014). To monitor the signalling activity, we determined the expression of well-established mitotic Wnt signalling targets, such as c-Myc and β-catenin (Acebron et al, 2014), using immunoblotting (Figs 1A and EV1C), and further validated our findings by enzyme-linked immunosorbent assays (ELISAs; Fig 1B) in both mitotic and asynchronised cells. As expected, we found common regulators for mitotic Wnt signalling and canonical Wnt signalling, such as Axin1, APC and DKK1, indicating the similarity between these two signalling pathways. Unexpectedly, we revealed that BCL9, a downstream nuclear regulator of β-catenin-mediated transcription, strongly regulated c-Myc and β-catenin proteins, especially in mitotic cells (Figs 1A and B, and EV1C). To exclude the off-target effect of BCL9 Smartpool siRNAs, we employed another independent siRNA pool (siGenome pool) for subsequent assays (Fig 1C and D, and Table EV1). Moreover, to exclude the nocodazole effect on mitotic Wnt target stability, we assessed natural mitotic cells generated by the shake-off method (Fig EV1D) and incubated them with cycloheximide (CHX) to inhibit protein translation. BCL9 depletion significantly inhibited mitotic Wnt signalling target proteins stability, notably in mitotic cells (Fig 1C and D), which was further confirmed in stable cells using previously reported BCL9 shRNA sequences (Fig EV1E; Mani et al, 2009). To explore whether BCL9 regulates mitotic Wnt signalling by inhibiting GSK3, we monitored GSK3 activity via a GFP-based biosensor, which responds well to Wnt3a in HeLa cells (Taelman et al, 2010; Acebron et al, 2014). Live-cell imaging analysis showed that BCL9 knockdown significantly impaired the Wnt3a-stabilised GSK3 biosensor, especially during G2M phase (Fig 1E and Movie EV1), indicating that BCL9 promotes mitotic Wnt signalling by inhibiting GSK3. To investigate whether BCL9 regulates mitotic Wnt target transcription, we analysed the transcriptome by RNA sequencing in the cells treated with the BCL9 siRNA pools described above and/or Wnt3a treatment for 4 h, a validated time point with peak expression of Axin2, a transcriptional target of Wnt signalling (Fig EV1F). We confirmed that this mitotic Wnt target gene expression was not markedly inhibited by BCL9 knockdown by transcriptome and real-time PCR assays (Table EV3 and Fig EV1G). Moreover, a fifteen Wnt signalling transcriptional targeting gene signature controlled by Axin1, APC and Wnt3a was identified (Fig 1F and Table EV3). Unexpectedly, BCL9 regulated none of these genes (Fig 1F and Table EV3), indicating that BCL9 has a potential role in Wnt signalling beyond transcription in HeLa cells. As BCL9 stabilises the β-catenin and c-Myc proteins, we employed a gene set enrichment analysis (GSEA) based on the transcriptome of cells with BCL9 knockdown to further evaluate the β-catenin and c-Myc activities (Figs 1G and EV1H). The results further confirmed that BCL9 knockdown significantly inhibited β-catenin and c-Myc activities. Click here to expand this figure. Figure EV1. BCL9 regulates mitotic Wnt signalling to stabilise proteins and promote tumorigenesis (related to Fig 1) The selection of 47 Wnt signalling regulators expressed in HeLa cells for siRNA study. Validation of siRNA knockdown efficiency by real-time PCR. Immunoblotting analysis of Wnt-stabilised protein targets β-catenin and c-Myc in mitosis (M) and asychronised (A) HeLa cells treated with siRNA. The synchronisation protocol to collect nature mitotic cells. Immunoblotting analysis of Wnt-stabilised proteins expression in BCL9 stable knockdown mitotic or G1S HeLa cells. Time-dependent induction of the expression of the Wnt transcriptional target Axin2 in HeLa cells by Wnt3a protein (100 ng/ml). Gene expression by real-time PCR of four mitotic Wnt targets in non-sychronised or mitotic cells. A: asynchronised cell; M: mitotic cells. Gene enrichment analysis of c-Myc activity in different dataset by using BCL9 regulated gene. Knockdown of BCL9 decreases G1 cell size. Forward scatter (FSC-H) profile of G1-gated HeLa cells treated with BCK9 or scramble siRNA 72 h post-transfection. Oncogenic soft agar colony formation and wound healing analysis in BCL9 stable knockdown cells. Effect of BCL9 knockdown on xenograft weight in tumorigenesis assay on day 40 post-tumour inoculation. Significance was measured by two-tailed unpaired t-test, *P < 0.05, **P < 0.01. All data are means ± SD, n = 6 for shS1 vs. shB1, n = 5 for shS1 vs. shB2. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Identification of BCL9 as a novel mitotic Wnt signalling regulator to stabilise proteins and promote oncogenesis A. Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators. B. ELISA validation of selected Wnt/STOP signalling regulators (n = 3). C, D. (C) Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n = 3). E. Representative pictures (left panel) and the normalised biosensor intensity based on live-cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n = 10). Scale bars represent 10 μm. F. Venn diagram analysis of the number of Axin1, APC, and Wnt3a signalling and/or BCL9-regulated transcriptional targets. G. Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9. H. The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n = 6 for shS1 vs. shB1, n = 5 for shS1 vs. shB2. I. Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm. Data information: Significance was measured by two-tailed unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001. All data are the mean ± SD. See also Fig EV1 and Tables EV1, EV2 and EV3. Source data are available online for this figure. Source Data for Figure 1 [embj201899395-sup-0011-SDataFig1.jpg] Download figure Download PowerPoint To address BCL9's roles in HeLa cells, we investigated the effect of BCL9 on cell size which could be regulated by mitotic Wnt signalling (Acebron et al, 2014), and we confirmed that BCL9 knockdown decreased the cell size in G1 phase HeLa cells (Fig EV1I). Moreover, we constructed stable BCL9 knockdown cells and found that loss of BCL9 substantially impaired soft agar colony formation, wound healing migration (Fig EV1J) and tumorigenesis of the cells (Figs 1H and EV1K). Protein expression of c-Myc, β-catenin or Axin2 was further examined in xenograft by immunohistochemistry (IHC) staining, which indicated that BCL9 knockdown inhibited c-Myc and β-catenin expression in mitotic cells and in interphase cells to a lesser extent but not Axin2 expression (Fig 1I). These results indicated that BCL9 regulates mitotic Wnt signalling beyond the activation of Wnt signalling transcription to promote cancer cells growth and tumorigenesis. BCL9 regulates the mitotic spindle and cell division partially by modulating mitotic Wnt signalling Wnt signalling components have been implicated in mitosis via regulation of the mitotic spindle orientation, chromosome alignment and/or precise cell division as shown previously (Hadjihannas et al, 2006; Kaplan et al, 2001; Kikuchi et al, 2010; Ong et al, 2010; Niehrs, 2012; Poulton et al, 2013; Stolz et al, 2015). To examine BCL9's roles in mitosis, we first synchronised cells (Fig EV2A) and analysed BCL9 or cyclin B1 protein (Fig 2A) and RNA (Fig EV2B) level from G1 to M phase. Notably, a significant band shift in addition to the BCL9 major band was detected specifically in mitosis and was verified by phosphorylated histone H3 (p-H3S10) and flow cytometric assays (Fig 2A). These assays showed that there were mitotic modifications on BCL9, which were most likely phosphorylation. After incubation of mitotic lysate with lambda protein phosphatase (λ-PPase), the shifted band disappeared (Fig EV2C), indicating mitotic phosphorylation on BCL9. To identify the potential kinase(s) involved, we first investigated CDK1, a master kinase in mitosis. A CDK1-specific inhibitor (RO3306) completely abolished the phosphorylation of both endogenous and exogenous BCL9 (Fig EV2D), indicating that CDK1 potentially regulates mitotic BCL9 phosphorylation. To confirm that CDK1 phosphorylating BCL9 is a direct effect of CDK1, we perform in vitro kinase assay by incubating CDK1/cyclin B1 and BCL9 proteins with or without ATP (Fig EV2E). We found that indeed CDK1 could directly induce BCL9 band shift (Fig EV2E), indicating CDK1 phosphorylating BCL9 protein directly. Click here to expand this figure. Figure EV2. BCL9 is phosphorylated and localises on mitotic spindle (related to Fig 2) Flow cytometry cell cycle analysis of thymidine synchronised HeLa cells. Cyclin B1 and BCL9 gene expression in synchronised cells. Immunoblotting analysis of lambda phosphatase effect on BCL9 phosphorylation in pro-metaphase (Pro-M), G1S and asynchronised HeLa cells (left panel), or the effect of phosphatase inhibitor okadaic acid, alone or combined with lambda phosphatase, on BCL9 phosphorylation in Pro-M HeLa cells (right panel). 3–8% TA gel refers to NuPAGE 3–8% Tris–acetate protein gels. Immunoblotting analysis of CDK1-specific inhibitor RO3306 inhibiting both endogenous (left panel) and exogenous (right panel) BCL9 phosphorylation. 3–8% TA gel refers to NuPAGE 3–8% Tris-acetate protein gels. CDK1/cyclin B1 kinase assay to detect the direct phosphorylation band shift of BCL9 protein purified by wheat germ translation system. 3–8% TA gel refers to NuPAGE 3–8% Tris–acetate protein gels. Immunofluorescence staining of BCL9 subcellular localisation during cell cycle by a mouse monoclonal antibody in HeLa cells. Scale bars represent 5 μm. Immunofluorescence staining of BCL9 subcellular localisation during cell cycle in RPE1 and HCCLM3 cells. Scale bars represent 5 or 10 μm as indicated in each figure. A diagram of instruction for how to calculate the spindle angle. Radial histograms showing the distribution of spindle angles in cells treated with Wnt3a or DKK1 purified proteins (n = 23). Source data are available online for this figure. Download figure Download PowerPoint Figure 2. The novel role of BCL9 in maintaining mitotic Wnt signalling to regulate proper cell division Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9. Immunofluorescence confocal analysis of BCL9 localisation on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase. Immunofluorescence confocal analysis of BCL9 localisation on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment. Confocal microscope 3D analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm. Confocal (left panel) and live-cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. Twenty-three H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live-cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. Twenty-three H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar) or growth well in 30 h (pink bar) by live-cell imaging analysis in monitored H2BGFP cells with different treatments. Data information: Significance was measured by two-tailed unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001. All data are the mean ± SD. See also Fig EV2. Source data are available online for this figure. Source Data for Figure 2 [embj201899395-sup-0012-SDataFig2.jpg] Download figure Download PowerPoint As Wnt components often localise to the mitotic spindle and/or centrosome (Niehrs, 2012), BCL9 subcellular localisation was evaluated by two commercial antibodies in HeLa cells (Figs 2B and EV2F). BCL9 was detected on the spindle and spindle pole during mitosis (Figs 2B and EV2F), and this localisation was further confirmed in two additional human cell lines (Fig EV2G). BCL9 was also detected on the centrosome by methanol fixation (Fig 2C). These data indicated that BCL9 might have a function in regulating mitotic spindle. To explore this role, we knocked down BCL9 and investigated the spindle orientation (SO; Figs 2D and EV2H), a notable Wnt-associated spindle function (Niehrs, 2012; Habib et al, 2013). By calculating two spindle pole distances in the x-axis, y-axis and z-axis, we could determine the angle between, which reflected the SO (Fig EV2H). These data indicated that BCL9 knockdown induced aberrant SO by increasing the spindle angle compared to scramble knockdown (Fig 2D), suggesting that BCL9 regulates SO. To determine the effect on cell division, we used H2B-GFP cells or immunofluorescence (IF) staining for live-cell imaging or confocal analysis. BCL9 knockdown significantly induced aberrant cell division with misaligned chromosomes (MCs), bridging chromosomes (BCs) and subsequent micronuclei (MN) formation in interphase cells (Fig 2E and Movie EV2). To examine the interpla