Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation

Abstract

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.

Figure 1

Loss of Polo activity results in a SAC-independent mitotic arrest with decreased MCC levels. (A) Mitotic index quantification based on H3^Ser10Ph staining in asynchronous-cultured cells and after 24-h colchicine incubation. Data represent mean±s.d., n>3000 cells for each condition, from three independent experiments. (B) Mitotic progression of S2 cells expressing EGFP–Cyclin B was monitored by time-lapse microscopy. Selected stills of live-cell imaging for the indicated conditions are shown. Time is in min:sec. (C) EGFP–Cyclin B degradation profiles of experiments in (B). Data represent the mean±s.d. of at least 10 cells for each experimental condition. EGFP–Cyclin B fluorescence at NEB was set to 100% except in cells treated with BI2536 or depleted of Polo. Due to technical constraints resulting from loss of Polo activity, we were not able to detect NEB in those cells and, therefore set EGFP–Cyclin B fluorescence in the first frame to 100%. (D) Ubiquitination analysis of EGFP–Cyclin B immunoprecipitates from total cell lysates obtained from control and cells depleted of the indicated proteins. Cultured cells were treated with MG132 for 2 h. Immunoprecipitates (IP) were probed by immunoblotting for conjugated ubiquitin and corresponding total cell lysates (Input) for the indicated proteins. (E) Immunoprecipitation of Cdc20 from total cell lysates obtained from control cells, cells depleted of Polo and cells treated with BI2536. IP and corresponding inputs were probed by immunoblotting for the indicated proteins. (F) Immunolocalization of Mad2. Insets show higher-magnification views of kinetochore pairs in the boxed areas. Graphs display the quantification of Mad2 kinetochore levels. Mad2 fluorescence intensities were determined relative to CID. Data represent mean±s.d. and mean values obtained for control cells were set to 100%. n>20 cells for each condition. (E, F) Cells were treated with MG132 for 1 h followed by 2 h of colchicine incubation. BI2536 was added 30 min prior to microtubule depolymerization with colchicine. Scale bars correspond to 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 2

Polo is required for Mps1 and Ndc80 recruitment to unattached kinetochores. Immunolocalization of Mps1, Polo (A) and Ndc80 (B). Insets show higher-magnification views of kinetochore pairs in the boxed areas. (C) Quantification of experiments in (A) and (B). Mps1, Polo and Ndc80 fluorescence intensities were determined relative to CID. Data represent mean±s.d. and mean values obtained for control cells were set to 100%. ***P<0.001, one-way ANOVA Turkey’s multiple comparison test. n>20 cells for each condition. (D) Variation of Mps1 and Ndc80 kinetochore levels with Polo levels at kinetochores from control and Polo-depleted cells in (A) and (B). (E) Immunoblot showing Mps1 phosphorylation status (top) and quantification of p-Mps1 (phosphorylated Mps1) normalized to Cyclin B signals. Data represent mean±s.d. and were obtained from four independent experiments. AP: alkaline phosphatase. (F) Immunolocalization of Aurora B and p-Ser10-Histone3 (H3^S10Ph). Kinetochore pairs in boxed areas are represented magnified and the respective intensity profiles of Aurora B and CID labelling along the dotted line shown. (A–F) Cells were treated with MG132 for 1 h followed by 2 h of colchicine incubation. When required, binucleine 2 was added to the cultures 30 min before treatment with colchicine. Scale bars represent 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 3

Constitutively active Polo promotes Ndc80 and Mps1 kinetochore recruitment independently of Aurora B activity. (A) Immunolocalization of Mps1 (top) and Ndc80 (bottom) in cells expressing EGFP–Polo^WT and EGFP–Polo^T182D upon treatment with DMSO, binucleine 2 or Ndc80 and Nuf2 RNAi. Insets show higher-magnification views of kinetochore pairs in the boxed areas. (B) Quantification of experiment in (A). Mps1 and Ndc80 fluorescence intensities at kinetochores were determined relative to CID. Data represent mean±s.d. **P<0.01; ***P<0.001, One-way ANOVA Turkey’s multiple comparison test. n>20 cells for each condition. (C) Cell lysates were analysed by immunoblotting to assess Mps1 phosphorylation status. AP: alkaline phosphatase. (D) Immunofluorescence analysis of asynchronous S2 cells stained for Polo–T182 phosphorylation (Polo^T182Ph) and CID. An early prometaphase cell with unaligned chromosomes and a late prometaphase cell with a discernible metaphase plate and one unaligned chromosome (yellow *) are shown. Kinetochore pairs in boxed areas are magnified and the respective fluorescence intensity profiles of Polo^T182Ph and CID are shown. (E) Immunolocalization of Mps1 in metaphase cells expressing EGFP–Polo^WT and EGFP–Polo^T182D treated with MG132 for 2 h. Kinetochore pairs in boxed areas are shown in higher magnification and the respective intensity profiles of Mps1 and CID labelling are represented. (F) Quantification of Polo^T182Ph and Mps1 kinetochore levels from experiments in (D) and (E), respectively, plotted against interkinetochore distance. Polo^T182Ph and Mps1 fluorescence intensities at kinetochores were determined relative to CID. (G) Mitotic progression of S2 cells expressing EGFP–Polo^WT or EGFP–Polo^T182D was monitored by time-lapse microscopy. Selected stills of live-cell imaging of control and RNAi-treated cells are shown. Time is in min. (H) Quantification of the mitotic timing — from nuclear envelope breakdown to anaphase onset — for cells expressing EGFP–Polo^WT or EGFP–Polo^T182D. (A–C) Cells were treated with MG132 for 1 h followed by 2-h incubation with colchicine. Binucleine 2 was added to cultures 30 min prior to colchicine treatment. Scale bars represent 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 4

Mps1 kinetochore localization controls BubR1 hyperphosphorylation. (A) Immunolocalization of BubR1 and Mps1. Insets show higher-magnification views of kinetochore pairs in the boxed areas. (B) Quantification of the experiment in (A). BubR1 and Mps1 fluorescence intensities at kinetochores were determined relative to CID. Data represent mean±s.d. and the mean values obtained for control cells were set to 100%. ***P<0.001, Student’s t-test. n >20 cells for each condition. (C) FRAP analysis of EGFP–BubR1 in control and Mps1-depleted cells after bleaching of single kinetochores. Graph shows average fluorescence intensities±s.e., n=18 cells/condition. Recovery half-life for both the slow and fast phase is depicted. (D) Kinetochore localization of BubR1 in third-instar larvae neuroblasts from w¹¹¹⁸ and ald^G4422homozygous. Insets show higher-magnification views of selected kinetochore pairs. (E) Selected stills of live-cell imaging showing the mitotic progression of control w¹¹¹⁸ and mutant ald^D4422 neuroblasts expressing mRFP-Tubulin and BubR1-GFP. Time is shown in min:sec. (F) Immunoprecipitation of BubR1 and Mps1 from total cell lysates obtained from control cells, Mps1-depleted cells and cells co-depleted of Ndc80 and Nuf2. Immunoprecipitates (IP) and corresponding total cell lysates (Input) were probed by immunoblotting for the indicated proteins. The phosphorylation status of BubR1 was examined (Input) and the graph displays the quantification of p-BubR1 blots normalized for Cyclin B signals. AP: alkaline phosphatase. Data are mean±s.d. of three independent experiments. (A and F) Cultured cells were treated with MG132 for 1 h followed by a 2-h period of colchicine incubation. Scale bars represent 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 5

Mps1 kinetochore localization is required to promote 3F3/2 phosphoepitope formation. (A) Immunolocalization of 3F3/2 phosphoepitope. Kinetochore pairs in boxed areas are shown at higher magnification. (B) Immunofluorescence analysis of Polo-T182 phosphorylation. The insets show higher-magnification views of kinetochore pairs in the boxed areas. Cultured cells were treated with MG132 for 1 h followed by 2 h of colchicine incubation. Binucleine 2 was added to cultures 30 min before microtubule depolymerization with colchicine. (C) Quantification of experiment in (A). 3F3/2 signal intensities at kinetochores were determined relative to CID. Data represent mean±s.d. and the mean values obtained for control cells were set to 100%. 0.01<*P<0.05; ***P<0.001, One-way ANOVA, Turkey’s multiple comparison test. n>20 cells for each condition. (D) Quantifications of experiment in (B). Polo^T182Ph fluorescence intensities at kinetochores were determined relative to CID. Data represent mean±s.d. and mean values obtained for control cells were set to 100%. ***P<0.001, One-way ANOVA, Turkey’s multiple comparison test. n>20 cells for each condition. (E) Immunolocalization of 3F3/2 phosphoepitope in S2 cells expressing EGFP–Polo^T182D treated with MG132 for 2 h. Kinetochore pair in boxed area is shown at higher magnification. 3F3/2 kinetochore levels were determined relative to CID and plotted against interkinetochore distance. Scale bars represent 5 μm.

Figure 6

Mps1-dependent BubR1 hyperphosphorylation is required for recruitment of Cdc20 to kinetochores and MCC formation. (A) Immunolocalization of Cdc20. Selected kinetochore pairs in the boxed areas are shown at higher-magnification. (B) Distribution of Cdc20 levels at unattached kinetochores for the experiments in (A). Cdc20 fluorescence intensities at kinetochores were determined relative to CID. n>20 cells for each condition. (C) Immunoprecipitation of Cdc20 and BubR1 from control cells lysates were probed by immunoblottimg for the indicated proteins. (D, E) Immunoprecipitation of Cdc20 (D) and BubR1 (E) from total cell lysates obtained from cells depleted of the indicated proteins. Immunoprecipitates (IP) and corresponding total cell lysates (Input) were probed by immunoblotting for the indicated proteins. Quantification of relative levels of protein bound to Cdc20 (C) or BubR1 (D) is shown. Co-immunoprecipitated protein signals were adjusted to the corresponding input signal and normalized for immunoprecipitated Cdc20 or BubR1. Graph bars in Cdc20 IP represent mean±s.d. and were obtained from three independent experiments. Values obtained for Cdc20 and BubR1 IP in control cells were set to 100%. Graph bars in BubR1 IP result from a single IP experiment. (A–E) Cultured cells were treated with MG132 for 1 h followed by 2 h of colchicine incubation. Binucleine 2 was added to cultures 30 min before microtubule depolymerization. Scale bars represent 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 7

Overexpression of Mps1 promotes accumulation of 3F3/2 phosphoepitope and Cdc20 at kinetochore regardless of chromosome biorientation. (A) 3F3/2, Cdc20 and Mps1 kinetochore localization in metaphase chromosomes of non-induced (−CuSO₄) and in overexpressing EGFP–Mps1 cells (+CuSO₄). CID signal was used as kinetochore reference. Selected kinetochore pairs are shown at higher magnification and the respective intensities profiles represented. (B) Variation of 3F3/2 and Cdc20 kinetochore levels with the interkinetochore distance in control cells and in cells overexpressing Mps1. (C) Immunoprecipitation of Cdc20 from total cell lysates obtained from control cells and cells overexpressing Mps1. Immunoprecipitates and corresponding total cell lysates were probed by immunoblotting for the indicated proteins. AP: alkaline phosphatase. For (C) cultured cells were treated with MG132 for 1 h followed by 2 h of colchicine incubation. Scale bars represent 5 μm. Source data for this figure is available on the online supplementary information page.

Figure 8

Schematic representation of the proposed model for SAC signalling controlled through a kinase network. At kinetochores/centromeres, Polo and Aurora B functional interdependency creates a positive feedback loop that promotes efficient recruitment of Mps1 kinase to unattached kinetochores allowing its prompt activation. At kinetochores, Mps1 activity is responsible for conformational exchange of soluble O-Mad2 to C-Mad2 (Hewitt et al, 2010) and controls BubR1 hyperphosphorylation. This then promotes the local association of Cdc20 with BubR1 thereby increasing the concentration of BubR1–Cdc20 complex at the site where C-Mad2 is being generated. The incorporation of C-Mad2 in the complex increases the stability of BubR1—Cdc20 interaction to allow efficient MCC assembly and sustained SAC function.