1. Volume 12, Issue 1, Pages 66-78 (July 2015)
Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells 
Conrad von Schubert, Fabien Cubizolles, Jasmine M. Bracher, Tale Sliedrecht, Geert J.P.L. Kops, Erich A. Nigg 
Cell Reports 
Volume 12, Issue 1, Pages (July 2015)
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2. Cell Reports 2015 12, 66-78DOI: (10.1016/j.celrep.2015.06.007)
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3. Figure 1
Human Plk1 and Mps1 Cooperatively Regulate Checkpoint Establishment and Maintenance
(A) Asynchronously growing RPE-1 cells were treated with the anti-MT drug nocodazole and solvent only (control), the Mps1 inhibitor Reversine, the Plk1 inhibitor TAL, or both inhibitors combined and analyzed by time-lapse imaging. Scatterplots show time in mitosis; 10–30 cells per conditions. aver, average; h, hours.
(B) RPE-1 cells pretreated with MG132 were incubated for 1 hr in the presence or absence of nocodazole and indicated inhibitors. Cells were analyzed by immunofluorescence (IF) microscopy, and human CREST antiserum was used to identify KTs. Left: chromosome alignment states at the end of incubation; percentages indicate the frequencies of shown spindle morphologies. Right: scatterplot depicts Mad1 staining intensity at KTs; 28–39 cells per condition. Rev, Reversine; rel. signal int., relative signal intensity.
(C) RPE-1 cells expressing endogenously EGFP-tagged Mad2 (Figure S1I) were synchronized in mitosis by incubation with nocodazole, followed by the addition of the indicated kinase inhibitors and time-lapse imaging. Left: stills from live recordings (Movie S1). Middle: traces illustrating loss of EGFP-Mad2 from KTs; n = 8 cells per condition, shaded areas represent SEM. Right: quantification of the time in mitosis; n = 41–49 cells per condition. int., intensity. Scale bars, 5 μm.
∗∗∗p < (from Student’s t tests). See also Figure S1I and Movie S1.
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4. Figure 2 Plk1 Inhibition Delays SAC Activation in Unperturbed Mitosis
Left: asynchronously growing RPE-1 cells were incubated with kinase inhibitors or solvent for 2 hr and analyzed by IF, using the indicated antibodies. Mitotic phases were determined as described in Results (see also Figure S2A).
Right: KT recruitment of C-Mad2 was measured at different stages of mitosis; scatterplot shows results for n = 377–456 KT pairs from 10–13 cells per condition. fluor. int., fluorescence intensity. Scale bar, 5 μm. ∗∗∗p < (from Student’s t tests).
See also Figure S2A.
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5. Figure 3 Plk1 Inhibition Acts on the KT Branch of the SAC
Asynchronous RPE-1 cells expressing endogenously Venus-tagged Cyclin B1 were incubated with nocodazole and indicated inhibitors, followed by time-lapse imaging.
(A) Scatterplots depict levels of Cyclin B1-Venus at the indicated cell cycle stages; time is indicated relative to nuclear translocation of Cyclin B1-Venus (n = 20–29 cells per condition). Prometaph., prometaphase.
(B) Upper: micrographs represent still images from live recordings (Movie S2); time stamps indicate time relative to nuclear translocation of Cyclin B1-Venus. Scale bar, 5 μm. Lower: tracings illustrating levels of Cyclin B1 before nuclear envelope breakdown (time = 0) as well as kinetics of subsequent degradation (n = 7–12 cells per condition). Shaded areas represent SEM. Rev, Reversine; int., intensity.
See also Movie S2.
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6. Figure 4
The Effect of Plk1 Inhibition on SAC Signaling Does Not Involve Delocalization or Reduced Catalytic Activity of Aurora B
In (A)–(C) and (E), RPE-1 cells were preincubated for 2 hr with MG132 and then treated with nocodazole and the indicated kinase inhibitors for 1 hr.
(A) Histogram shows the phosphorylation state of histone H3 Thr3 and centromere levels of Aurora B as determined by IF (n = 25–35 cells per condition); micrographs are shown in Figure S4A. rel. signal int., relative signal intensity.
(B) Left: EGFP centromere signals captured from living cells expressing endogenously EGFP-tagged Aurora B (Figure S4B). Right: scatterplot showing EGFP signals for n = 120 centromeres from 15 cells per condition. Rev., Reversine.
(C) Histogram shows Mad1 KT levels as determined by IF analysis (n = 31–39 cells per condition; micrographs are shown in Figure S4C).
(D) Asynchronously growing HeLa cells stably expressing a chromatin-targeted FRET sensor for Aurora B activity were incubated with nocodazole and kinase inhibitors and monitored by time-lapse ratio imaging (Movie S3). Left: heatmap representation of reporter phosphorylation; time is indicated relative to mitotic entry. Right: traces illustrating the results from n = 14–22 cells per condition; shaded areas represent SEM.
(E) Histogram shows the quantification of IF signals produced by phospho-specific antibodies detecting Aurora B targets. Signals were quantified over chromosome arms for phospho-Ser10 on histone H3 and over centromeres for phospho-Thr7 on CENP-A (n = 24–29 cells per condition; micrographs are shown in Figure S4E).
Scale bars, 5 μm. Error bars indicate SEM. ∗∗∗p < (from Student’s t tests); ns, not significant. See also Figure S4 and Movie S3.
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7. Figure 5 Plk1 and Mps1 Both Target KNL-1 MELT Motifs
(A) Autoradiography shows the results of in vitro peptide spotting kinase assays. Peptides containing the MELT motif (shaded areas) were synthesized carrying the Thr phosphoacceptor (T, arrowhead) or an Ala (A) in the corresponding position and incubated with γ-32P-ATP in the presence (+Plk1) or absence (no enzyme) of recombinant Plk1. Additional peptides carrying Ala in place of potentially interfering phosphoacceptors were also analyzed. Equal synthesis of peptides was monitored by hydration of membranes prior to side-chain de-protection (peptide).
(B) The motif around KNL-1 Thr875 was used to design a MELT-based FRET sensor (Figure S5C) targeted to chromatin by H2B fusion. To determine the specificity of MELT phosphorylation, Thr875 was substituted with Ala. HeLa S3 cells expressing the FRET reporters were enriched in mitosis with nocodazole and additionally treated with MG132 and 100 nM BI-2536 before CFP/FRET ratios were determined by live-cell microscopy. Left: heatmap representation of reporter phosphorylation. Right: scatterplot shows emission ratios from n = 17–31 cells per condition.
(C) RPE-1 cells were synchronized in metaphase using MG132 and subsequently treated with nocodazole and kinase inhibitors. Left: KNL-1 phosphorylation site occupancy, as determined by IF staining with an antibody specific for phospho-Thr875. Right: histogram shows quantification of P-Thr875 IF staining (n = 50–62 cells per condition). Rev., Reversine; rel. signal int., relative signal intensity. Error bars indicate SEM.
(D and E) Plk1 inhibition reduces Bub3:BubR1 complex recruitment to KTs. RPE-1 cells (D) or RPE-1 cells expressing endogenously EGFP-tagged Bub3 (E; Figure S5H) were treated as described in (C) and analyzed by IF, using the indicated antibodies; scatterplots show results from quantitative IF (n = 65–69 cells per condition; micrographs are shown in Figure S5I).
Scale bars, 5 μm. ∗∗∗p < (from Student’s t tests). See also Figure S5.
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8. Figure 6 Plk1 Targets Mps1 Autophosphorylation Sites In Vitro
(A) Mixtures of recombinant Plk1 and Mps1 were incubated for 2 min with γ-32P-ATP and with kinase inhibitors as indicated. Phosphorylation of the Plk1 substrate casein was monitored for control. Phosphorylation of Mps1 and casein was detected by autoradiography (32P), levels of Plk1 and Mps1 were monitored by western blotting (WB), and casein was visualized by Ponceau S (PS) staining. Rev., Reversine.
(B) Autoradiography and WB illustrate the time course of in vitro Mps1 phosphorylation in the presence or absence of Plk1 and Reversine (left). Quantitative data (right) were averaged from two distinct experiments.
(C) Autoradiography shows the results of peptide spotting kinase assays performed as described in the legend for Figure 5A. Peptides were synthesized to represent phosphorylation sites within Mps1 that conform to the Plk1/Mps1 consensus or were previously annotated as Mps1 autophosphorylation sites (marked by asterisks). T/S, Thr/Ser phosphoacceptor; A, Ala in position of the phosphoacceptor.
(D) Mixtures of recombinant Plk1 and kinase-dead Mps1 were subjected to in vitro kinase assays before phosphorylations on Mps1 (pS/pT) were mapped by mass spectrometry. Phosphopeptides were identified either with Mascot/Scaffold (M/S) or MaxQuant/Andromeda (MQ/A) with a decoy FDR of 0.01 on peptide spectrum matches as significance cutoff (Table S1). Asterisks indicate Mps1 autophosphorylation sites.
(E) Schematic summarizes Plk1-dependent phosphorylations on Mps1, as detected in the assays shown in (C) and (D), as well as Table S1. Annotated Mps1 auto-phosphorylation sites are highlighted in blue.
See also Table S1.
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9. Figure 7 Plk1 Enhances Mps1 Kinase Activity and KT Dissociation
(A) Mps1 null cells ectopically expressing GFP-tagged Mps1 were treated with nocodazole, MG132, and kinase inhibitors (100 nM BI-2536, 500 nM Reversine [Rev], 5 μM ZM ) before GFP-Mps1 KT levels were recorded by live-cell microscopy. Top: scatterplot shows intensity of GFP signals (n = 157–241 KTs from 45–50 cells per condition). Bottom: representative GFP KT signals.
(B) Cells described in (A) were incubated with nocodazole, MG132, and kinase inhibitors, and GFP-Mps1 KT levels were monitored by 500-ms time-lapse microscopy (Movie S5). After 2 s, a single KT pair was bleached (top left), and fluorescence recovery was measured (bottom). Top right: traces illustrate fluorescence recovery (n = 17–20 KT pairs per condition); shaded areas represent SEM. t1/2, half-time; plat., plateau.
(C) Asynchronously growing cells, as used in (A and B), were incubated with nocodazole and kinase inhibitors before fluorescence at KTs was monitored by time-lapse microscopy (Movie S6). Left: stills show data obtained from −60 min (G2 phase) to +10 min (M phase) relative to the time of mitotic entry (t = 0). Arrowheads indicate premature KT association of GFP-Mps1. Right: scatterplot shows GFP-Mps1 intensity at KTs (50 KTs from ten cells per condition).
(D) RPE-1 cells were synchronized in metaphase using MG132 and subsequently treated with nocodazole and kinase inhibitors. Mps1 activation state was assessed by staining with an antibody directed at phospho-Thr676. Histogram shows staining intensity (n = 75–79 cells per condition; micrographs are shown in Figure S7B). Error bars indicate SEM. rel. signal int., relative signal intensity.
(E) Asynchronously growing RPE-1 cells were incubated for 2 hr with kinase inhibitors in the absence of nocodazole and then stained for Mps1 activation loop phosphorylation (P-Thr676). IF staining (right) shows KT levels of P-Thr676; scatterplot (left) indicates P-Thr676 staining intensities at KTs, determined at the indicated mitotic stages (n = 19–38 cells per condition). Mitotic phases were classified as described in the legend for Figure 2. fluor. int., fluorescence intensity.
(F) Schematic illustrating the proposed involvement of Plk1 in SAC signaling at KTs. P, phosphorylation.
Scale bars, 5 μm. ∗p = ; ∗∗p = ; ∗∗∗p < (from Student’s t tests). See also Figure S7B and Movies S5 and S6.
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