Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation

Abstract

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Keywords: Mps1; Ska complex; Ska1; kinetochore; mass spectrometry; microtubule; mitosis; mitotic spindle; phosphorylation; protein kinase.

Figure 1. Kinetochore-associated Mps1 destabilizes microtubule attachment independently of Aurora B

(A) Strategy for chemically controlled targeting of Mps1 to metaphase kinetochores. FRB-mCherry-Mps1 was co-expressed with Mis12-GFP-FKBP in HeLa cells stably depleted of endogenous FKBP (Ballister et al., 2014). Kinetochore localization occurred within minutes of rapamycin addition (Fig. S1A). (B and C) Targeting Mps1 to kinetochores disrupts preformed K-fibers. Cells expressing FRB-mCherry-Mps1 and Mis12-GFP-FKBP were cultured with or without rapamycin for 20 minutes, then incubated on ice for 10 minutes prior to fixation and immunostaining as shown. Ndc80 was used to detect kinetochores. Integrated MT intensity was quantified in three separate experiments (n = 40 control and 57 rapamycin-treated cells) and compared using the Mann-Whitney U test. Error bars indicate SEM. Scale bars, 5 μm. (D and E) Kinetochore-targeted Mps1 disrupts metaphase bi-orientation independently of Aurora B. Cells expressing FRB-mCherry-Mps1 and Mis12-GFP-FKBP were cultured in MG132 for 20 minutes to induce metaphase arrest. Thereafter, cells were maintained in MG132 ± reversine (rev) or ZM447439 (ZM) for 60 minutes to pre-inhibit Mps1 or Aurora B, and then imaged during a final 30-min treatment with rapamycin or solvent (DMSO). The fraction of cells with no, low (≤ 4 affected chromosomes), medium (> 4 chromosomes), or high levels of misalignment (no discernable metaphase plate) was determined in five separate experiments (n = 90 to 180 cells per condition; representative images shown in panel D and Fig. S1D) and compared using two-way ANOVA and Sidak post hoc tests. Error bars indicate SEM. (F and G) Cells expressing FRB-mCherry-Mps1 and Mis12-GFP-FKBP were treated as in Fig. 1D, then fixed and stained with antibodies to Aurora B-phosphorylated Dsn1 (pS109-Dsn1; (Welburn et al., 2010)) or GFP (to detect Mis12-GFP-FKBP). Representative images (main panels) and magnified views of kinetochores (insets) are shown. Dsn1 phosphorylation was quantified from ≥20 kinetochores of 10 cells per condition and compared using one-way ANOVA and Kruskal-Wallis post hoc tests. Error bars indicate SEM. See also Figure S1.

Figure 2. Global phosphoproteomics reveals sites of Mps1-regulated phosphorylation at the outer kinetochore

(A) SILAC-based strategy for systematic discovery of Mps1 substrates and effectors. Mps1^wt and Mps1^as human retinal pigment epithelial cells (Maciejowski et al., 2010) were grown in medium containing light (Arg⁰ and Lys⁰) or heavy (Arg¹⁰Lys⁸) amino acids, synchronized in mitosis with nocodazole, then treated with or without 3MB-PP1 (plus nocodazole and MG132) for 2 hours. Tryptic phosphopeptide samples were prepared by strong cation exchange (SCX) fractionation and enrichment by immobilized metal affinity chromatography (IMAC), followed by LC-MS analysis and MaxQuant data processing (Oppermann et al., 2012). (B) Summary of large-scale phosphoproteomics data collected for Mps1^wt and Mps1^as cells. Four biological replicates were analyzed for each cell line. Only phosphosites that could be quantified and assigned to specific serine, threonine or tyrosine residues with a localization probability of at least 0.75 (class I sites) were counted (Olsen et al., 2006). (C) Gene ontology (GO) term enrichment analysis on Mps1-regulated phosphoproteins. P-values were computed by Fisher’s exact test and corrected using the Benjamini-Hochberg FDR. (D) Functional association networks were retrieved from the STRING database and visualized in Cytoscape (Shannon et al., 2003). Mps1-regulated phosphoproteins quantified in at least two experimental runs per cell line are highlighted in red (decreased phosphorylation) and green (increased phosphorylation), while those quantified in fewer runs are in pink (decreased phosphorylation). Proteins and complexes with Mps1-dependent kinetochore localization (Hewitt et al., 2010; Kwiatkowski et al., 2010; Maciejowski et al., 2010; Santaguida et al., 2010) are marked in orange. APC/C^Cdc20 substrates securin (PTTG1) and cyclin B1 (CCNB1) are in yellow. All other proteins in the network are in blue. See also Figure S2 and Tables S1–S3.

Figure 3. Mps1 substrate phosphorylation is sensitive to microtubule attachment and tension

HeLa cells expressing LAP-tagged Mps1 or Ska3 (A and B) or GFP-tagged Rod (C) were synchronized in mitosis with nocodazole, shaken off, and transferred into medium containing the indicated compounds for two hours. Inhibitors selective for Mps1 (reversine and IN-1) (Kwiatkowski et al., 2010; Santaguida et al., 2010), Aurora B (ZM447439) (Ditchfield et al., 2003), and Plk1 (BI-2536) (Lenart et al., 2007) have been described. Tagged proteins were purified from mitotic lysates using S-protein-agarose (A–B) or GFP antibody-coupled magnetic beads (C) and analyzed by Western blotting. Phosphospecific and total protein band intensities were quantified and used to compute relative phosphorylation ratios, expressed as a fraction of the no-kinase-inhibitor control (lane 1). Consistent results were obtained in at least two separate experiments. (D–F) RPE cells were treated with the indicated compounds for two hours, then fixed and permeabilized concurrently (D–E) or pre-extracted before fixation and immunostaining (F). Maximum-intensity projections of deconvolved z-stacks and magnified views of individual kinetochores are shown. Images are representative of three experiments. (G) Fluorescence intensities were measured from 200 to 250 kinetochores per condition and compared using two-way ANOVA and Sidak post hoc tests. Consistent results were obtained in three experiments. Error bars indicate SEM. See also Figure S3.

Figure 4. B56-PP2A phosphatases counteract the attachment-destabilizing activity of Mps1

(A and B) PP2A-B56 reduces Mps1-dependent phosphorylation at prometaphase kinetochores. RPE cells were transfected with a nontargeting control siRNA (siCTRL) or an siRNA pool targeting all B56 subunits (siB56). Maximum-intensity projections of deconvolved z-stacks (main panels) and magnified views of individual kinetochores (insets) are representative of two experiments. Plk1 enrichment at kinetochores was used as a positive control for PP2A-B56 depletion (Foley et al., 2011). Fluorescence intensities were measured from 59 to 113 kinetochores per condition and compared using two-way ANOVA and Sidak post hoc tests. Error bars indicate SEM. (C and D) Mps1 inhibition stabilizes kinetochore-microtubule attachments in PP2A-B56-deficient cells. Control and B56-depleted cells were treated with MG132 for 20 minutes, followed by addition of 200 nM reversine (rev) or solvent (DMSO) for 50 minutes. Specimens were incubated on ice for 10 minutes before fixation, processed for IFM, and scored visually. Maximum-intensity projections and magnified views of individual kinetochores (insets) are representative of three experiments. The fraction of cells with few/no cold-stable microtubules was determined in three experiments (n ≥ 40 cells per condition per experiment) and compared using one-way ANOVA and Sidak post hoc tests. Error bars indicate SEM from three experiments.

Figure 5. Mps1 destabilizes kinetochore-microtubule attachments by phosphorylating the hinge region of the Ska complex

(A) Structures of the Ska1 microtubule binding domain (MTBD) (Abad et al., 2014) and the Ska1/2/3 core complex (Jeyaprakash et al., 2012). Inset displays a magnified view of the hinge region, with serine 34 on Ska3 labeled in red. (B) HeLa cells bearing the indicated LAP-Ska3 transgenes were transfected with a 3′ UTR-specific siRNA (siSKA3) or luciferase-specific siRNA (siGL2) as a negative control, then harvested 72 hr later and analyzed by Western blotting. Chk1 was used a loading control. Results are representative of two experiments. (C) HeLa cells transgenic for LAP-Ska3 were synchronized in mitosis with nocodazole and harvested immediately (wt, S34A, and S34D; lanes 1–3), or after further treatment with MG132 (wt + MG, lane 4) or MG132 and reversine (wt + rev, lane 5) for 2 hours. LAP-Ska3 was precipitated with S-protein-agarose and analyzed by Western blotting to assess its phosphorylation state and binding to Ska1. Results are representative of three independent experiments. (D) SKA3-depleted HeLa cells were treated with nocodazole for 2 hr and analyzed by IFM to detect LAP-Ska3 (green) or CREST (red). Images are representative of three experiments. Kinetochore targeting of LAP-Ska3 is reported as a percentage ± SEM of the wildtype control. (E and F) Control and SKA3-depleted HeLa cells were treated with MG132 for 50 minutes, incubated on ice for 10 minutes, and analyzed by IFM as shown. Images shown are representative of three experiments. Cold-stable microtubules were quantified from 8 to 12 cells per condition and compared using one-way ANOVA and Kruskal-Wallis post hoc test. No cold-stability defect was observed in the absence of Ska3 depletion, indicating that the S34D allele acts recessively.

Figure 6. Regulated phosphorylation and dephosphorylation of the Ska complex hinge are required for accurate chromosome segregation

(A) HeLa cells transgenic for LAP-Ska3 and H2B-RFP were transfected with siRNAs and followed by spinning disk microscopy. Time relative to nuclear envelope breakdown (NEBD) is indicated on each image and representative of at least three experiments per condition. (B) The length of M phase (from NEBD to mitotic exit or cell death) was quantified from 104 to 208 cells in three experiments and compared using one-way ANOVA and Kruskal-Wallis post hoc test. (C) Cells analyzed in (B) were also scored for their eventual fates (persistent mitotic arrest, cell death, or anaphase with or without lagging chromosomes). (D and E) HeLa cells reconstituted with wildtype or mutant LAP-Ska3 were treated with monastrol for 2 hours. Following washout, cells were incubated in MG132 for 60 minutes, then fixed and analyzed by IFM. Images are representative of two experiments (n = 40 to 100 cells analyzed per condition; P-values computed using Fisher’s exact test). (F and G) Live imaging of LAP-Ska3 and H2B-RFP in cells reaching metaphase after endogenous SKA3 depletion. Inter-kinetochore distances were measured from 105 to 130 sister kinetochore pairs (6 to 10 cells per condition) and compared using one-way ANOVA and Kruskal-Wallis post hoc test.

Figure 7. Phosphomimetic hinge mutation decreases the Ska complex’s microtubule residency time and impedes its lattice-to-end conversion on disassembling microtubules

(A) Kymographs of GFP-labeled wildtype and hinge-mutant Ska complexes on GMP-CPP stabilized microtubules, demonstrating 1-D diffusion on the lattice. (B) Cumulative plot of microtubule residency times for SKA^wt (n=223), SKA^S34A (n=208), and SKA^S34D complexes (n=207). Half-lives (t_0.5) of each complex were compared using a Mann-Whitney U test. (C) Kymographs of GFP-labeled Ska complexes on dynamic X-rhodamine labeled microtubules, demonstrating end-tracking behavior. Note that microtubule seeds were labeled in the far-red channel and hence not visible. (D) Magnified views of lattice-to-end conversion events (arrowheads), at which Ska complexes switched from 1-D diffusion to processive tracking of the depolymerizing plus end. (E) Scheme for quantitative analysis of lattice-to-end conversion events. The initial intensity of end-tracking Ska complexes during the first 10 seconds of end-tracking (left panel) and their change in intensity relative to pre-conversion Ska complexes (right panel) were determined as shown. (F) Median intensities of SKA^wt (n=29), SKA^S34A (n=27), and SKA^S34D particles (n=19) that initiate end tracking. Statistical comparisons were made using a Mann Whitney U test. (G) Integrated post-conversion (ΣI_post) and pre-conversion (ΣI_pre) intensities were determined for SKA^wt (n=18), SKA^S34A (n=14), and SKA^S34D particles (n=13). Ratios below 1 indicate loss of Ska complexes during lattice-to-end conversion. Statistical comparisons were made using a Mann Whitney U test. (H) Hinge phosphorylation promotes the Ska complex’s dissociation from microtubules and impedes its conversion from lattice diffusion to processive end-tracking during microtubule depolymerization. See also Figure S4.