The Ska complex promotes Aurora B activity to ensure chromosome biorientation

Abstract

Chromosome biorientation and accurate segregation rely on the plasticity of kinetochore-microtubule (KT-MT) attachments. Aurora B facilitates KT-MT dynamics by phosphorylating kinetochore proteins that are critical for KT-MT interactions. Among the substrates whose microtubule and kinetochore binding is curtailed by Aurora B is the spindle and kinetochore-associated (Ska) complex, a key factor for KT-MT stability. Here, we show that Ska is not only a substrate of Aurora B, but is also required for Aurora B activity. Ska-deficient cells fail to biorient and display chromosome segregation errors underlying suppressed KT-MT turnover. These defects coincide with KNL1-Mis12-Ndc80 network hypophosphorylation, reduced mitotic centromere-associated kinesin localization, and Aurora B T-loop phosphorylation at kinetochores. We further show that Ska requires its microtubule-binding capability to promote Aurora B activity in cells and stimulates Aurora B catalytic activity in vitro. Finally, we show that protein phosphatase 1 counteracts Aurora B activity to enable Ska kinetochore accumulation once biorientation is achieved. We propose that Ska promotes Aurora B activity to limit its own microtubule and kinetochore association and to ensure that KT-MT dynamics and stability fall within an optimal balance for biorientation.

Figure 1.

The Ska complex is required for error-free chromosome segregation and regulation of KT-MT dynamics. (A) Stills from live-imaging videos of HeLa S3 H2B–GFP cells treated with control or Ska1 and Ska3 siRNAs for 36–40 h before filming. Arrows point at the lagging chromosome; arrowheads highlight chromosome fragments. (B) Percentage of anaphase cells with lagging chromosomes (CHRs). Bars represent mean ± 95% CI (n ≥ 300 anaphase cells per condition from four independent experiments). (C) Nuclear integrity of siControl or siSka1+3 cells in interphase 48 h after siRNA transfection. (D) Percentage of micronucleated interphase cells. Bars represent mean ± 95% CI (n = 1,500 cells per condition from three experiments). (E) siControl or siSka1+3 transfected cells were treated after 48 h with the Mps1 inhibitor reversine for 20–25 min to force anaphase entry, followed by 5-min incubation at 4°C to depolymerize non-KT-MTs. Cells were fixed and stained with the indicated antibodies. Shown are optical sections of a siControl cell (left) with a bioriented sister KT pair in the enlarged crop before anaphase onset, and siSka1+3 cells (right) with merotelic KTs highlighted with arrows before and after anaphase onset. (F) Live-cell fluorescence images before and after photoactivation of HeLa K cells stably expressing PA-GFP–α-tubulin/H2B–mRFP treated with control or Ska3 siRNAs for 48 h. Note that depletion of a single Ska subunits leads to complete loss of the complex (see Fig. S1 G). (G) Quantification of the fluorescence intensity decay of the activated regions over time. Relative GFP intensities were fitted to a double exponential equation corresponding to fast (non-KT-MT) and slow (KT-MT) MT populations (see Materials and methods for details and Table S1 for sample sizes and fitting parameters). Shaded areas represent the 95% CI. Percentage of the non-KT-MTs (H) and half-lives of non-KT-MTs and KT-MTs (I) obtained from G. Bars represent mean ± 95% CI. Asterisks show statistical significance (Student’s t test, unpaired). ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01. Bars: 5 µm; (E, bottom) 1 µm.

Figure 2.

The Ska complex is required for Aurora B activity in cells. Immunofluorescence images (A, C, E, and G) and quantification (B, D, F, and H) of relative Hec1-pS44, KNL1-pS24, MCAK, and histone H3-pS10 intensities, respectively, in HeLa S3 cells treated for 48 h with control or Ska1 and Ska3 siRNAs. The Aurora B inhibitor ZM was added to siControl cells 1 h before fixation (n = 10–20, n = 15–20, n = 10–77, and n = 47–61 cells per condition, respectively, from one to three experiments). (I) HeLa S3 cells were depleted of endogenous Ska1 and Ska3 or treated with control siRNAs, synchronized by a double thymidine arrest/release, and rescued (Res.) by transfection with siRNA-resistant mCherry (mCh.)-Ska1 and Myc-Ska3 or empty mCherry- and Myc-Vectors (Vector), as control, and stained with the indicated antibodies. (J) Relative H3-pS10 intensities in cells treated as in I (n = 44–59 cells from two experiments). Res., rescue with mCh.-Ska1 and Myc-Ska3. Live-cell images of HeLa K cells stably expressing a CENP-B–fused (K and L) or H2B-fused (M and N) Aurora B FRET sensor treated as in A. The FRET sensors were completely dephosphorylated in siControl cells treated with ZM 30 min before imaging. Shown are emission ratio images (TFP/YFP or CFP/YFP) and images for sensor localization (TFP or CFP emission). (L) Scatter plot showing TFP/YFP emission ratios calculated for individual aligned KTs in siControl and siSka1+3 cells (n = 500 KTs from 50 cells each from two experiments) or mostly unaligned KTs in ZM-treated cells (n = 100 KTs from 10 cells from one experiment). (N) CFP/YFP emission ratios calculated for individual siControl and siSka1+3 cells with aligned chromosomes (n = 33–55 cells from three experiments) or ZM-treated cells with mostly unaligned chromosomes (n = 33 cells from one experiment). Horizontal lines depict mean. Asterisks show statistical significance (Student’s t test, unpaired). ****, P ≤ 0.0001; ns, nonsignificant. Bars, 5 µm.

Figure 3.

The Ska complex promotes Aurora B kinase activity at KTs independently of Aurora B centromere targeting. HeLa S3 cells treated for 48 h with control, Ska1 and Ska3, or control siRNAs and ZM for 1 h were fixed and stained for Aurora B and H3-pT3 (A) or AurB-pT232 (D). (B) Quantification of Aurora B centromere levels (n = 40 cells per condition from three experiments). (C) Relative histone H3-pT3 intensities in cells treated as in A (n = 40 cells per condition from three experiments). (E) Relative AurB-pT232 and Aurora B intensities in cells treated as in D (n = 30–40 per condition from two experiments). (F–I) HeLa S3 cells were cotransfected with CB^DBD-INCENP–GFP and control or Ska1 and Ska3 siRNAs for 48 h. ZM was added to siControl cells 1 h before fixation. Immunofluorescence images (F) and quantification (G) of Aurora B and CB^DBD-INCENP–GFP centromere (CEN) over chromosome (CHR) arm signal ratios (n = 25–44 cells per condition from two or more experiments). Immunofluorescence images (H) and quantification (I) of AurB-pT232 over CB^DBD-INCENP–GFP signal ratios (n = 19–86 cells per condition from one to three experiments). Asterisks show statistical significance (Student’s t test, unpaired). ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05; ns, nonsignificant. Bars: 5 µm; (D, insets) 1 µm.

Figure 4.

The MT-binding capability of the Ska complex is required for Aurora B activity. Immunofluorescence images (A, C, E, G, and I) and quantification (B, D, F, H, and J) of relative Hec1-pS44, KNL1-pS24, MCAK, histone H3-pS10, and AurB-pT232 intensities, respectively, in HeLa cells stably expressing GFP-tagged and siRNA-resistant wild-type Ska1 (WT) or the MT-binding deficient mutants Ska1^{R155A, R236A, R245A} (ARG) and Ska1^1-131 (ΔMTBD), respectively, treated for 48 h with control or Ska1 siRNAs (n = 25, n = 40–55, n = 17–26, n = 35–40, and n = 81–105 cells per condition, respectively, from one to four experiments). Note that spindle association of GFP-Ska1^WT is not visible in all cells because of the simultaneous cell permeabilization and fixation. Horizontal lines depict mean. Asterisks show statistical significance (Student’s t test, unpaired). ****, P ≤ 0.0001; ***, P ≤ 0.001. Bars, 5 µm.

Figure 5.

The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP^790–919–His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[³²P]ATP. (B) Quantification of histone H3 ³²P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[³²P]ATP. Aurora B autophosphorylation is visualized by autoradiography (³²P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP^790–919–His or BSA, as control, before addition of histone H3 and γ-[³²P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).

Figure 6.

PP1 opposes Aurora B activity to enable Ska complex enrichment at bioriented KTs. (A) Asynchronously growing HeLa S3 cells were arrested in metaphase with MG132 (MG) or in early prometaphase with STLC for 2–3 h and treated for 30 min with the indicated combinations of OA and ZM. DMSO treatment served as negative control. Cells were fixed and stained with the indicated antibodies. (B) Quantification of relative Ska3 KT intensities in cells treated as in A (n = 41–51 from two experiments for MG-pretreated cells, and n = 18–20 cells from one experiment for STLC-pretreated cells). (C) HeLa S3 cells were transfected with siRNA-resistant wild-type (WT) mCherry-KNL1 or PP1-binding-deficient mCherry-KNL1^RVSF/AAAA together with GFP-PP1γ, before depletion of endogenous KNL1. Cells were fixed and stained with the indicated antibodies. (D) Quantification of relative Ska3 KT intensities in cells treated as in C (n = 8–11 cells per condition from one experiment). (E) Model for the regulation of KT-MT attachment dynamics during chromosome biorientation by the feedback loop between the Ska complex and Aurora B (see Discussion for details). (F) Scheme for feedback control of Ska KT/MT and Aurora B activity levels (see Discussion for details). Horizontal lines depict mean. NEBD, nuclear envelope breakdown. Asterisks show statistical significance (Student’s t test, unpaired). ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; ns, nonsignificant. Bars, 5 µm.