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Kinetochore–microtubule error correction is driven by differentially regulated interaction modes

Nature Cell Biology volume 17, pages 421–433 (2015)Cite this article

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An Erratum to this article was published on 27 March 2015

This article has been updated

Abstract

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore–microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

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Figure 1: The KT–MT attachments are turned over repeatedly when the Dam1 C-terminus and Ndc80 N-tail are deleted.
Figure 2: The KT attaches normally to the MT lateral surface but detaches afterwards, when the Dam1 C-terminus and the Ndc80 N-tail are deleted.
Figure 3: The KT often detaches from the MT end when the Dam1 C-terminus and the Ndc80 N-tail are deleted.
Figure 4: The Dam1 C-terminus helps Stu2 to rescue an MT.
Figure 5: Aurora B phospho-mimicking Dam1 and Ndc80 mutants show repeated turnover of KT–MT attachments.
Figure 6: Aurora B phospho-mimicking Dam1 and Ndc80 mutants show normal lateral KT–MT attachment, but subsequent detachment from the MT end.
Figure 7: Aurora B facilitates detachment of an unreplicated MC from the MT end without changing the kinetics of its lateral MT attachment.
Figure 8: Non-phosphorylatable Dam1 and Ndc80 mutants at aurora B sites reduce the detachment rate of a KT from the MT plus end.

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Change history

  • 11 March 2015

    In the version of this Article originally published online, the lines connecting the data points were missing from the chart on the left of Fig. 8c. This has been corrected in all versions of the Article.

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Acknowledgements

We thank M. Gierlinski and the Tanaka and Novak groups for discussion, E. Griffis and L. Clayton for reading the manuscript and A. Musacchio for advising on ndc80-CH-K6A mutations. We thank J-F. Maure for constructing the T7427 strain and dam1C-4D mutant, N. Kobayashi for making and testing the ipl1-321–aid mutant, R. Ciosk, J. E. Haber, M. Kanemaki, U. K. Laemmli, K. Nasmyth, K. E. Sawin, R. Y. Tsien, F. Uhlmann, EUROSCARF and the Yeast Resource Centre for reagents and S. Swift and A. F.M. Ibrahim for technical help. This work was supported by the Wellcome Trust (096535, 083524, 097945), the Medical Research Council (84678), EC FP7 MitoSys (241548), an ERC advanced grant (322682), Cancer Research UK (A6996) and the Human Frontier Science Program (RGP0035-2009). M.K. received a BBSRC studentship. T.U.T. is a Wellcome Trust Principal Research Fellow.

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  1. Maria Kalantzaki & Akihisa Mino

    Present address: Present addresses: MRC Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK (M.K.); Boston Children’s Hospital, Joslin Diabetes Center, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, USA (A.M.).,

Authors and Affiliations

  1. Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street Dundee DD1 5EH, UK,

    Maria Kalantzaki, Etsushi Kitamura, Akihisa Mino & Tomoyuki U. Tanaka

  2. Department of Biochemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, South Parks Road Oxford OX1 3QU, UK,

    Tongli Zhang & Béla Novák

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Contributions

M.K., E.K. and T.U.T. designed experiments and interpreted results. M.K. and E.K. carried out experiments and analysed data. T.U.T., M.K., E.K., T.Z. and B.N. wrote the manuscript. A.M. gave technical support.

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Integrated supplementary information

Supplementary Figure 3 KTs show detachment from the spindle, followed by recapture, when Dam1 C-terminus and Ndc80 N-tail are deleted.

NDC80+DAM1+ (T9659), ndc80ΔN (T9298), dam1-TEVsites (T9258), ndc80ΔN dam1-TEVsites (T11454) cells with PGAL-TEV (except for T9298) MTW1-4×mCherry Venus-TUB1 PMET3-CDC20 were treated as in Fig. 1e, except that images were acquired every 30 s. A representative cell with Ndc80ΔN plus Dam1ΔCclv (0 min: start of image acquisition) is shown here. The graphs show percentages of cells that showed KT detachment from the spindle (usually followed by reattachment); n = 33, 38, 34 and 35 cells were analysed (from left to right). Experiments were performed twice (statistics source data are shown in Supplementary Table 2) and a representative experiment is shown here. p-values (two tailed) were obtained by Fisher’s exact test.

Supplementary Figure 4 Mutations at the Ndc80 calponin-homology domain lead to a defect in the lateral KT–MT attachment.

PCUP1-ubi-DHFR-ndc80 (ndc80-td) PGAL-CEN3-tetOs TetR-GFP YFP-TUB1 PMET3-CDC20 cells with NDC80+ (T10069), ndc80-CH-K6A (T7427) [expressed from NDC80 promoter] or no additional NDC80 construct (T7428), inserted at his3 locus, were treated with α-factor at 25 °C in methionine-dropout medium with raffinose, galactose and 2% CuSO4. After 3 h, cells were released to YPA medium with raffinose, galactose (to inactivate PGAL–CEN3) and methionine (to deplete Cdc20) at 35 °C (to degrade Ndc80-td). After 20 min, cells were transferred to synthetic complete medium with glucose (to re-activate PGAL-CEN3) and methionine, and images were acquired every 5 min (start of image acquisition; 0 min) at 35 °C. In the ndc80-CH-K6A mutant, six lysines were replaced with alanines within the calponin-homology domain. Representative images of T10069 and T7427 cells at 20 min (left) and the percentage of cells (n = 20–30 cells were analyse at each time point) with CEN3 that is unattached to MTs (right). The above result and Fig. 2 suggest the Ndc80 calponin-homology domain, but not the Dam1 C-terminus or the Ndc80 N-tail, is required for the initial lateral KT–MT attachment. Note that the KT on CEN3 before being caught on the lateral surface of a spindle MT (a MT extending from a spindle pole) often generates short MTs that are thought to facilitate a subsequent KT encounter with a spindle MT (ref. 57); such short KT-derived MTs were found similarly in the wild-type control, Dam1ΔCclv, Ndc80ΔN and double deletion (our unpublished result). Once CEN3is loaded on the lattice of a spindle MT, CEN3 showed sliding along this MT towards a spindle pole3; this sliding was also found similarly in wild-type control and the deletion mutants (our unpublished result).

Supplementary Figure 5 Dam1 and Stu2 interact physically in a two-hybrid assay and this interaction is abolished with Dam1ΔC and with Dam1C-4D[AurB].

(a) Stu2 protein and its C-terminus and N-terminus deletions are shown in the diagram. These deletions were used in b and c. (b,c) b shows that Dam1 and Stu2 interact physically in a two-hybrid assay and this interaction requires the Dam1 C-terminus. c shows that Dam1–Stu2 interaction in a two-hybrid assay is abolished with Dam1C-4D[AurB], that is, with phospho-mimicking mutations of the Dam1 C-terminus at Aurora B sites. Duo1 is a component of the Dam1 complex and serves as a control. Ras and Raf were also used as controls. AD and BD were as in Fig. 3g. (d) Phospho-mimicking mutants of the Dam1 C-terminus at Aurora B sites are defective in assisting Stu2 in rescuing a MT. Graph shows percentage of Stu2 transport events along a MT, leading or not leading, to MT rescue, as in Fig. 4b. DAM1+ (T11596) and dam1C-4D[AurB] dam1-aid(T11595) cells with TIR PGAL-CEN3-tetOs TetR-3xCFP STU2-4×mCherry GFP-TUB1 PMET3-CDC20 were treated as in Fig. 6a–g. n = 13 Stu2 transport events were analysed in each of T11595 and T11596. p-values (two tailed) were obtained by Fisher’s exact test. Data represent one out of two independent experiments.

Supplementary Figure 6 Phospho-mimicking mutants of the Dam1 C-terminus at Mps1 sites do not show KT detachment from the spindle.

We tested whether phospho-mimicking Dam1 mutants at Mps1 sites (dam1C-4D[Mps1]; T217, S218, S221 and S232 replaced with aspartates) show phenotypes similar to those of dam1C-4D[AurB]. dam1C-4D[Mps1](T11680) and dam1C-8D[AurB+Mps1] (T11642) cells with dam1-aid TIR PGAL-CEN3-tetOs TetR-3×CFP GFP-TUB1 PMET3-CDC20were treated and analysed as in Fig. 5c, d. The results of T9530 and T11326 in Fig. 5c, d are shown again for comparison. n = 65 and 59 cells were analysed for T11680 and T11642, respectively. Experiments were performed twice (statistics source data are shown in Supplementary Table 2) and a representative experiment is shown here. p-values (two tailed) were obtained by Fisher’s exact test. In contrast to dam1C-4D[AurB], dam1C-4D[Mps1] did not significantly increase the level of bi-orientation defects or CEN3 detachment from the spindle. Furthermore, the combination of phospho-mimicking mutations at both Aurora B and Mps1 sites (dam1C-8D[AurB+Mps1]) did not exacerbate the defects found in dam1C-4D[AurB]. Thus Mps1 phosphorylation of the Dam1 C-terminus may not suppress the function of this domain and may not contribute to error correction. Consistently, non-phosphorylatable mutants of Dam1 at these Mps1 sites did not show a defect in bi-orientation35 (our unpublished result).

Supplementary Figure 7 Non-phosphorylatable mutants of the Ndc80 N-tail and Dam1 C-terminus at Aurora B sites show normal lateral KT–MT interaction and slower bi-orientation establishment.

Cells in Fig. 8b were treated as in Fig. 6a–g; that is, CEN3 under the GAL promoter was inactivated on release from α factor treatment and then reactivated during metaphase arrest. Percentage of cells at each step of KT–MT interaction is shown (as in Fig. 2c–f). Note that, in these cells, we did not observe any CEN3 detachment from a MT or from the spindle after CEN3 was caught on the MT or on the spindle. n = 21 cells were analysed in each strain. Data represent one out of two independent experiments.

Supplementary Figure 8 Full scans of western blots (for Figs 1c and 5a).

Positions of protein size markers are shown at left of each blot.

Supplementary Table 1 Genotypes of yeast strains used in this study.
Full size table
Supplementary Table 2 Statistics source data.
Full size table

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Kalantzaki, M., Kitamura, E., Zhang, T. et al. Kinetochore–microtubule error correction is driven by differentially regulated interaction modes. Nat Cell Biol 17, 421–433 (2015). https://doi.org/10.1038/ncb3128

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