Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Multiple phosphorylations control recruitment of the KMN network onto kinetochores

A Publisher Correction to this article was published on 19 November 2018

This article has been updated

Abstract

To establish a functional kinetochore, the constitutive centromere-associated network (CCAN) forms a foundation on the centromere and recruits the KMN network, which directly binds to spindle microtubules. The CENP-C and CENP-T pathways in the CCAN recruit the KMN network to kinetochores, independently. The CENP-C pathway has been considered the major scaffold for the KMN network in vertebrate CCAN. However, we demonstrate that it is mainly the CENP-T pathway that recruits the KMN network onto the kinetochores and that CENP-T–KMN interactions are essential in chicken DT40 cells. By contrast, less Ndc80 binds to the CENP-C pathway in mitosis and the Mis12–CENP-C association is decreased during mitotic progression, which is consistent with the finding that the Mis12 complex–CENP-C binding is dispensable for cell viability. Furthermore, we find that multiple phosphoregulations of CENP-T and the Mis12 complex make the CENP-T pathway dominant. These results provide key insights into kinetochore dynamics during mitotic progression.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Mis12C-binding domain of CENP-C is dispensable for mitotic progression and completion in DT40 cells.
Fig. 2: Ndc80C-binding domain of CENP-T is required for viability and mitotic progression in DT40 cells.
Fig. 3: Mis12 and Ndc80 levels on kinetochores change during mitotic progression.
Fig. 4: Mis12C binding to CENP-T is essential in DT40 cells, and the KMN network binding to CENP-T functions independently of the CENP-T C terminus.
Fig. 5: Cdk1 phosphorylation on Dsn1 reduces the affinity of the Mis12C to the Ndc8C.
Fig. 6: Forced binding of the Mis12C to CENP-C suppresses the deficiency of Ndc80C–CENP-T binding.
Fig. 7: CENP-T is a major pathway to recruit the KMN network during mitotic progression in DT40 cells.

Similar content being viewed by others

Data availability

Flow cytometry data have been provided as Supplementary Table 1. Source data for Figs. 1e,f, 2d,e, 3b–e, 4c, 5d, 6c and 7a,b and Supplementary Figs. 2a,b,e, 5e and 7f have been provided as Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Change history

  • 19 November 2018

    In the version of this Article originally published, the ‘ON’ and ‘OFF’ labels in panel c of Fig. 6 were incorrect. For the Tet treated cells (+Tet) in both image panels, CENP-T should have been ‘OFF’ and CENP-T Δ90 should have been ‘ON’. For the cells untreated with Tet (–Tet) in both graph panels, CENP-T Δ90 should have been ‘ON’. This has now been amended.

References

  1. Fukagawa, T. & Earnshaw, W. C. The centromere: chromatin foundation for the kinetochore machinery. Dev. Cell 30, 496–508 (2014).

    Article  CAS  Google Scholar 

  2. McKinley, K. L. & Cheeseman, I. M. The molecular basis for centromere identity and function. Nat. Rev. Mol. Cell Biol. 17, 16–29 (2016).

    Article  CAS  Google Scholar 

  3. Hara, M. & Fukagawa, T. Critical foundation of the kinetochore: the constitutive centromere-associated network (CCAN). Prog. Mol. Subcell. Biol. 56, 29–57 (2017).

    Article  CAS  Google Scholar 

  4. Hara, M. & Fukagawa, T. Kinetochore assembly and disassembly during mitotic entry and exit. Curr. Opin. Cell Biol. 52, 73–81 (2018).

    Article  CAS  Google Scholar 

  5. Pesenti, M. E., Weir, J. R. & Musacchio, A. Progress in the structural and functional characterization of kinetochores. Curr. Opin. Struct. Biol. 37, 152–163 (2016).

    Article  CAS  Google Scholar 

  6. Nagpal, H. & Fukagawa, T. Kinetochore assembly and function through the cell cycle. Chromosoma 125, 645–659 (2016).

    Article  CAS  Google Scholar 

  7. Black, B. E. & Cleveland, D. W. Epigenetic centromere propagation and the nature of CENP-A nucleosomes. Cell 144, 471–479 (2011).

    Article  CAS  Google Scholar 

  8. Westhorpe, F. G. & Straight, A. F. Functions of the centromere and kinetochore in chromosome segregation. Curr. Opin. Cell Biol. 25, 334–340 (2013).

    Article  CAS  Google Scholar 

  9. Suzuki, A. et al. Spindle microtubules generate tension-dependent changes in the distribution of inner kinetochore proteins. J. Cell Biol. 193, 125–140 (2011).

    Article  CAS  Google Scholar 

  10. Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. & Desai, A. The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127, 983–997 (2006).

    Article  CAS  Google Scholar 

  11. DeLuca, J. G. et al. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 127, 969–982 (2006).

    Article  CAS  Google Scholar 

  12. Alushin, G. M. et al. The Ndc80 kinetochore complex forms oligomeric arrays along microtubules. Nature 467, 805–810 (2010).

    Article  CAS  Google Scholar 

  13. Santaguida, S. & Musacchio, A. The life and miracles of kinetochores. EMBO J. 28, 2511–2531 (2009).

    Article  CAS  Google Scholar 

  14. Foltz, D. R. et al. The human CENP-A centromeric nucleosome-associated complex. Nat. Cell Biol. 8, 458–469 (2006).

    Article  CAS  Google Scholar 

  15. Okada, M. et al. The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat. Cell Biol. 8, 446–457 (2006).

    Article  CAS  Google Scholar 

  16. Weir, J. R. et al. Insights from biochemical reconstitution into the architecture of human kinetochores. Nature 537, 249–253 (2016).

    Article  CAS  Google Scholar 

  17. Saitoh, H. et al. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70, 115–125 (1992).

    Article  CAS  Google Scholar 

  18. Kwon, M. S., Hori, T., Okada, M. & Fukagawa, T. CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol. Biol. Cell 18, 2155–2168 (2007).

    Article  CAS  Google Scholar 

  19. Klare, K. et al. CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores. J. Cell Biol. 210, 11–22 (2015).

    Article  CAS  Google Scholar 

  20. Nishino, T. et al. CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold. Cell 148, 487–501 (2012).

    Article  CAS  Google Scholar 

  21. Nagpal, H. et al. Dynamic changes in CCAN organization through CENP-C during cell-cycle progression. Mol. Biol. Cell 26, 3768–3776 (2015).

    Article  CAS  Google Scholar 

  22. McKinley, K. L. et al. The CENP-L-N complex forms a critical node in an integrated meshwork of interactions at the centromere–kinetochore interface. Mol. Cell 60, 886–898 (2015).

    Article  CAS  Google Scholar 

  23. Chittori, S. et al. Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N. Science 359, 339–343 (2018).

    Article  CAS  Google Scholar 

  24. Pentakota, S. et al. Decoding the centromeric nucleosome through CENP-N. eLife 6, e33442 (2017).

    Article  Google Scholar 

  25. Tian, T. et al. Molecular basis for CENP-N recognition of CENP-A nucleosome on the human kinetochore. Cell Res. 28, 374–378 (2018).

    Article  CAS  Google Scholar 

  26. Cheeseman, I. M., Hori, T., Fukagawa, T. & Desai, A. KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. Mol. Biol. Cell 19, 587–594 (2008).

    Article  CAS  Google Scholar 

  27. Basilico, F. et al. The pseudo GTPase CENP-M drives human kinetochore assembly. eLife 3, e02978 (2014).

    Article  Google Scholar 

  28. Hori, T., Okada, M., Maenaka, K. & Fukagawa, T. CENP-O class proteins form a stable complex and are required for proper kinetochore function. Mol. Biol. Cell 19, 843–854 (2008).

    Article  CAS  Google Scholar 

  29. Gascoigne, K. E. et al. Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes. Cell 145, 410–422 (2011).

    Article  CAS  Google Scholar 

  30. Hori, T., Shang, W. H., Takeuchi, K. & Fukagawa, T. The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly. J. Cell Biol. 200, 45–60 (2013).

    Article  CAS  Google Scholar 

  31. Kato, H. et al. A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. Science 340, 1110–1113 (2013).

    Article  CAS  Google Scholar 

  32. Falk, S. J. et al. Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. Science 348, 699–703 (2015).

    Article  CAS  Google Scholar 

  33. Guo, L. Y. et al. Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition. Nat. Commun. 8, 15775 (2017).

    Article  Google Scholar 

  34. Petrovic, A. et al. The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J. Cell Biol. 190, 835–852 (2010).

    Article  CAS  Google Scholar 

  35. Przewloka, M. R. et al. CENP-C is a structural platform for kinetochore assembly. Curr. Biol. 21, 399–405 (2011).

    Article  CAS  Google Scholar 

  36. Dimitrova, Y. N., Jenni, S., Valverde, R., Khin, Y. & Harrison, S. C. Structure of the MIND complex defines a regulatory focus for yeast kinetochore assembly. Cell 167, 1014–1027.e12 (2016).

    Article  CAS  Google Scholar 

  37. Petrovic, A. et al. Structure of the MIS12 complex and molecular basis of its interaction with CENP-C at human kinetochores. Cell 167, 1028–1040.e15 (2016).

    Article  CAS  Google Scholar 

  38. Hori, T. et al. CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135, 1039–1052 (2008).

    Article  CAS  Google Scholar 

  39. Takeuchi, K. et al. The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA. Nucleic Acids Res. 42, 1644–1655 (2014).

    Article  CAS  Google Scholar 

  40. Nishino, T. et al. CENP-T provides a structural platform for outer kinetochore assembly. EMBO J. 32, 424–436 (2013).

    Article  CAS  Google Scholar 

  41. Rago, F., Gascoigne, K. E. & Cheeseman, I. M. Distinct organization and regulation of the outer kinetochore KMN network downstream of CENP-C and CENP-T. Curr. Biol. 25, 671–677 (2015).

    Article  CAS  Google Scholar 

  42. Kim, S. & Yu, H. Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores. J. Cell Biol. 208, 181–196 (2015).

    Article  CAS  Google Scholar 

  43. Suzuki, A., Badger, B. L. & Salmon, E. D. A quantitative description of Ndc80 complex linkage to human kinetochores. Nat. Commun. 6, 8161 (2015).

    Article  Google Scholar 

  44. Huis In ‘t Veld, P. J. et al. Molecular basis of outer kinetochore assembly on CENP-T. eLife 5, e21007 (2016).

    Article  Google Scholar 

  45. Welburn, J. P. et al. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore–microtubule interface. Mol. Cell 38, 383–392 (2010).

    Article  CAS  Google Scholar 

  46. del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding. Science 323, 638–641 (2009).

    Article  Google Scholar 

  47. Ye, A. A., Cane, S. & Maresca, T. J. Chromosome biorientation produces hundreds of piconewtons at a metazoan kinetochore. Nat. Commun. 7, 13221 (2016).

    Article  CAS  Google Scholar 

  48. Volkov, V. A., Huis In ‘t Veld, P. J., Dogterom, M. & Musacchio, A. Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces. eLife 7, e36764 (2018).

    Article  Google Scholar 

  49. Hornung, P. et al. A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A. J. Cell Biol. 206, 509–524 (2014).

    Article  CAS  Google Scholar 

  50. Bock, L. J. et al. Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nat. Cell Biol. 14, 614–624 (2012).

    Article  CAS  Google Scholar 

  51. Schleiffer, A. et al. CENP-T proteins are conserved centromere receptors of the Ndc80 complex. Nat. Cell Biol. 14, 604–613 (2012).

    Article  CAS  Google Scholar 

  52. Lang, J., Barber, A. & Biggins, S. An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast. eLife 7, e37819 (2018).

    Article  Google Scholar 

  53. Drinnenberg, I. A., Henikoff, S. & Malik, H. S. Evolutionary turnover of kinetochore proteins: a ship of Theseus? Trends Cell Biol. 26, 498–510 (2016).

    Article  CAS  Google Scholar 

  54. Orr, B. & Sunkel, C. E. Drosophila CENP-C is essential for centromere identity. Chromosoma 120, 83–96 (2011).

    Article  CAS  Google Scholar 

  55. Moore, L. L. & Roth, M. B. HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres. J. Cell Biol. 153, 1199–1208 (2001).

    Article  CAS  Google Scholar 

  56. van Hooff, J. J., Tromer, E., van Wijk, L. M., Snel, B. & Kops, G. J. Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics. EMBO Rep. 18, 1559–1571 (2017).

    Article  Google Scholar 

  57. Buerstedde, J. M. et al. Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9, 921–927 (1990).

    Article  CAS  Google Scholar 

  58. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  59. Kline, S. L., Cheeseman, I. M., Hori, T., Fukagawa, T. & Desai, A. The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. J. Cell Biol. 173, 9–17 (2006).

    Article  CAS  Google Scholar 

  60. Okumura, E., Sekiai, T., Hisanaga, S., Tachibana, K. & Kishimoto, T. Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase. J. Cell Biol. 132, 125–135 (1996).

    Article  CAS  Google Scholar 

  61. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  62. Dosztanyi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).

    Article  CAS  Google Scholar 

  63. Yang, Z. R., Thomson, R., McNeil, P. & Esnouf, R. M. RONN: the bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 21, 3369–3376 (2005).

    Article  CAS  Google Scholar 

  64. Ward, J. J., Sodhi, J. S., McGuffin, L. J., Buxton, B. F. & Jones, D. T. Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 337, 635–645 (2004).

    Article  CAS  Google Scholar 

  65. Fukagawa, T., Pendon, C., Morris, J. & Brown, W. CENP-C is necessary but not sufficient to induce formation of a functional centromere. EMBO J. 18, 4196–4209 (1999).

    Article  CAS  Google Scholar 

  66. Bordoli, L. et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1–13 (2009).

    Article  CAS  Google Scholar 

  67. Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    Article  CAS  Google Scholar 

  68. Hoffman, D. B., Pearson, C. G., Yen, T. J., Howell, B. J. & Salmon, E. D. Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores. Mol. Biol. Cell 12, 1995–2009 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Y. Fukagawa and R. Fukuoka for technical assistance. We also thank T. Nishino, T. Maresca and I. Cheeseman for providing plasmid constructs. This work was supported by JSPS KAKENHI grant numbers 15H05972, 16H06279 and 17H06167 to T.F., JSPS KAKENHI grant number 16K18491 to M.H., JSPS KAKENHI grant number 16H01304 to M.A. and JSPS KAKENHI grant number to 17K07501 to T.H.

Author information

Authors and Affiliations

Authors

Contributions

M.H. designed and performed all of the experiments in this study. M.A. performed the binding studies of CENP-T and Dsn1 peptides to Spc24–Spc25 using fluorescence polarization and helped to prepare recombinant proteins. M.A. also performed the structure modelling. E.-i.O. purified CycB–Cdk1 from starfish oocytes. T.H. and T.F. generated some mutant DT40 cell lines. T.F. performed the tension sensor analysis, supervised all of the experiments and wrote the manuscript in collaboration with M.H., discussing with all authors.

Corresponding author

Correspondence to Tatsuo Fukagawa.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Deletion of the Mis12-binding domain of CENP-C does not affect cell cycle progression.

(a), Interphase cells of cKO-CENP-C with or without expression of GFP-CENP-C FL (FL) or -CENP-C ∆73 (∆73), which lacks Mis12C- binding domain. Chicken DT40 cells in which expression of wild-type CENP-C was conditionally turned off by tetracycline (Tet) addition (cKO-CENP-C cells) were treated with Tet (upper panels: +Tet or –Tet) for 48 h and harvested. The cells were fixed and stained with an anti-Mis12 antibody. CENP-T was also stained as a marker for kinetochores. cKO-CENP-C cells expressing GFP-CENP-C FL (FL) or - CENP-C ∆73 (∆73) were cultured in the presence of Tet (lower panels). The cells were harvested, fixed, and immunostained with an anti- Mis12 antibody. DNA was stained with DAPI. Representative images of interphase cells are shown. Scale bars indicate 10 µm. Interphase cells with Mis12 signals on kinetochores were quantified in the indicated cell lines (right graph). (b), Cell cycle in the CENP-C conditional knockout background lines. The indicated cell lines were incubated with BrdU for 20 min at the indicated time after Tet addition and harvested (+Tet). Following fixation, cells were stained with an anti-BrdU antibody and propidium iodide. Cell cycle distribution was analyzed by flow cytometry. cKO-CENP-C cells and Tet-untreated cells in each line were also examined as controls. Original flow cytometry data are provided in Supplementary Table 1. (c), Model for kinetochore with CENP-C ∆73 and CENP-T ∆90. WT: CENP-C and-T bind to the KMN-network (KNL1, Mis12, and Ndc80 complexes: KNL1C, Mis12C, and Ndc80C). CENP-C ∆73: Although deletion of CENP-C aa 1–73 prevents its Mis12C binding, the cells are viable, indicating that CENP-C binding to Mis12C is dispensable in chicken DT40 cells. CENP-T ∆90: The CENP-T N-terminus (aa 1–90), which binds to Ndc80C, is essential for viability of chicken DT40 cells, as CENP-T ∆90 does not suppress the CENP-T deficiency.

Supplementary Figure 2 Dynamics of KMN in cKO-CENP-T/ CENP-T ∆90 cells during mitotic progression and Ndc80/Hec1 binding to chicken CENP-T N-terminus.

(a), Mis12 levels on kinetochore in CENP-T conditional knockout (cKO-CENP-T) cells expressing CENP-T FL or ∆90 during mitotic progression. The cells were treated with Tet for 48 h and stained with anti-Mis12. Mis12 signals on kinetochores of each mitotic phase were quantified and normalized. Error bars show the mean and standard deviation. The sample sizes (n) of FL-LateG2/Pro, FL-Prometa, FL-Meta, FL-Ana, ∆90-LateG2/Pro, ∆90-Prometa, ∆90-Meta, ∆90-Ana and ∆90-arrested are 409, 433, 436, 401, 390, 439, 443, 438 and 434 kinetochores from 5 cells, respectively. (b), Ndc80/Hec1 levels on kinetochore in cKO-CENP-T cells expressing CENP-T FL or ∆90 during mitotic progression. The cells were stained with an anti- Ndc80/Hec1 antibody as in (a). Ndc80/Hec1 signals on kinetochores of each mitotic phase were quantified and normalized as in (a). Error bars show the mean and standard deviation. The sample sizes (n) of FL-LateG2/Pro, FL-Prometa, FL-Meta, FL-Ana, ∆90-LateG2/Pro, ∆90-Prometa, ∆90-Meta, ∆90-Ana and ∆90-arrested are 409, 437, 434,416, 384, 435, 428, 440 and 439 kinetochores from 5 cells, respectively. (c), Expression of CENP-T FL or the mutants in cKO-CENP-T cells. The cells were treated with Tet for 48 and were examined. Presented blots are representative results of two independent experiments. (d), Growth of cKO-CENP-T cells expressing CENP-T FL or the mutants. Their cell numbers were examined after Tet treatment and were normalized. (e), Ndc80/Hec1 levels on kinetochores in cKO-CENP-T cells expressing CENP-T FL or the mutants. Ndc80/Hec1 levels on kinetochores in the cell lines were quantified as in b and normalized. Error bars show the mean and standard deviation. The sample sizes (n) of FL, ∆30, ∆60, ∆90, ∆120–240 and T184A are 442, 432, 438, 438, 439 and 441 kinetochores from 5 cells, respectively. FL vs ∆30, ∆60 or ∆90: ****p < 0.0001; FL vs ∆120–240: *p = 0.0108; FL vs T184A: ***p = 0.0002 (two-tailed Welch’s t- test). All unprocessed scans of blots are provided in Supplementary Fig.8. Source data are provided in Supplementary Table 2.

Supplementary Figure 3 Schematic representation of CENP-C-CENP-T chimeric protein.

(a), Schematic representation of CENP-C-CENP-T chimeric protein. The Ndc80C-binding region of CENP- (aa 1–90) was replaced with the Mis12C-binding region of CENP-C (aa 1–73) (CC73-CT91–639). CC73-CT91–639 was expressed in CENP-T conditional knockout (cKO- CENP-T). (b), Schematic representation of CENP-T-CENP-C chimeric protein. The Mis12C-binding region of CENP-C (aa 1–73) was replaced with CENP-T aa 1–240, including the Ndc80C- and Mis12C-binding regions, fused with GFP (CT240-CC73–864). CT240-CC73–864 was expressed in cKO-CENP-T cells expressing CENP-T ∆90 mutant (cKO-CENP-T/CENP-T ∆90), as the CENP-T C-terminus is essential. In cKO-CENP-T/CENP-T ∆90 cells, CENP-T ∆90, but not wild-type CENP-T, was expressed after Tet addition (+Tet).

Supplementary Figure 4 Cdk1 phosphorylation facilitates CENP-T-Spc24/25 interaction.

(a), Pairwise sequence alignment of the Spc24/25 binding regions of chicken CENP-T (GgCENP-T: aa 63–90) and human Dsn1 (HsDsn1: aa 323–350). The purple arrows indicate the phosphorylation sites in each sequence. The conserved residues are highlighted in magenta. (b), The crystal structure of the chicken Spc24/25 globular domain (GgSpc24 and GgSpc25) in complex with GgCENP-T (PDB entry 3VZA). The phospho-mimetic T72D in GgCENP-T forms hydrogen bonds with the side chain of R74. (c), The homology model of the human Spc24/25 globular domain (HsSpc24 aa 136–197 and HsSpc25 aa 127–224) in complex with HsDsn1. The HsDSN1 residues sheared with GgCENP-T are colored in magenta. (d), In vitro pulldown assay with Spc24/25. Recombinant human Mis12 complex (HsMis12C) associated with human Spc24/25 globular domain [HsSpc24/25: StrepII-tagged HsSpc24 (aa 57–197) and His-tagged HsSpc25(aa 70–224)] were purified (HsMis12/HsSpc24/25) and incubated with various amounts of MBP-fused human CENP-T (HsCENP-T) aa 1–375 fragment in which Ser201 was replaced with Ala with ATP in the presence or absence of active CyclinB-Cdk1 (CycB-Cdk1) (Input). HsSpc24/25 was pulled-down using Strep-Tactin beads (HsSpc24/25 pull-down). The protein complexes were separated by SDS-PAGE and stained with Coomassie brilliant blue. Asterisk shows a protein from Strep-Tactin beads. Presented results are representative results of two independent experiments. (e), Cdk1 phosphorylation increases the binding affinity of CENP-T, but decrease the binding affinity of Mis12C to Ndc80C. All unprocessed scans of gels are provided in Supplementary Fig.8.

Supplementary Figure 5 Increased binding of the Ndc80 complex to CENP-C does not suppress deficiency of CENP-T pathway.

(a), Schematic representation of chicken Dsn1 and its mutants. Dsn1 aa 326–349 that binds to the Spc24/25 complex (Spc24/25) was replaced with Spc24/25-binding region of CENP-T (aa 65–88) (Dsn1[326–349;CENP-T 65–88]) or was deleted (Dsn1∆326–349). The black box shows a conserved basic motif (aa 93–114). (b), Expression of GFP-fused Dsn1 (GFP-Dsn1) WT or the mutants in cKO-Dsn1 cells. GFP-Dsn1 WT or mutants were expressed in Dsn1-conditional knockout (cKO-Dsn1) cells. The cells were treated with for 48 h were examined. cKO-Dsn1 cells (–) were examined as controls. Presented blots are representative results of two independent experiments. (c), Growth of cKO-Dsn1 cells expressing GFP-Dsn1 WT or the mutants. Their cell numbers were examined after Tet (+Tet) and were normalized to those at 0 h for each line. Untreated cells were also examined (–Tet). (d), Expression of GFP-Dsn1 or Dsn1[326–349;CENP- T 65–88] in CENP-T conditional knock-out (cKO-CENP-T) chicken DT40 cells expressing CENP-T ∆90 mutant (cKO-CENP-T/CENP-T ∆90). cKO-CENP-T/CENP-T ∆90 cells expressing GFP-Dsn1 WT or [326–349;CENP-T 65–88] were treated with Tet for 48 h and were examined. cKO-CENP-T/CENP-T ∆90 cells (–) were examined as controls. Presented blots are representative results of two independent experiments. (e), Ndc80/Hec1 localization in cKO-CENP-T/CENP-T ∆90 cells expressing GFP-Dsn1 WT or [326–349;CENP-T 65–88]. The cells were treated with Tet for 48 h and stained with an anti-Ndc80/Hec1 antibody. DNA was stained with DAPI. Ndc80/Hec1 signals on kinetochores were quantified and normalized to the average of Ndc80/Hec1 signals in cKO-CENP-T/CENP-T ∆90 cells expressing GFP- Dsn1 WT. Error bars show the mean and standard deviation (WT: n = 960 kinetochores from 10 cells, [326–349;CENP-T 65–88]: n = 964 kinetochores from 10 cells). ****p < 0.0001 (two-tailed Welch’s t-test). (f), Growth of cKO-CENP-T/CENP-T ∆90 cells expressing GFP-Dsn1 WT or [326–349;CENP-T 65–88]. Their cell numbers were examined as in (c). Scale bar, 10 µm. All unprocessed scans of blots are provided in Supplementary Fig.8. Source data are provided in Supplementary Table 2.

Supplementary Figure 6 Deletion of the basic domain in Dsn1 increases binding affinity of Mis12 complex to CENP-C.

(a), Schematic representation of chicken Dsn1 (GgDsn1) and the basic domain in human Dsn1 (HsDsn1) and GgDsn1. Aurora B (AurB) phosphorylation sites in HsDsn1 (Ser100 and Ser109) are marked. (b), Model for regulation of the Mis12 complex (Mis12C) -binding to CENP-C through AurB phosphoryation of the basic domain. Deletion of the basic domain stabilizes the Mis12C-binding to CENP-C1. (c), Growth of Dsn1 cKO-Dsn1 expressing GFP-fused Dsn1 (GFP-Dsn1) WT or the basic domain deletion mutant (∆93–114). Their cell numbers were examined after Tet treatment and were normalized. (d), Dsn1 localization in interphase cells. cKO-Dsn1 cells expressing GFP-Dsn1 WT or ∆93–114 were treated with Tet for 48 h and stained DNA with DAPI and CENP-T with an anti-CENP-T antibody. Interphase cells, which have kinetochore localization of GFP-Dsn1, were quantified (right graph). Alanine substitution of potential AurB sites in GgDsn1 (Ser97, Ser101, Ser102, and Ser111) prevents GFP-Dsn1 localization to the interphase centromeres. (e), Immunoprecipitation with Dsn1 or CENP-C. cKO-Dsn1 cells expressing GFP-Dsn1 WT or ∆93–114 (∆) were cultured with Tet. The proteins were extracted from the chromatin fraction (Input) and immunoprecipitated with an anti-GFP antibody (GFP) or anti-CENP-C antiserum (CC) (IP). Control IgG (Cont) and preimmune serum (Pre) were used as controls. Unbound fractions were also examined (Flow-through: FT). Presented blots are representative results of two independent experiments. (f), Expression of CENP-T and Dsn1 proteins in cKO-CENP-T cells expressing GFP-Dsn1 WT or ∆93–114. The cells were treated with for 48 h and were examined. Presented blots are representative results of two independent experiments. (g), Growth of cKO-CENP-T cells expressing GFP-Dsn1 WT or ∆93–114. Their cell numbers were examined as in (c. h), Model for kinetochore in cKO-CENP-T/CENP-T ∆90 cells expressing the Dsn1 mutants. Deletion of the basic domain of Dsn1 suppresses CENP-T ∆90 via stable binding of Mis12C to CENP-C (∆93–114, see Fig. 6). However, increased Ndc80C affinity to Mis12C does not CENP-T ∆90 ([326–349;CENP-T 65–88], see Supplementary Fig.5). Scale bar, 10 µm. All unprocessed scans of blots are provided in Supplementary Fig.8.

Supplementary Figure 7 Design for CENP-T and -C Tension sensors.

(a), Schematic representation of a tension sensor system using chicken talin Rod (TR: aa 482–889) and chicken vinculin head domain (VH: aa 1–258). TR has vinculin-binding domains shown in purple that bind to VH. When tension is applied to TR, vinculin-binding domain in TR is exposed, resulting in VH-binding. Using GFP-fused VH, the force applied to TR was visualized as the accumulation of GFP on TR. (b), Design of CENP-T tension sensor. CENP-T has disordered region predicted with algorithms [IUPred, RONN and DISOPRED2–4]. The predicted disordered region (aa 241–529) is exchanged with TR and fused with TagRFP (241–529:TR). (c), Design of CENP-C tension sensor. Disordered regions in CENP-C as predicted with algorithms shown in b (aa 324–557 or 421–557) are exchanged with TR and fused with TagRFP (325–556:TR or 433–556:TR). (d), Expression of tension sensor proteins. Proteins were extracted from detergent- insoluble fractions and examined. Parental cells were examined as a control. Endogenous CENP-C or CENP-T proteins were indicated as CENP- C endo. or CENP-T endo., respectively. Presented blots are representative results of two independent experiments. (e), Design of CENP-C and -T chimeric tension sensor. CENP-C aa 1–73 in CENP-C [433–556:TR] was replaced with CENP-T aa 1–240 (CENP-C[1–73;1–240CT/422–556;TR]). (f), VH-GFP localization to kinetochore in cells expressing CENP-C[1–73;1–240CT/422–556;TR]. CENP-C[1–73;1–240CT/422–556;TR] was expressed in DT40 cells expressing VH-GFP. The cells were treated with or without nocodazole (Noc + or–), fixed, and stained with an anti-CENP-T antibody. DNA was stained with DAPI. GFP signals on kinetochores were quantified and normalized to the average GFP signals in the Noc-untreated cells. Error bars show the mean and standard deviation (Noc –: n = 876 kinetochores from 10 cells, Noc +: n = 590 kinetochores from 10 cells). ****p < 0.0001 (two-tailed Welch’s t-test). All unprocessed scans of blots are provided in Supplementary Fig.8. Source data are provided in Supplementary Table 2.

Supplementary Figure 8 Unprocessed blots, autoradiographs and gel images.

The boxes indicate pictures shown in Fig. 1b,d. The boxes indicate pictures shown in Fig. 2b and 4b. The boxes indicate pictures shown in Fig. 4e,h,j. The boxes indicate pictures shown in Fig. 5a,c. The boxes indicate pictures shown in Fig. 5f and 6a. The boxes indicate pictures shown in Supplementary. Fig 2c and 4d. The boxes indicate pictures shown in Supplementary. Fig 5b,d. The boxes indicate pictures shown in Supplementary. Fig 6e,f and 7d.

Supplementary information

Supplementary information

Supplementary Figures 1–8, Supplementary Table legends and Supplementary References.

Reporting Summary

Supplementary Table 1

Flow cytometry for cell cycle distribution analysis.

Supplementary Table 2

Statistics source data.

Supplementary Table 3

Lists of antibodies used in this study.

Supplementary Table 4

Lists of antibodies used in this study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hara, M., Ariyoshi, M., Okumura, Ei. et al. Multiple phosphorylations control recruitment of the KMN network onto kinetochores. Nat Cell Biol 20, 1378–1388 (2018). https://doi.org/10.1038/s41556-018-0230-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-018-0230-0

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing