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Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Abstract

Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore–microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.

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Figure 1: Cells depleted of Cdt1 after S phase do not complete cell division.
Figure 2: G2-specific Cdt1 inhibition induces mitotic arrest.
Figure 3: Cdt1 transiently localizes to kinetochores during prometaphase and metaphase.
Figure 4: Hec1 is required for Cdt1 kinetochore localization.
Figure 5: Cdt1 targeting to kinetochores depends on the flexible loop region of Hec1.
Figure 6: Cdt1 and the Hec1 loop domain are required to satisfy the spindle-assembly checkpoint.
Figure 7: Cdt1 and the Hec1 loop domain are required for stable kMT attachments.
Figure 8: Cdt1 and the Hec1 loop domain are required for proper Ndc80 conformation in vivo.

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Acknowledgements

We thank A. Desai for providing Knl1, Nsl1, Dsn1 and Spindly antibodies, A. Musacchio (Max Planck Institute of Molecular Physiology, Dortmund, Germany) for anti-Mad1, Zwint1 and ZW10 antibodies, B. Stillman (Cold Spring Harbor Laboratory, New York, USA) for anti-Orc6 antibody, S. Taylor (University of Manchester, UK) for anti-Bub1 and BubR1 antibodies, T. Stukenberg (University of Virginia at Charlottesville, Virginia, USA) for anti-Spc24 antibody, T. Yen (Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA) for anti-CENP-E antibody and I. Cheeseman (Whitehead Institute of Biomedical Research and MIT, Cambridge, Massachusetts, USA) for anti-Ska3 antibody. We are grateful to A. Desai (University of California San Diego, La Jolla, CA, USA), D. Cheerambathur, T. Stukenberg, K. Slep and T. Maresca for helpful discussions and to J. Mick for generating Hec1 constructs. We would also like to thank other members of the Salmon, Cook, A. Desai and J. Nevins laboratories for their support during this project. J.G.C. was supported by NIH K01 CA094907 and NIH GM083024, E.D.S. was supported by NIH GM24364 and J.G.D. was supported by NIH GM088371 and a grant from the Pew Scholars Program in the Biomedical Sciences.

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D.V. and J.G.C. designed and carried out experiments, analysed data and wrote the manuscript. S.C., L.J.R.S. and K.T.R. carried out experiments and analysed data. D.A.D.C. and J.G.C. conducted the two-hybrid screen. K.R.N. and S.C. characterized the arrest of Cdc6- and Cdt1-depleted normal fibroblasts. X.W. and D.V. conducted the Delta analyses. J.G.D., E.D.S. and J.G.C. designed experiments, analysed data and wrote the manuscript. All authors proofread the manuscript.

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Correspondence to E. D. Salmon or Jeanette Gowen Cook.

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Varma, D., Chandrasekaran, S., Sundin, L. et al. Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment. Nat Cell Biol 14, 593–603 (2012). https://doi.org/10.1038/ncb2489

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