Kinetochores are specialized multi-protein complexes that play a crucial role in maintaining genome stability1. They bridge attachments between chromosomes and microtubules during mitosis and they activate the spindle assembly checkpoint (SAC) to arrest division until all chromosomes are attached2. Kinetochores are able to efficiently integrate these two processes because they can rapidly respond to changes in microtubule occupancy by switching localized SAC signalling ON or OFF2,3,4. We show that this responsiveness arises because the SAC primes kinetochore phosphatases to induce negative feedback and silence its own signal. Active SAC signalling recruits PP2A-B56 to kinetochores where it antagonizes Aurora B to promote PP1 recruitment. PP1 in turn silences the SAC and delocalizes PP2A-B56. Preventing or bypassing key regulatory steps demonstrates that this spatiotemporal control of phosphatase feedback underlies rapid signal switching at the kinetochore by: allowing the SAC to quickly transition to the ON state in the absence of antagonizing phosphatase activity; and ensuring phosphatases are then primed to rapidly switch the SAC signal OFF when kinetochore kinase activities are diminished by force-producing microtubule attachments.
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Santaguida, S. & Musacchio, A. The life and miracles of kinetochores. EMBO J. 28, 2511–2531 (2009).
Foley, E. A. & Kapoor, T. M. Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat. Rev. Mol. Cell Biol. 14, 25–37 (2013).
Kops, G. J. & Shah, J. V. Connecting up and clearing out: how kinetochore attachment silences the spindle assembly checkpoint. Chromosoma 121, 509–525 (2012).
Funabiki, H. & Wynne, D. J. Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122, 135–158 (2013).
Bomont, P., Maddox, P., Shah, J. V., Desai, A. B. & Cleveland, D. W. Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F. EMBO J. 24, 3927–3939 (2005).
Dick, A. E. & Gerlich, D. W. Kinetic framework of spindle assembly checkpoint signalling. Nat. Cell Biol. 15, 1370–1377 (2013).
Santaguida, S., Tighe, A., D’Alise, A. M., Taylor, S. S. & Musacchio, A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 (2010).
Espeut, J., Cheerambathur, D. K., Krenning, L., Oegema, K. & Desai, A. Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore. J. Cell Biol. 196, 469–482 (2012).
Vanoosthuyse, V. & Hardwick, K. G. A novel protein phosphatase 1-dependent spindle checkpoint silencing mechanism. Curr. Biol. 19, 1176–1181 (2009).
Meadows, J. C. et al. Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors. Dev. Cell 20, 739–750 (2011).
Rosenberg, J. S., Cross, F. R. & Funabiki, H. KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint. Curr. Biol. 21, 942–947 (2011).
Pinsky, B. A., Nelson, C. R. & Biggins, S. Protein phosphatase 1 regulates exit from the spindle checkpoint in budding yeast. Curr. Biol. 19, 1182–1187 (2009).
Gutierrez-Caballero, C., Cebollero, L. R. & Pendas, A. M. Shugoshins: from protectors of cohesion to versatile adaptors at the centromere. Trends Genet. 28, 351–360 (2012).
Foley, E. A., Maldonado, M. & Kapoor, T. M. Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase. Nat. Cell Biol. 13, 1265–1271 (2011).
Xu, P., Virshup, D. M. & Lee, S. H. B56-PP2A regulates motor dynamics for mitotic chromosome alignment. J. Cell Sci. (2014)10.1242/jcs.154609
Akopyan, K. et al. Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Mol. Cell 53, 843–853 (2014).
Suijkerbuijk, S. J., Vleugel, M., Teixeira, A. & Kops, G. J. Integration of kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments. Dev. Cell 23, 745–755 (2012).
Kruse, T. et al. Direct binding between BubR1 and B56-PP2A phosphatase complexes regulate mitotic progression. J. Cell Sci. 126, 1086–1092 (2013).
Xu, P., Raetz, E. A., Kitagawa, M., Virshup, D. M. & Lee, S. H. BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression. Biol. Open 2, 479–486 (2013).
Hewitt, L. et al. Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 core complex. J. Cell Biol. 190, 25–34 (2010).
Liu, D. et al. Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase. J. Cell Biol. 188, 809–820 (2010).
Vleugel, M. et al. Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation. J. Cell Biol. 203, 943–955 (2013).
Primorac, I. et al. Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling. eLife 2, e01030 (2013).
Zhang, G., Lischetti, T. & Nilsson, J. A minimal number of MELT repeats supports all the functions of KNL1 in chromosome segregation. J. Cell Sci. 127, 871–884 (2014).
Yamagishi, Y., Yang, C. H., Tanno, Y. & Watanabe, Y. MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat. Cell Biol. 14, 746–752 (2012).
London, N., Ceto, S., Ranish, J. A. & Biggins, S. Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr. Biol. 22, 900–906 (2012).
Shepperd, L. A. et al. Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint. Curr. Biol. 22, 891–899 (2012).
Krenn, V., Overlack, K., Primorac, I., van Gerwen, S. & Musacchio, A. KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats. Curr. Biol. 24, 29–39 (2014).
Posch, M. et al. Sds22 regulates aurora B activity and microtubule-kinetochore interactions at mitosis. J. Cell Biol. 191, 61–74 (2010).
Espert, A. et al. PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing. J. Cell Biol. 206, 833–842 (2014).
Bollen, M., Peti, W., Ragusa, M. J. & Beullens, M. The extended PP1 toolkit: designed to create specificity. Trends Biochem. Sci. 35, 450–458 (2010).
Barr, F. A., Elliott, P. R. & Gruneberg, U. Protein phosphatases and the regulation of mitosis. J. Cell Sci. 124, 2323–2334 (2011).
Liu, D., Vader, G., Vromans, M. J., Lampson, M. A. & Lens, S. M. Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates. Science 323, 1350–1353 (2009).
Nijenhuis, W. et al. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B. J. Cell Biol. 201, 217–231 (2013).
Santaguida, S., Vernieri, C., Villa, F., Ciliberto, A. & Musacchio, A. Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction. EMBO J. 30, 1508–1519 (2011).
Pines, J. Cubism and the cell cycle: the many faces of the APC/C. Nat. Rev. Mol. Cell Biol. 12, 427–438 (2011).
Morin, V. et al. CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint. Curr. Biol. 22, 289–295 (2012).
D’Angiolella, V., Mari, C., Nocera, D., Rametti, L. & Grieco, D. The spindle checkpoint requires cyclin-dependent kinase activity. Genes Dev. 17, 2520–2525 (2003).
Vazquez-Novelle, M. D. et al. Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase. Curr. Biol. 24, 638–645 (2014).
Welburn, J. P. et al. Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface. Mol. Cell 38, 383–392 (2010).
Gascoigne, K. E. et al. Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes. Cell 145, 410–422 (2011).
Saurin, A. T., van der Waal, M. S., Medema, R. H., Lens, S. M. & Kops, G. J. Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. Nat. Commun. 2, 316 (2011).
We thank M. Vleugel and M. Omerzu for help with the pMELT-KNL1 antibody, and I. Cheeseman (Whitehead Institute, USA), A. Musacchio (MPI-Dortmund, Germany), A. de Antoni (IFOM, Italy), P. Parker (CRUK London Research Institute, UK) and S. Taylor (University of Manchester, UK) for reagents. We thank the Kops, Lens, Saurin, Swedlow and Griffis laboratories for discussions. This work is supported by the European Research Council (ERC-StG KINSIGN to G.J.P.L.K.), by the Netherlands Organisation for Scientific Research (NWO-Vici 865.12.004 to G.J.P.L.K.), by the KWF Kankerbestrijding (UU-2012-5427 to G.J.P.L.K.), by TiPharma (T3-503 to G.J.P.L.K.) and by financial support to A.T.S. (from the Ninewells Cancer Campaign, Leng Charitable Trust and Tenovus Scotland Tayside).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Representative images of HA-B56 isoform localisation in nocodazole-arrested mitotic cells. (b) Immunoblot of whole cell lysates from nocodazole-arrested LAP-BUBR1WT or LAP-BUBR1ΔKARD cells two days after doxycycline addition (Methods), which is representative of 2 independent experiments. (c) Representative images and (d) quantifications of relative kinetochore intensities of indicated antigens from nocodazole-arrested LAP-BUBR1WT or LAP-BUBR1ΔKARD-expressing cells. (e) Line plots displaying mean intensities (±SEM) of indicated antigens from 30 kinetochore pairs from 6 cells, which is representative of 2 independent experiments. (f) Time-lapse analysis of duration of mitotic arrest in nocodazole-treated LAP-BUBR1WT or LAP-BUBR1ΔKARD-expressing cells that entered mitosis in the presence of the MPS1 inhibitor AZ-3146 (2.5 μM). (g–j) Time-lapse analysis as in (f), except cells, additionally transfected with mock or PP2A-B56 siRNA, entered mitosis in the presence of the indicated concentrations of either reversine or AZ-3146. (k) Representative images and quantification of relative kinetochore intensity of SGO1 in nocodazole-arrested Flp-in HeLa treated with mock or SGO1 siRNA. Images show centromeric B56α depletion and premature loss of sister chromatid cohesion following SGO1 depletion. (l,m) Time-lapse analysis of duration of mitotic arrest in Flp-in HeLa cells transfected with mock or SGO1 siRNA in the absence (g) or presence (h) of nocodazole and reversine (500 nM). Kinetochore intensities are relative to LAP-BUBR1WT (d) or mock treated Flp-in HeLa (k) cells, and at least 10 cells were quantified for each condition per experiment. Quantifications show the mean data (±SD) from 3 (d) or 5 (k) independent experiments (Supplementary Table 2). Asterisks indicate significance (Student t-test, unpaired). ∗∗∗∗:p < 0.0001. Insets display magnifications of the boxed regions. Bars, 5 μm.
(a) Representative images and (b–d) quantification of relative kinetochore intensities of indicated antigens in nocodazole-arrested Flp-in HeLa cells transfected with mock or PP2A-B56 siRNA (b), or with KNL1 siRNA and induced to express LAP-KNL1WT or LAP-KNL12SA (c,d). (e) KNL1, GFP, Transferrin Receptor Protein 1 (TFR1) immunoblot of whole-cell lysates from nocodazole-arrested Flp-in HeLa cells transfected with mock or KNL1 siRNA and induced to express the indicated LAP-KNL1 mutants. Boxes on left show molecular mass standard. Mw, molecular weight. Westerns are representative of 2 independent experiments. (f) Quantification of relative kinetochore intensities of LAP-KNL1 in nocodazole-arrested Flp-in HeLa cells expressing KNL1WT or indicated KNL1 mutants. Insets display magnifications of the boxed regions. Bar graphs display mean fold-change in kinetochore intensities (±SD) relative to mock transfected (b) or LAP-KNL1WT cells (c,d,f), from 3 (c,d), 4 (b), or 7 (f) independent experiments with at least 10 cells quantified for each condition per experiment (Supplementary Table 2). Asterisks indicate significance (Student t-test, unpaired). NS: not significant, ∗:p < 0.05,∗∗∗∗:p < 0.0001. DNA (DAPI) is shown in blue. Bars, 5 μm.
Supplementary Figure 3 Aurora B activity and KNL1-PP1 interaction controls SAC silencing downstream of PP2A-B56.
(a–f) Time-lapse analysis of duration of mitotic arrest in nocodazole-treated Flp-in HeLa cells transfected with mock siRNA (a,c), PP2A-B56 siRNA (b,d), or expressing LAP-BUBR1WT (e) or LAP-BUBR1ΔKARD (f). Cells entered mitosis in the presence of DMSO, reversine (125–500 nM), AZ-3146 (2.5–0.63 μM), and ZM-447439 (2 μM), as indicated. (g) MAD2, CDC20, Tubulin and BUBR1 immunoblots (IB) of whole-cell lysates and CDC20 immunoblots of immunopurified (IP) MAD2 from mitotic Flp-in HeLa cells transfected with KNL1 siRNA and expressing the indicated LAP-KNL1 mutants. Nocodazole-arrested cells were treated with MG132 and DMSO or reversine (500 nM). Band intensity of CDC20 in MAD2 IPs is indicated. Boxes on left show molecular mass standard. Mw, molecular weight. Westerns are representative of 3 independent experiments. (h) Time-lapse analysis of duration of mitotic arrest in nocodazole-treated Flp-in HeLa cells transfected with KNL1 siRNA and expressing the indicated LAP-KNL1 constructs. Cells entered mitosis in the presence of 500 nM of reversine. Cells were additionally transfected with mock, MAD2 or BUBR1 siRNAs. Graphs show cumulative data from 50 cells per treatment from one experiment, which is representative of 2 independent experiments.
Supplementary Figure 4 Kinetochore PP1 antagonises SAC protein accumulation at kinetochores without affecting kinetochore kinase activity.
(a) Quantification of relative kinetochore intensities of indicated antigens from nocodazole-treated Flp-in HeLa cells expressing BUBR1WT or BUBR1ΔKARD. (b) Quantification of chromosomal alignment in Flp-in HeLa cells expressing LAP-KNL1WT or LAP-KNL12A and transfected with mock or PP2A-B56 siRNA. At least 100 cells were quantified for each condition per experiment and data is the mean of 3 independent experiments (Supplementary Table 2). (c) Quantification of relative kinetochore intensities of indicated antigens from nocodazole-treated Flp-in HeLa cells expressing LAP-KNL1WT or LAP-KNL12SA and treated with mock or B56 siRNA, as indicated. (d) GST and p-MELT immunoblots of purified GST-KNL1-M3 or GST-KNL1-A3 incubated in a kinase reaction −/+ MPS1 for 60 mins to phosphorylate the MELT motifs. KNL1-M3 contains a KNL1818−1051 fragment that incorporates 3 MELT motifs and the A3 version is the same fragment but with critical residues in each MELT motif mutated to alanine22. Westerns are representative of 2 independent experiments. (e–j) Representative images (e,g,i) and quantification (f,h,j) of relative kinetochore intensities of indicated antigens from nocodazole-treated Flp-in HeLa cells expressing various LAP-KNL1 mutants and treated with nocodazole, DMSO, reversine (500 nM) or MG132, as indicated. (k,l) Representative images of indicated antigens on metaphase aligned kinetochores in Flp-in HeLa cells expressing various LAP-KNL1 mutants. (m) Quantification of relative kinetochore intensities of indicated antigens from nocodazole-treated Flp-in HeLa cells expressing various KNL1 mutants. The p-Aurora B (Thr232) and p-MPS1 (Thr676) antibodies are directed against the activation loop phosphorylation sites. Only early prometaphase cells were characterised for p-MPS1 (Thr676) antibody, for reasons stated previously40. The p-CENP-T (Ser47) site is a characterised kinetochore CDK1 substrate41. All kinetochore intensities are relative to the untreated wild type controls in each experiment. At least 10 cells were quantified for each condition per experiment and quantifications show the mean data (±SD) from 3 (a,f,j,m), 4 (c) or 5 (h) independent experiments (see Supplementary Table 2). DNA (DAPI) is shown in blue. Insets are magnifications of the boxed regions. Asterisks indicate significance (Student t-test, unpaired). NS: not significant, ∗:p < 0.05,∗∗:p < 0.01,∗∗∗:p < 0.001,∗∗∗∗:p < 0.0001. Bars, 5 μm.
Supplementary Figure 5 Negative feedback and SAC silencing is unaffected by high nocodazole or SGO1 depletion.
(a,b) Quantifications of relative kinetochore intensities of indicated antigens from Flp-in HeLa cells (a), LAP-BUBR1WT or LAP-BUBR1ΔKARD-expressing cells (b) treated with a high dose of nocodazole (3.3 μM)35. Different mitotic phases where determined by nuclear morphology; Prophase (Pro), early mitosis (EM; defined as dispersed lightly condensed chromatin) and late mitosis (LM; highly condensed chromatin balls typical of nocodazole-arrested cells). Insets display magnifications of the boxed regions. All kinetochore intensities are relative to the maximum signal in each experiment, except in b, which are relative to the prophase signal in LAP-BUBR1WT cells. The bar graphs show the mean data (±SD) from at least 10 cells for each condition from 1 experiment, which is representative of 2 experiments. (c) Quantification of relative LAP-BUBR1 kinetochore intensities from time-lapse images of LAP-BUBR1WT cells transfected with mock or SGO1 siRNA arrested in prometaphase with nocodazole and MG132, prior to treatment with reversine (1 μM) and ZM-447439 (2 μM) for the indicated times. At least 15 cells were quantified for each cell line per experiment and quantifications show the mean data (±SD), relative to the 0 min timepoint, from 3 independent experiments. Bars, 5 μm.
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Nijenhuis, W., Vallardi, G., Teixeira, A. et al. Negative feedback at kinetochores underlies a responsive spindle checkpoint signal. Nat Cell Biol 16, 1257–1264 (2014). https://doi.org/10.1038/ncb3065
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