Abstract
The spindle assembly checkpoint (SAC) is essential in mammalian mitosis to ensure the equal segregation of sister chromatids1,2. The SAC generates a mitotic checkpoint complex (MCC) to prevent the anaphase-promoting complex/cyclosome (APC/C) from targeting key mitotic regulators for destruction until all of the chromosomes have attached to the mitotic apparatus1,3,4. A single unattached kinetochore can delay anaphase for several hours5, but how it is able to block the APC/C throughout the cell is not understood. Present concepts of the SAC posit that either it exhibits an all-or-nothing response6 or there is a minimum threshold sufficient to block the APC/C (ref. 7). Here, we have used gene targeting to measure SAC activity, and find that it does not have an all-or-nothing response. Instead, the strength of the SAC depends on the amount of MAD2 recruited to kinetochores and on the amount of MCC formed. Furthermore, we show that different drugs activate the SAC to different extents, which may be relevant to their efficacy in chemotherapy.
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References
Musacchio, A. & Salmon, E. D. The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393 (2007).
Lara-Gonzalez, P., Westhorpe, F. G. & Taylor, S. S. The spindle assembly checkpoint. Curr. Biol. 22, R966–R980 (2012).
Pines, J. Cubism and the cell cycle: the many faces of the APC/C. Nat. Rev. Mol. Cell Biol. 12, 427–438 (2011).
Sudakin, V., Chan, G. K. & Yen, T. J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J. Cell Biol. 154, 925–936 (2001).
Rieder, C. L., Cole, R. W., Khodjakov, A. & Sluder, G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130, 941–948 (1995).
De Antoni, A. et al. The Mad1/MAD2 complex as a template for MAD2 activation in the spindle assembly checkpoint. Curr. Biol. 15, 214–225 (2005).
Weaver, B. A. et al. Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss. J. Cell Biol. 162, 551–563 (2003).
Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517 (1991).
Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519–531 (1991).
Kulukian, A., Han, J. S. & Cleveland, D. W. Unattached kinetochores catalyze production of an anaphase inhibitor that requires a MAD2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16, 105–117 (2009).
Tang, Z., Bharadwaj, R., Li, B. & Yu, H. MAD2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1, 227–237 (2001).
Nilsson, J., Yekezare, M., Minshull, J. & Pines, J. The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat. Cell Biol. 10, 1411–1420 (2008).
Westhorpe, F. G., Tighe, A., Lara-Gonzalez, P. & Taylor, S. S. p31comet-mediated extraction of MAD2 from the MCC promotes efficient mitotic exit. J. Cell Sci. 124, 3905–3916 (2011).
Chao, W. C., Kulkarni, K., Zhang, Z., Kong, E. H. & Barford, D. Structure of the mitotic checkpoint complex. Nature 484, 208–213 (2012).
Luo, X., Tang, Z., Rizo, J. & Yu, H. The MAD2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol. Cell 9, 59–71 (2002).
Kops, G. J., Weaver, B. A. & Cleveland, D. W. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785 (2005).
Di Fiore, B. & Pines, J. How cyclin A destruction escapes the spindle assembly checkpoint. J. Cell Biol. 190, 501–509 (2010).
den Elzen, N. & Pines, J. Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase. J. Cell Biol. 153, 121–136 (2001).
Geley, S. et al. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153, 137–148 (2001).
Wolthuis, R. et al. Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A. Mol. Cell 30, 290–302 (2008).
Gascoigne, K. E. & Taylor, S. S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 14, 111–122 (2008).
Orth, J. D. et al. Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics. Cancer Res. 71, 4608–4616 (2011).
Brito, D. A. & Rieder, C. L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr. Biol. 16, 1194–1200 (2006).
Huang, H. C., Mitchison, T. J. & Shi, J. Stochastic competition between mechanistically independent slippage and death pathways determines cell fate during mitotic arrest. PLoS One 5, e15724 (2010).
Hagting, A., Karlsson, C., Clute, P., Jackman, M. & Pines, J. MPF localization is controlled by nuclear export. EMBO J. 17, 4127–4138 (1998).
Yang, Z., Kenny, A. E., Brito, D. A. & Rieder, C. L. Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments. J. Cell Biol. 186, 675–684 (2009).
Kapoor, T. M., Mayer, T. U., Coughlin, M. L. & Mitchison, T. J. Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J. Cell Biol. 150, 975–988 (2000).
Waters, J. C., Chen, R. H., Murray, A. W. & Salmon, E. D. Localization of MAD2 to kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141, 1181–1191 (1998).
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).
Buffin, E., Lefebvre, C., Huang, J., Gagou, M. E. & Karess, R. E. Recruitment of MAD2 to the kinetochore requires the Rod/Zw10 complex. Curr. Biol. 15, 856–861 (2005).
Mansfeld, J., Collin, P., Collins, M. O., Choudhary, J. S. & Pines, J. APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat. Cell Biol. 13, 1234–1243 (2011).
Kim, S. H., Lin, D. P., Matsumoto, S., Kitazono, A. & Matsumoto, T. Fission yeast Slp1: an effector of the MAD2-dependent spindle checkpoint. Science 279, 1045–1047 (1998).
Izawa, D. & Pines, J. MAD2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. J. Cell Biol. 199, 27–37 (2012).
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).
Jelluma, N. et al. Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment. Cell 132, 233–246 (2008).
Maciejowski, J. et al. Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling. J. Cell Biol. 190, 89–100 (2010).
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).
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).
Hagan, R. S. et al. p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment. Mol. Biol. Cell 22, 4236–4246 (2011).
Varetti, G., Guida, C., Santaguida, S., Chiroli, E. & Musacchio, A. Homeostatic control of mitotic arrest. Mol. Cell 44, 710–720 (2011).
Michel, L. S. et al. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature 409, 355–359 (2001).
Berdougo, E., Terret, M. E. & Jallepalli, P. V. Functional dissection of mitotic regulators through gene targeting in human somatic cells. Methods Mol. Biol. 545, 21–37 (2009).
Clute, P. & Pines, J. Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1, 82–87 (1999).
Wu, J. Q. & Pollard, T. D. Counting cytokinesis proteins globally and locally in fission yeast. Science 310, 310–314 (2005).
Johnston, K. et al. Vertebrate kinetochore protein architecture: protein copy number. J. Cell Biol. 189, 937–943 (2010).
Acknowledgements
We are grateful to P. Jallepalli for reagents and advice, F. Cubizolles and E. Nigg for generating the pAAV–Venus–MAD2 plasmid, and to A. Khodjakov and all of our laboratory members for critical discussions. We would also like to thank D. Gerlich and A. Dick for sharing results before publication. This work was supported by a Programme grant from Cancer Research UK and a project grant from the BBSRC to J.P. J.P. acknowledges core funding provided by the Wellcome Trust (092096) and CRUK (C6946/A14492).
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P.C. and J.P. designed the experiments, P.C. and O.N. generated the knock-in cell lines and R.W. isolated the fluorescent cells by FACS. PC carried out the experiments, analysed the data and interpreted the results with J.P. P.C. and J.P. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Identification and characterization of Cyclin A2-Venus RPE1 clones.
(a) Genomic PCR screen for correctly targeted Cyclin A2-Venus integrants following flow cytometry selection. Primer sets consisted of an oligonucleotide annealing outside the targeting construct (either 5′ (#1) or 3′ (#4)) and oligonucleotides annealing to the ORF of Venus (2 or 3). (b) PCR analysis with different primer combinations of a positive clone compared with parental RPE1 cells. (c) Single-cell Cyclin A2-Venus destruction assays of unsynchronised RPE1 Cyclin A2-Venus cells (n = 8 cells). Error bars indicate s.d. and are representative of two independent experiments. (d) Single-cell Cyclin A2 destruction assays of asynchronously growing RPE1 Cyclin A2-Venus cells treated with DMA (10 μM) and MPS1 inhibitor reversine (0.5 μM) or Mad2 siRNA (20 μM) for 48 hours (n = 6 cells for each condition). Images in c) and d) were acquired at 3 minutes intervals and the total cell fluorescence was quantified. Fluorescence intensities were normalized to the level at nuclear envelope breakdown (NEBD) and aligned at metaphase and NEBD respectively. Error bars indicate s.d. and are representative of two independent experiments. (e) Scatter plot of the time between nuclear envelope breakdown (NEBD) and exit from mitosis as judged by the disappearance of the Cyclin B1-Venus signal in Cyclin B1-Venus cells were treated simultaneously with DMA (10μM) and the indicated concentration of AZ3146. The black line in each condition represents the mean. n = 50 cells per condition. Representative of three independent experiments. Scale bar = 10 μm.
Supplementary Figure 2 Identification and characterization of Cyclin B1-Venus RPE1 clones.
(a) Genomic PCR screen for correctly targeted Cyclin B1-Venus-neoR integrants following antibiotic selection. Primer sets consisted of an oligonucleotide annealing in the neomycin resistance cassette (1) and another outside the targeting construct (either 5′ (#5) or 3′ (#4)), and oligonucleotides annealing to the ORF of Venus (2 or 3). (b) PCR analysis of neomycin resistant pools compared with parental RPE1 cells. (c) PCR analysis of a Cyclin B1-Venus RPE1 clone following Cre-recombination compared to RPE1 control cells. (d) Serum-starved RPE1 Cyclin B1-Venus cells were released from quiescence by adding FBS and the levels of Cyclin B1 and Cyclin B1-Venus were followed by immunoblotting. Note the similar kinetics of appearance of the untagged and tagged proteins. Molecular mass markers on the left. (e) Proteolysis of Cyclin B1 and Cyclin B1-Venus after release from a DMA-induced mitotic block. Note that both Cyclin B1 and Cyclin B1-Venus accumulate during the block. Molecular mass markers on the left. (f) Analysis of Cyclin B1-Venus levels by time-lapse fluorescence imaging during mitosis under conditions that either activate or eliminate the SAC. Representative of 15 cells in each condition and from at least two independent experiments. (g) Scatter plot of the time between nuclear envelope breakdown (NEBD) and exit from mitosis as judged by the disappearance of the Cyclin B1-Venus signal in Cyclin B1-Venus cells were treated simultaneously with nocodazole and the indicated concentration of AZ3146 (n = 46 cells for nocodazole control; n = 21 cells for 0.31 μM; n = 27 cells for 0.62 μM; n = 19 cells for 1.25 μM; n = 36 cells for 2.5 μM and n = 55 cells for 5 μM). The black line in each condition represents the mean. Representative of at least two independent experiments.
Supplementary Figure 3 Characterization of Venus-Mad2 RPE1 clones and estimation of the number of molecules of Venus-Mad2 in a cell and at kinetochores.
(a) Genomic PCR screen for correctly targeted Venus-Mad2 integrants following flow cytometry selection. Primer sets consisted of an oligonucleotide annealing outside the targeting construct (either 5′ (#1) or 3′ (#4)) and an oligonucleotide annealing to the ORF of Venus (2 or 3). (b) PCR analysis with different primer combinations of a positive clone compared with parental RPE1 cells. Note that in lane 1+4 the upper band corresponds to the targeted allele whereas the bottom band corresponds to the wild-type allele. The asterisk shows a hybrid product caused by the annealing of DNA from the two other bands. (c) Localisation of Venus-Mad2 in prometaphase and metaphase. Candidate Venus-Mad2 RPE1 cells expressing ectopic CENP A-Cerulean were imaged by spinning-disk confocal microscopy. Note the co-localisation of the two signals in prometaphase that disappears in metaphase once all the kinetochores are attached. (d) Asynchronous RPE1 cells were treated similarly to Fig.3c, fixed with methanol and processed for immunofluoresence. Mad2 foci that colocalised with ACA staining were counted (n = 20 cells for nocodazole; n = 7 cells for Taxol and n = 10 cells for DMA) and compared to the data obtained by live-cell imaging (Fig. 3d,e). Error bars indicate s.d.. and are representative of at least two independent experiments. (e) Quantification of Venus-Mad2 molecules in protein extracts by immunoblotting using an anti-Mad2 antibody and recombinant His6-Mad2. Mitotic Venus-Mad2 RPE1 cells were collected by mitotic shake-off after nocodazole treatment, counted and processed for immunoblotting. Protein extracts were prepared from known numbers of cells. Molecular mass markers on the left. (f) Calibration curve for the Mad2 antibody obtained by quantifying band intensities in (e). The dotted line represents the linear regression. The inset graph shows the frequency distribution of Venus-Mad2 fluorescence in cells treated with nocodazole and analyzed by confocal microscopy (n = 100 cells). Note that by knowing the mean number and mean fluorescence of Venus-Mad2 molecules per cell we can calculate the mean fluorescence per molecule of Venus-Mad2. (g) A maximum intensity projection of 50 z -sections taken at 0.5 μm intervals was used to identify individual kinetochores. (h) An inner circle (red) was drawn around the single kinetochore. An outer circle (blue) just around the inner circle was used to measure the local background around the kinetochore. (i) Since kinetochore signals span more than one 0.5 μm z-section, sum intensity projections were used to measure the integrated fluorescence intensity. (j) Equation used to obtain the total fluorescence intensity (Ftot) of a single kinetochore. This takes into account the local background and the respective areas of measurements. Note that all integrated fluorescence intensities were initially corrected for camera noise by subtracting fluorescence intensities of regions adjacent to cells. Scale bar = 10μm.
Supplementary Figure 4 Size-exclusion chromatography of RPE1 cells treated with Taxol and nocodazole.
Extracts from (a) Taxol- and (b) nocodazole-treated RPE1 cells were fractionated by size-exclusion chromatography and analysed by quantitative immunoblotting with the indicated antibodies. Signal intensities (right panels) are relative to the highest value for each protein. Representative of three independent experiments. Molecular mass markers on the left. (c) Absolute amount of checkpoint proteins per Cdc20 in the MCC fractions of both Taxol- and nocodazole-treated RPE1 cells (n = 3). Taxol values were set to 1. Error bars indicate s.e.m. and are representative of three experiments.
Supplementary Figure 5 Model.
(a) Each unattached kinetochore has an influence on the rate of MCC formation in a cell. (b) We hypothesise that Cdc20 can be inhibited by the SAC in two ways: by sequestration and inactivation by the MCC, or by binding of the MCC away from the normal co-activator site on the APC/C. To recognize its metaphase substrates the APC/C needs first to bind to a free Cdc20 molecule to generate the bi-partite destruction box receptor. Assuming constant MCC turn over, dependent on p31Comet and APC15 pathways, the higher the concentration of MCC is in a cell, the higher the probablilty (p) that it will bind APC/C complexes. Consequently, this will decrease the probability that APC/C complexes can bind to free Cdc20. (c) In unperturbed cells, the stability of metaphase APC/C substrates is largely due to the initial strong “pulse” of MCC formation at NEBD. In drug-treated cells, the continuous presence of unattached kinetochores brings MCC concentration to “supra-physiological” levels (assuming constant MCC turn over), keeping the APC/C inhibited. Drug-treated cells will slip out of mitosis at a rate that is inversely related to the number of improperly attached kinetochores that recruit Mad2. In nocodazole there are very few MTs, therefore Mad2 is recruited to most kinetochores and maintains strong inhibition of the APC/C. In DMA, MTs attach to kinetochores but are efficiently detached by the error-correction machinery. In Taxol, most kinetochores attach to MTs and are only inefficiently detached by the error correction machinery, generating a smaller number of MCC per unit time than in nocodazole or DMA-treated cells, which allows a higher level of APC/C activity and faster slippage.
Supplementary Figure 6 Complete scans of all Western blot analyses presented in Figs 1–5 and Supplementary Figs S1–s5.
Cropped regions are indicated by dashed boxes. 700 nm or 800 nm channels indicate scans of the same blot using secondary antibodies coupled to fluorophores that are excited by 680 nm or 800 nm light, respectively.
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Video of the RPE1 Cyclin A2-Venus knock-in cell line going through mitosis.
A Cyclin A2-Venus RPE1 cell imaged on a single plane and at 3 minute intervals. 40X objective. Representative of >50 cells from more than three experiments. (MOV 516 kb)
Video of the RPE1 Cyclin B1-Venus knock-in cell line going through mitosis.
A Cyclin B1-Venus RPE1 cell imaged on a single plane and at 15 seconds intervals. 60X objective. Representative of >50 cells from more than three experiments. (MOV 920 kb)
Video of the RPE1 Venus-Mad2 knock-in cell line going through mitosis.
A Venus-Mad2 RPE1 cell imaged on a single plane and at 15 seconds intervals. 40X objective. Representative of >50 cells from more than three experiments. (MOV 2013 kb)
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Collin, P., Nashchekina, O., Walker, R. et al. The spindle assembly checkpoint works like a rheostat rather than a toggle switch. Nat Cell Biol 15, 1378–1385 (2013). https://doi.org/10.1038/ncb2855
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DOI: https://doi.org/10.1038/ncb2855
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