Abstract
The mitotic spindle assembly checkpoint (SAC) delays anaphase onset until all chromosomes have attached to both spindle poles1,2. Here, we investigated SAC signalling kinetics in response to acute detachment of individual chromosomes using laser microsurgery. Most detached chromosomes delayed anaphase until they had realigned to the metaphase plate. A substantial fraction of cells, however, entered anaphase in the presence of unaligned chromosomes. We identify two mechanisms by which cells can bypass the SAC: first, single unattached chromosomes inhibit the anaphase-promoting complex/cyclosome (APC/C) less efficiently than a full complement of unattached chromosomes; second, because of the relatively slow kinetics of re-imposing APC/C inhibition during metaphase, cells were unresponsive to chromosome detachment up to several minutes before anaphase onset. Our study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes. Detailed knowledge about SAC signalling kinetics is important for understanding the emergence of aneuploidy and the response of cancer cells to chemotherapeutics targeting the mitotic spindle.
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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, R966–980 (2012).
Kitajima, T. S., Ohsugi, M. & Ellenberg, J. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes. Cell 146, 568–581 (2011).
Magidson, V. et al. The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. Cell 146, 555–567 (2011).
Lampson, M. A. & Cheeseman, I. M. Sensing centromere tension: aurora B and the regulation of kinetochore function. Trends Cell Biol. 21, 133–140 (2011).
Rieder, C. L., Cole, R. W., Khodjakov, A. & Sluder, G. The checkpointdelaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J. Cell Biol. 130, 941–948 (1995).
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).
Rieder, C. L. & Maiato, H. Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. Dev. Cell 7, 637–651 (2004).
Manchado, E. et al. Targeting mitotic exit leads to tumor regression in vivo: modulation by Cdk1, Mastl, and the PP2A/B55alpha,delta phosphatase. Cancer Cell 18, 641–654 (2010).
Gascoigne, K. E. & Taylor, S. S. How do anti-mitotic drugs kill cancer cells? J. Cell Sci. 122, 2579–2585 (2009).
Huang, H. C., Shi, J., Orth, J. D. & Mitchison, T. J. Evidence that mitotic exit is a better cancer therapeutic target than spindle assembly. Cancer Cell 16, 347–358 (2009).
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).
Reddy, S. K., Rape, M., Margansky, W. A. & Kirschner, M. W. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921–925 (2007).
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).
Uzunova, K. et al. APC15 mediates CDC20 autoubiquitylation by APC/C(MCC) and disassembly of the mitotic checkpoint complex. Nat. Struct. Mol. Biol. 19, 1116–1123 (2012).
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).
Foster, S. A. & Morgan, D. O. The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation. Mol. Cell 47, 921–932 (2012).
Varetti, G., Guida, C., Santaguida, S., Chiroli, E. & Musacchio, A. Homeostatic control of mitotic arrest. Mol. Cell 44, 710–720 (2011).
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).
Lenart, P. et al. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr. Biol. 17, 304–315 (2007).
Liu, D., Davydenko, O. & Lampson, M. A. Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing. J. Cell Biol. 198, 491–499 (2012).
Kapoor, T. M. et al. Chromosomes can congress to the metaphase plate before biorientation. Science 311, 388–391 (2006).
Stevens, D., Gassmann, R., Oegema, K. & Desai, A. Uncoordinated loss of chromatid cohesion is a common outcome of extended metaphase arrest. PLoS One 6, e22969 (2011).
Hagting, A. et al. Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. 157, 1125–1137 (2002).
Held, M. et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nat. Methods 7, 747–754 (2010).
Vazquez-Novelle, M. D. & Petronczki, M. Relocation of the chromosomal passenger complex prevents mitotic checkpoint engagement at anaphase. Curr. Biol. 20, 1402–1407 (2010).
Mirchenko, L. & Uhlmann, F. Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset. Curr. Biol. 20, 1396–1401 (2010).
Gerlich, D., Koch, B., Dupeux, F., Peters, J. M. & Ellenberg, J. Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication. Curr. Biol. 16, 1571–1578 (2006).
Shindo, N., Kumada, K. & Hirota, T. Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition. Dev. Cell 23, 112–123 (2012).
Gordon, D. J., Resio, B. & Pellman, D. Causes and consequences of aneuploidy in cancer. Nat. Rev. Genet. 13, 189–203 (2012).
Holland, A. J. & Cleveland, D. W. Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis. Nat. Rev. Mol. Cell Biol. 10, 478–487 (2009).
Cimini, D. et al. Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J. Cell Biol. 153, 517–527 (2001).
Thompson, S. L. & Compton, D. A. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–672 (2008).
Glover, D. M. Mitosis in the Drosophila embryo–in and out of control. Trends Genet. 7, 125–132 (1991).
Sluder, G. Role of spindle microtubules in the control of cell cycle timing. J. Cell Biol. 80, 674–691 (1979).
Schmitz, M. H. & Gerlich, D. W. Automated live microscopy to study mitotic gene function in fluorescent reporter cell lines. Methods Mol. Biol. 545, 113–134 (2009).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Acknowledgements
The authors thank M. Petronczki and L. Vazquez-Novelle for critical comments on the manuscript, A. Hyman and I. Poser for providing cells expressing Mad2–EGFP, the IMBA-IMP BioOptics core facility, the ETH Light Microscopy Center and the MBL Central Microscopy Facility for excellent technical support, M. Terasaki, J. M. Peters, P. Meraldi for helpful discussions, and C. Sommer for statistical consultation. Research in the Gerlich laboratory has received financial support from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreements no 241548 (MitoSys) and no 258068 (Systems Microscopy), from an ERC Starting Grant (agreement no 281198), from the EMBO Young Investigator Programme, from the Swiss National Science Foundation, from the Austrian Science Fund (FWF)-funded project ‘SFB Chromosome Dynamics’, and from a Summer Research Award of the Marine Biology Laboratory Woods Hole (Laura and Arthur Colwin Endowed Summer Research Fellowship Fund). A.E.D. is a fellow of the Zurich PhD Program in Molecular Life Sciences and has received funding from a PhD fellowship by the Boehringer Ingelheim Fonds and from a Peter Müller fellowship.
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A.E.D. designed and conducted experiments and analysed data. D.W.G. conceived the project, analysed data, and wrote the manuscript with help from A.E.D.
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Integrated supplementary information
Supplementary Figure 1 Validation of the mouse Mad2-EGFP-expressing cell line and further analysis of correlative laser microsurgery, time-lapse imaging, and immunofluorescence staining of the spindle.
(a) Prolonged mitosis in presence of 100 ng/ml nocodazole. HeLa cells stably expressing H2B-mCherry and securin-mEGFP were transfected with non-targeting control siRNA and imaged live after 24 h in presence of 100 ng/ml nocodazole. Time = 0:00 h:min at prometaphase onset. (b) Mitotic slippage after depletion of Mad2. Imaging was as in (a), but this cell was transfected with siRNA targeting human Mad2 (siHsMad2), 24 h prior to imaging. Bars 5 μm. (c) Mitotic slippage induced by transfection of siHsMad2 is suppressed in HeLa cells expressing mouse Mad2-EGFP. Imaging was as in (a, b) using cells stably expressing H2B-mCherry and securin-mEGFP, or H2B-mCherry and mouse-Mad2-EGFP. Mitotic slippage within 3 h after mitotic entry was scored based on the condensation status of H2B-mCherry (n = 30 for each condition). (d) The cell shown in Fig. 1e was cut in metaphase with a pulsed 915 nm laser at the area indicated by the white line, and imaged by 3D-confocal live-cell microscopy. (e) The cell shown in (d) was fixed 2:20 min:s after laser microsurgery and stained for α-tubulin. Bars: 10 μm. (f) Quantification of Mad2-EGFP levels on both sister kinetochores of 18 laser-displaced chromosomes with two Mad2 positive kinetochores.
Supplementary Figure 2 Fate of non-cancerous retinal pigmental epithelium cells, immortalized by stable overexpression of hTERT (hTERT-RPE1), after laser-induced chromosome detachment.
hTERT-RPE1 cells stably expressing H2B-mCherry were cut with a pulsed 915 nm laser at the area indicated with the white lines and imaged by 3D-confocal live-cell microscopy for 40 min. Time = 0:00 min:s at the first image acquired immediately after laser microsurgery. (a) A representative control cell was cut in a cytoplasmic region away from the spindle so that no chromosome was detached. (b) A representative cell in which a single chromosome was displaced from the metaphase plate by laser microsurgery, which subsequently recongresses before anaphase onset. (c) As in (b), but this cell enters anaphase in presence of an unaligned chromosome. Bars: 10 μm. (d) Fate trajectories of 36 cells, of which 13 control cells were cut adjacent to the spindle without perturbing the metaphase plate, and 23 cells were cut on the spindle to displace one or few chromosomes from the metaphase plate.
Supplementary Figure 3 Analysis of securin-mEGFP degradation.
(a) Original image frames of a HeLa cell stably expressing securin-mEGFP and H2B-mCherry. (b) Raw measurements of total mEGFP-securin fluorescence (black curve; scale is indicated to the right of the plot), overlaid on background-subtracted and bleach-corrected curve normalized to prometaphase (green, scale on left y-axis). Solid line indicates pre-anaphase stages, dashed line indicates post-anaphase stages. Light gray line indicates background, measured in a region adjacent to the cell. (c) Acquisition photobleaching was measured in a mitotic cell treated with 200 ng/ml nocodazole, with fast time-lapse to minimize the effect of mitotic slippage degradation (time-lapse: 2.7 s/frame). An exponential function (pink curve) was fitted to 5 bleach measurements (black curves). This function was determined separately for each experimental condition to correct for acquisition photobleaching as indicated in the online methods. (d) Raw total fluorescence measurements for the data shown in Fig. 5a (control cells, non-targeting siRNA). (e) Raw total fluorescence measurements for the data shown in Fig. 5a (Mad2 RNAi). (f) Raw measurements for the data shown in Fig. 5b.
Supplementary Figure 4 Kinetics of APC/C inhibition after chromosome detachment for 11 cells.
HeLa cells expressing H2B-mCherry and securin-mEGFP were imaged by 3D-confocal microscopy from prophase until metaphase. At different time points during metaphase (indicated by black star), the spindle was cut by a pulsed 915 nm laser to detach individual chromosomes. Time = 0 min at the first image acquired immediately after laser microsurgery. Red indicates time points where individual chromosomes were dealigned from the metaphase plate. Dashed lines indicate anaphase, with (red) or without (green) unaligned chromosomes. The gaps in the curve indicate pauses during time-lapse imaging to reduce light exposure. (a) Background-subtracted total fluorescence was normalized to prometaphase as shown in Fig. 4c,d. The first two panels represent the cells shown in Fig. 4. (b) Raw measurements of total fluorescence intensity in mean intensity projections of z-stacks. Gray curves indicate measurements outside cells used for background subtraction.
Supplementary Figure 5 Four additional examples for securin-mEGFP degradation in correlation with unaligned chromosomes induced by low dose nocodazole.
HeLa cells expressing H2B-mCherry and securin-mEGFP were imaged and analyzed as in Fig. 5f,g. Dark red indicates time intervals with >5 unaligned chromosomes, light red indicates time intervals with 2–5 unaligned chromosomes, orange indicates one unaligned chromosome, green indicates alignment of all chromosomes. Solid lines indicate pre-anaphase stages, dashed lines indicate anaphase stages. (a–d) Background-subtracted and bleach corrected total fluorescence normalized to prometaphase. (a) Cell treated with 6 ng/ml nocodazole. (b) 25 ng/ml nocodazole. (c, d) 12 ng/ml nocodazole. (e-h) Background-subtracted raw measurements of the cells shown in a-d.
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Mitotic progression in control cell that was cut in the cytoplasm.
A metaphase cell expressing H2B-mCherry and mEGFP-α-tubulin was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. The cut was positioned so that the mitotic spindle remained intact. (MOV 2443 kb)
Mitotic progression after acute detachment of a metaphase chromosome.
A metaphase cell expressing H2B-mCherry and mEGFP-α-tubulin was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line to detach a chromosome from the metaphase plate. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. (MOV 4038 kb)
Anaphase entry in presence of a unaligned chromosome.
A metaphase cell expressing H2B-mCherry and mEGFP-α-tubulin was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line to detach a chromosome from the metaphase plate. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. (MOV 1316 kb)
Mad2-EGFP recruitment after laser-mediated chromosome detachment.
A metaphase cell expressing H2B-mCherry and mouse Mad2-EGFP was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line to detach a chromosome from the metaphase plate. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. (MOV 3842 kb)
Securin-mEGFP degradation after laser-mediated chromosome detachment.
A metaphase cell expressing H2B-mCherry and securin-mEGFP was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line to detach a chromosome from the metaphase plate. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. In this cell, the chromosome recongresses to the metaphase plate before anaphase entry. (MOV 8624 kb)
Securin-mEGFP degradation in a cell that enters anaphase in presence of an unaligned chromosome.
A metaphase cell expressing H2B-mCherry and securin-mEGFP was imaged by 3D-confocal microscopy and cut by a pulsed 915 nm laser at the region indicated by the white line to detach a chromosome from the metaphase plate. Time = 00:00 min:s at the first image acquired immediately after laser microsurgery. This cell enters anaphase in presence of the dealigned chromosome. (MOV 1725 kb)
Securin-mEGFP degradation in a cell exposed to 12 ng/ml nocodazole.
A cell expressing H2B-mCherry and securin-mEGFP was imaged by 3D-confocal microscopy from prophase until anaphase. Time = 00:00 min:s at prometaphase onset. (MOV 3350 kb)
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Dick, A., Gerlich, D. Kinetic framework of spindle assembly checkpoint signalling. Nat Cell Biol 15, 1370–1377 (2013). https://doi.org/10.1038/ncb2842
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DOI: https://doi.org/10.1038/ncb2842
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