System-level feedbacks make the anaphase switch irreversible Enuo Hea, Orsolya Kapuya, Raquel A. Oliveirab, Frank Uhlmannc, John J. Tysond, and Béla Nováka,1 aOxford Centre for Integrative Systems Biology, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; bDepartment of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; cChromosome Segregation Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, United Kingdom; and dDepartment of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Edited by Tim Hunt, Cancer Research UK, South Mimms, United Kingdom, and approved May 3, 2011 (received for review February 7, 2011) The mitotic checkpoint prevents a eukaryotic cell from commencing The switch is irreversible, because the cell is locked in the post- to separate its replicated genome into two daughter cells (ana- transition state even after the inducing signal disappears. This phase) until all of its chromosomes are properly aligned on the systems view of irreversibility is based on computational modeling metaphase plate, with the two copies of each chromosome attached (4–6) and is supported by experimental data (7–10) for some of to opposite poles of the mitotic spindle. The mitotic checkpoint is these transitions (G1/S, G2/M, and T/G1). exquisitely sensitive in that a single unaligned chromosome, 1 of The separation of sister chromatids at anaphase is an apparent a total of ∼50, is sufficient to delay progression into anaphase; exception to our feedback view of irreversible cell-cycle tran- however, when the last chromosome comes into alignment on the sitions. In this case, it seems that the M/A transition is irreversible metaphase plate, the mitotic checkpoint is quickly satisfied, and for thermodynamic reasons rather than network dynamic con- the replicated chromosomes are rapidly partitioned to opposite siderations. Sister chromatids are held together at metaphase by poles of the dividing cell. The mitotic checkpoint is also curious in cohesin rings that oppose the pulling forces of the mitotic spindle the sense that, before metaphase alignment, chromosomes that are on the bioriented chromosomes (11). At the M/A transition, not being pulled in opposite directions by the mitotic spindle acti- a protease (called separase) is activated, which cleaves the cohesin vate the checkpoint, but during anaphase, these same tensionless rings, thereby allowing the sister chromatids to be pulled apart by chromosomes can no longer activate the checkpoint. These and spindle forces (12). Cohesin cleavage by proteolysis is a thermo- other puzzles associated with the mitotic checkpoint are addressed dynamically spontaneous reaction, and the metaphase alignment SYSTEMS BIOLOGY by a proposed molecular mechanism, which involves two positive of chromosomes cannot be recreated simply by resynthesizing feedback loops that create a bistable response of the checkpoint to cohesin proteins. Thermodynamically spontaneous forces have chromosomal tension. pulled the sister chromatids apart, and they will not come back together again spontaneously, even if the inducing signal is re- bistability | cell cycle | irreversible transition | mitotic checkpoint | moved and the cohesin rings are resealed. We consider this to be spindle assembly checkpoint the first puzzle about the M/A transition. (Puzzle 1) Is the M/A transition irreversible for thermodynamic he cell cycle is an ordered sequence of events by which cells reasons, unlike the other three cell-cycle transitions, which are Treplicate their chromosomes (S phase) and partition the irreversible because of regulatory feedback controls? identical sister chromatids to opposite poles of the mitotic spindle (M phase). In growing cells, temporal gaps separate S phase from The M/A transition is puzzling in other respects as well. Its M phase (G1-S-G2-M-G1- etc.). Progression through the cell guard, the mitotic checkpoint, is activated in prometaphase by cycle is characterized by irreversible transitions at the boundaries kinetochores that are not under tension, because the cohesin- of these four phases: G1/S, G2/M, and M/G1. The M/G1 transi- bound chromatids have not yet achieved biorientation on the tion takes place in two steps: metaphase/anaphase (M/A; parti- mitotic spindle (13, 14). In the pretransition state, chromosomes tioning of sister chromatids) and telophase/G1 (T/G1; mitotic exit that are not under tension send a strong signal to the mitotic fi and return to G1 and cytokinesis). Speci c, transient biochemical checkpoint to block cell-cycle progression. As soon as all of the signals trigger these transitions, which are irreversible in the sense chromosomes are properly aligned on the spindle, the mitotic that, after the triggering signal disappears, the cell does not revert checkpoint is lifted, separase is activated, cohesin rings are to the previous cell-cycle phase but is continually ratcheted for- cleaved, and sister chromatids are pulled apart. ward through the G1-S-G2-M sequence. The three major irreversible transitions are guarded by check- (Puzzle 2) Why is it that, before the M/A transition, zero point mechanisms that delay or block the transitions until con- tension prevents progression into anaphase, but after the tran- ditions are favorable to progress to the next phase of the cell cycle sition, when tension is decreasing because the cohesin rings (1). At the restriction point, cells check that they have the proper are broken, the control network is locked in a posttransition growth factor signals and that their DNA is undamaged before state and does not revert to the pretransition state (15, 16)? they leave G1 and enter S phase. At the G2/M checkpoint, they check that DNA replication is completed before entering mitosis. Puzzle 2 has been bolstered by recent experimental manipu- Cells may pass the mitotic checkpoint only if the mitotic spindle is lations (17–19) that make the M/A transition reversible by in- fully assembled and all chromosomes are properly aligned on the terfering with the tension-sensing mechanism. metaphase plate with sister chromatids attached to opposite poles of the spindle. We have argued that the irreversibility of these transitions is Author contributions: R.A.O., F.U., J.J.T., and B.N. designed research; E.H. and O.K. based on system-level feedbacks in the molecular regulatory performed research; E.H., O.K., R.A.O., and F.U. analyzed data; and J.J.T. and B.N. wrote mechanisms of the checkpoints (2, 3). In particular, positive (or the paper. double-negative) feedback circuits in these regulatory networks The authors declare no conflict of interest. create one-way toggle switches with two alternative stable steady This article is a PNAS Direct Submission. states: the pre- and posttransition states. The checkpoints hold 1To whom correspondence should be addressed. E-mail: [email protected]. cells in the pretransition steady state until a triggering signal is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. generated to induce a switch to the posttransition steady state. 1073/pnas.1102106108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102106108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A third feature of the mitotic checkpoint has been acknowl- active separase cleaves cohesins, and the mitotic spindle pulls edged as a puzzle for many years. sister chromatids to the opposing poles. Because APCCdc20 activation at anaphase results in cohesin (Puzzle 3) How is it that a single unattached kinetochore is cleavage and loss of tension at kinetochores, the error-correction enough to block the M/A transition? mechanism and the mitotic checkpoint are in danger to be re- activated during anaphase (15, 16).However,microtubule- For a human cell, having 45 of 46 chromosomes properly Cdc20 aligned on the metaphase plate is not enough to satisfy the mi- kinetochore attachments are stable, and APC stays active totic checkpoint (20). All 46 chromosomes must be bioriented during normal anaphase, suggesting the existence of an inacti- and under tension. The mitotic checkpoint can reliably distin- vating mechanism that suppresses reactivation of the mitotic guish between the numbers 45/46 = 0.98 and 46/46 = 1.00. checkpoint during anaphase. A crucial change taking place at the M/A transition is a drop in Biochemical control systems are not usually associated with such CycB precision. Of course, one might turn these numbers around and activity of cyclin B-dependent kinase (CDK ) and an increase claim that the mitotic checkpoint is telling the difference be- in activity of its counteracting protein phosphatase (CAPP). Four – CycB tween (46 − 45)/46 = 0.02 and (46 − 46)/46 = 0 and that there is papers (17 19, 28) conclude that this abrupt drop in CDK / a big difference between nonzero and zero. However, this ar- CAPP ratio during anaphase blocks reactivation of the error- gument comes with its own puzzle. From this perspective, during correction mechanism and the mitotic checkpoint. Cohesins prometaphase, the signal from the mitotic checkpoint drops engineered with cleavage sites for tobacco etch virus (TEV) protease were expressed in Drosophila embryos (18) and budding from 1.00 to 0.02, but even the weakest signal is able to hold Cdc20 the checkpoint in the state of no additional progression. Hence, yeast cells (17) arrested in metaphase by APC inactivation. By inducing TEV protease in these metaphase-arrested cells, the kinetic processes that are trying to disengage the mitotic fi checkpoint must be very weak themselves. In that case, another cohesins are cleaved, and a pseudoanaphase is initiated. At rst, puzzle is apparent. sister chromatids move to opposite poles, but later, they start to oscillate between the two poles (18). TEV-induced cohesin (Puzzle 4) How is it that even a weak signal can keep the cleavage is accompanied by reaccumulation of checkpoint pro- mitotic checkpoint engaged, but when the signal drops to zero, teins to kinetochores in both flies and yeast (17, 18).