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The eukaryotic cell cycle comprises an ordered series of events, orchestrated by the activity of cyclin-dependent kinases (Cdks), leading from chromosome replication during S-phase to their segregation in mitosis. The unidirectionality of cell cycle transitions is fundamental for successful completion of this cycle. It is thought that irrevocable proteolytic degradation of key cell cycle regulators makes cell cycle transitions irreversible, thereby enforcing directionality1-3. Here, we have experimentally examined the contribution of cyclin proteolysis to the irreversibility of mitotic exit, the transition from high mitotic Cdk activity back to low activity in G1. We show that forced cyclin destruction in mitotic budding yeast cells efficiently drives mitotic exit events. However, these remain reversible after termination of cyclin proteolysis, with recovery of the mitotic state and cyclin levels. Mitotic exit becomes irreversible only after longer periods of cyclin degradation, due to activation of a double-negative feedback loop involving the Cdk inhibitor Sic1 (refs 4​,​5). Quantitative modelling suggests that feedback is required to maintain low Cdk activity and to prevent cyclin resynthesis. Our findings demonstrate that unidirectionality of mitotic exit is not the consequence of proteolysis but of systems level feedback required to maintain the cell cycle in a new stable state.

The mitotic checkpoint prevents a eukaryotic cell from commencing to separate its replicated genome into two daughter cells (anaphase) until all of its chromosomes are properly aligned on the metaphase plate, with the two copies of each chromosome attached to opposite poles of the mitotic spindle. The mitotic checkpoint is exquisitely sensitive in that a single unaligned chromosome, 1 of a total of ∼50, is sufficient to delay progression into anaphase; however, when the last chromosome comes into alignment on the metaphase plate, the mitotic checkpoint is quickly satisfied, and the replicated chromosomes are rapidly partitioned to opposite poles of the dividing cell. The mitotic checkpoint is also curious in the sense that, before metaphase alignment, chromosomes that are not being pulled in opposite directions by the mitotic spindle activate the checkpoint, but during anaphase, these same tensionless chromosomes can no longer activate the checkpoint. These and other puzzles associated with the mitotic checkpoint are addressed by a proposed molecular mechanism, which involves two positive feedback loops that create a bistable response of the checkpoint to chromosomal tension.bistability | cell cycle | irreversible transition | mitotic checkpoint | spindle assembly checkpoint T he cell cycle is an ordered sequence of events by which cells replicate their chromosomes (S phase) and partition the identical sister chromatids to opposite poles of the mitotic spindle (M phase). In growing cells, temporal gaps separate S phase from M phase (G1-S-G2-M-G1-etc.). Progression through the cell cycle is characterized by irreversible transitions at the boundaries of these four phases: G1/S, G2/M, and M/G1. The M/G1 transition takes place in two steps: metaphase/anaphase (M/A; partitioning of sister chromatids) and telophase/G1 (T/G1; mitotic exit and return to G1 and cytokinesis). Specific, transient biochemical signals trigger these transitions, which are irreversible in the sense that, after the triggering signal disappears, the cell does not revert to the previous cell-cycle phase but is continually ratcheted forward through the G1-S-G2-M sequence.The three major irreversible transitions are guarded by checkpoint mechanisms that delay or block the transitions until conditions are favorable to progress to the next phase of the cell cycle (1). At the restriction point, cells check that they have the proper growth factor signals and that their DNA is undamaged before they leave G1 and enter S phase. At the G2/M checkpoint, they check that DNA replication is completed before entering mitosis. Cells may pass the mitotic checkpoint only if the mitotic spindle is fully assembled and all chromosomes are properly aligned on the metaphase plate with sister chromatids attached to opposite poles of the spindle.We have argued that the irreversibility of these transitions is based on system-level feedbacks in the molecular regulatory mechanisms of the checkpoints (2, 3). In particular, positive (or double-negative) feedback circuits in t...