Irreversibility of mitotic exit is the consequence of systems-level feedback

Abstract

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 the successful completion of this cycle. It is thought that irrevocable proteolytic degradation of key cell-cycle regulators makes cell-cycle transitions irreversible, thereby enforcing directionality. 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, owing 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 the 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.

Figure 1. Clb2 destruction promotes reversible mitotic exit events

a, Scheme depicting the experimental design and the predicted outcomes if cyclin proteolysis does, or does not, make mitotic exit irreversible. b, APC^Cdh1(m11)-driven Clb2 destruction is reversible and leads to reversible Cdk substrate dephosphorylation. Cdh1(m11) was induced in metaphase arrested cells for 30 minutes, and APC^Cdh1(m11) activity terminated after 50 minutes by inactivation of the cdc16-123 allele at 37°C. Cdh1(m11) was detected by Western blotting against its N-terminal HA epitope, Sli15, Ask1 and Ase1 were detected via C-terminal Pk₆ epitopes. Tub1 served as a loading control. c, Clb2 degradation and re-accumulation are accompanied by spindle breakdown and re-assembly. As b, but cells were processed for indirect immunofluorescence to visualise the spindle pole body (SPB) component γ-tubulin (Tub4), mitotic spindles (tubulin) and nuclear DNA (stained with DAPI). Scale bar, 5 μm. d, FACS analysis of DNA content of the cells in c confirms their mitotic arrest throughout the timecourse.

Figure 2. Irreversibility of mitotic exit requires feedback loop activation

a, Irreversible mitotic exit after longer periods of APC^Cdh1(m11) activity depends on Sic1. As Fig. 1, but Cdh1(m11) expression was not terminated and cdc16-123 was inactivated after 60 minutes. Mating pheromone α-factor (5 μg/ml) was added to prevent progression through the next cell cycle. FACS analysis of DNA content reveals completion of cytokinesis in cells containing Sic1. b-c, Limited activation of feedback loop components during reversible mitotic exit. b, Swi5 retains cytoplasmic localisation and c, Sic1 accumulation is incomplete during reversible APC^Cdh1(m11)-driven mitotic exit. For comparison, cells were released from metaphase arrest into synchronous mitotic exit by Cdc20 re-induction. Swi5 was visualised by indirect immunofluorescence. Levels of Clb2 and Sic1 were analysed by Western blotting. Scale bar, 5 μm.

Figure 3. Sic1 turns mitotic exit irreversible

a, Ectopic expression of Cdc14 or Sic1(m3) advances irreversibility of mitotic exit. Cdh1(m11) was expressed without or together with Cdc14 or Sic1(m3) for 30 minutes, before APC^cdc16-123 was inactivated after 50 minutes. α-factor was added as in Fig. 2a. b, Sic1 promotes irreversible mitotic exit in the absence of APC activity after chemical Cdk inhibition. Cdk (cdc28-as1) was inhibited by addition of 5 μM 1NM-PP1 in metaphase arrested SIC1 and sic1Δ cells, depleted for Cdc20 and APC^cdc16-123 inactivated at 37°C. After 10 or 50 minutes, 1NM-PP1 was washed out while APC^cdc16-123 remained inactive. Levels and gel mobility of the indicated proteins were analysed by Western blotting. Tub1 or Act1 served as loading controls.

Figure 4. Computational analysis of mitotic exit

a, Wiring diagram for Clb2/Cdk (abbreviated Clb2) and Sic1 regulation during budding yeast mitotic exit. AA, amino acids. b-d, Numerical simulations of protein levels and activities with a mathematical model (see Supplementary Fig. 6) during Cdh1(m11)-induced mitotic exit and e-g, after chemical Cdk inhibition. Clb2 represents the total level of Clb2/Cdk complexes, including inactive complexes bound to Sic1, Clb2a its associated kinase activity, MCM1 the active form of the Clb2 transcription factor complex Fkh2/Ndd1/Mcm1, APC the level of active APC^Cdh1(m11). b,e, Reversible exit: APC^Cdh1(m11) activity is terminated after 50 minutes, or Cdk inhibition by 1NM-PP1 released after 10 minutes. c,f, Irreversible exit: APC^Cdh1(m11) activity continues for 60 minutes, or Cdk inhibition for 50 minutes. d,g, Reversible exit in sic1Δ cells: as c,f, but Sic1 synthesis is zero.