Fission Yeast and Minimal Cell Cycle

ARTICLEdoi:10.1038/nature09543

Driving the cell cycle with a minimalCDK control networkDamien Coudreuse1 & Paul Nurse1

Control of eukaryotic cell proliferation involves an extended regulatory network, the complexity of which has made itdifficult to understand the basic principles of the cell cycle. To investigate the core engine of the mitotic cycle we havegenerated a minimal control network in fission yeast that efficiently sustains cellular reproduction. Here we demonstratethat orderly progression through the major events of the cell cycle can be driven by oscillation of an engineeredmonomolecular cyclin-dependent protein kinase (CDK) module lacking much of the canonical regulation. We showfurther that the CDK oscillator acts as the primary organizer of the cell cycle, imposing timing and directionality to asystem of two CDK activity thresholds that define independent cell cycle phases. We propose that this simple corearchitecture forms the basic control of the eukaryotic cell cycle.

Progression through the eukaryotic cell cycle is driven by CDKs,which form bipartite complexes with different cyclins1. Changes inactivity of these complexes depend on oscillations in levels of thecyclins, the synthesis and degradation of which are regulated through-out the cell cycle, while combinatorial associations of CDKs andcyclins are thought to generate the distinct substrate specificitiesrequired to bring about the different cell cycle transitions2,3. Controlof the respective expression and subcellular localization of centralCDK machinery subunits constitutes a primary layer of cell cycleregulation4. In addition, CDK activity is modulated by specific inhi-bitors and by changes in phosphorylation of the catalytic subunit inresponse to inputs such as nutrient availability, cell size, and activa-tion of checkpoint mechanisms1. Some of these controls form feed-back loops that generate sharp changes in activity with hystereticproperties, contributing to the unidirectionality of the cell cycle5–9.Integration of all these parameters ensures orderly progressionthrough the mitotic cycle and appropriate responses to perturbations.

The complexity of eukaryotic cell cycle control has made it difficultto fully understand its basic principles, as demonstrated by the plas-ticity reported for certain key cell cycle effectors10–13. To investigatethe core engine of the mitotic cycle, we have generated a minimalcontrol network in the fission yeast Schizosaccharomyces pombe. Weshow that oscillation of a single monomolecular CDK module in theabsence of many of the known regulatory inputs and feedbacks issufficient to sequentially trigger the major cell cycle events. Wedemonstrate further that the core cycle can be built on a circuit oftwo CDK activity thresholds defining independent states with noinherent directionality, upon which sequence and timing are imposedby a single CDK oscillator.

A minimal cell cycle in fission yeastThe fission yeast cell cycle is controlled by a single CDK, Cdc2,required for both the G1/S and G2/M transitions14,15. DNA replicationand mitosis are triggered by association of Cdc2 with the B-typecyclins Cig2 and Cdc1316–20, respectively, with two additional cyclins,Cig1 and Puc1, having more minor roles in G117,21–25. In addition,Cdc13/Cdc2 activity in G2 blocks reinitiation of DNA replication26,27.

To simplify the cell cycle control machinery, a cassette expressing afusion of cdc13 and cdc2 under the control of the cdc13 regulatory

elements was integrated into the genome (cdc13-L-cdc2; Fig. 1a). Thisminimal CDK system differs from that operative in wild-type cells inseveral ways: (1) the regulatory and catalytic subunits are subject tothe same transcriptional, translational and degradation programs, arealways present in a 1:1 ratio, and always co-localize; (2) the rise inCDK activity is not triggered by a separate cyclin concentrationthreshold; (3) fusing the kinase with a specific cyclin is likely to pre-vent association with other cyclins and renders modulators of bindingbetween the two subunits irrelevant. The fusion protein was activeand additive to the function of the endogenous CDK machinery(Supplementary Fig. 1). We next deleted the genomic copies of cdc2

1Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.

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Figure 1 | A Cdc13-L-Cdc2 fusion in fission yeast. a, Schematicrepresentation of the cdc13-L-cdc2 fusion. Numbers are open reading framecoordinates. L, linker. Pcdc13, cdc13 promoter and 59 untranslated region(UTR). b–d, Labels are indicated in box. b, Western blots probed for Cdc13 andtubulin. E, endogenous Cdc13 (56 kDa). F, Cdc13-L-Cdc2 (91 kDa). A singleband was also observed using an anti-Cdc2 antibody (data not shown).c, Blankophor staining of exponentially growing cells. Scale bar, 10mm. d, DNAcontent analysis of cells in c (see Methods). e, Exponentially growing cdc13-L-cdc2-YFP D2D13DCCP cells at 25 uC. YFP and DNA imaging (Hoechst) ofindividual cells arranged according to their cell cycle stage. Dashed lines showcell outlines. Scale bar, 5mm.

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and cdc13 (D2D13). In this background, Cdc13-L-Cdc2 was detectedas a single full-length protein (Fig. 1b) and cdc13-L-cdc2 D2D13 cellswere almost identical to wild type, having a normal generation timeand only a small increase in cell length at division (Fig. 1c andTable 1). DNA content analysis showed the same profile as wild type(Fig. 1d and Methods), indicating that the relative durations of thedifferent cell cycle phases were maintained.

To demonstrate that this minimal machinery autonomously drivescell cycle progression, two alterations were introduced within thefusion protein (Supplementary Fig. 2a): (1) Cdc13(C379Y) (herereferred to as Cdc13ts), generating a temperature-sensitive protein14;and (2) Cdc2(F84G) (here referred to as Cdc2as; as, analogue sensi-tive), rendering the kinase sensitive to chemical inhibition28,29.Treatment with inhibitor (ATP analogue NmPP1) or shift to restrictivetemperature arrested cdc13ts-L-cdc2as D2D13 cells in G2 (Supplemen-tary Fig. 2b). Next, we additionally deleted cig2, which encodes themajor S-phase cyclin. Absence of Cig2 had no effect on the timing ofDNA replication (Supplementary Fig. 3a), but impairing function ofeither moiety of the fusion protein in G1 cells delayed S-phase onset(Supplementary Fig. 3b–e). These data show that both kinase andcyclin moieties of the CDK module are required to trigger the G1/Sand G2/M transitions.

The fission yeast genome contains 13 cyclin-like genes. Proteinsequence comparisons showed that Cig1, Cig2 and Puc1, the onlyother cyclins known to have mitotic cell cycle functions in complexeswith Cdc2, were the only mitotic cyclins that clustered with Cdc13(Supplementary Fig. 4). To simplify the network further, we thereforedeleted cig1, cig2 and puc1 (DCCP) in cdc13-L-cdc2 D2D13 cells. Thisstrain had no apparent cell cycle defects (Fig. 1b–d and Table 1). Inthis background, the CDK module oscillated in abundance, peaking atthe end of G2 and disappearing at mitotic exit, and recapitulated thenormal cell cycle changes in Cdc13 subcellular localization30 (Fig. 1eand Supplementary Fig. 5).

These results demonstrate that a single monomolecular CDK modulelacking several regulatory features of the endogenous machinery is suf-ficient to trigger the two major cell cycle transitions and sustains aneffective mitotic cycle. Other endogenous cyclin/CDK complexes thatmay have more peripheral roles in cell cycle regulation, including Mcs2/Mcs631 and Pas1/Pef132, cannot substitute for the CDK fusion and so donot have direct roles in driving the onsets of S and M phases.

Oscillations in CDK activityNext we investigated how a single CDK module distinguishes betweenG1/S and G2/M. Using cdc13-L-cdc2as D2D13DCCP cells (Table 1 andSupplementary Fig. 6), we asked whether progression through S andM in the absence of much of the canonical regulation is primarilymediated by distinct thresholds of a single CDK activity.

First, different inhibitor concentrations were added to synchronizedcells in early G2. Timing of mitosis was delayed in a dose-dependentmanner, with concentrations of 300 nM and above preventing mitosis

(Fig. 2a and Supplementary Fig. 7a–d). Cells delayed in G2 elongatedand accumulated the fusion protein (Supplementary Fig. 8a, b). Wesurmised that a critical ratio of CDK module to inhibitor concentrationmust be reached to allow mitotic onset. Consistent with this, a popu-lation of large cells incubated with inhibitor in early G2 entered mitosisearlier than similarly treated small cells, but at the same size (Sup-plementary Fig. 8c–g). Moreover, cells of asynchronous cultures treatedfor 8 h with different NmPP1 concentrations had longer sizes at divi-sion, proportional to the amount of inhibitor (Supplementary Fig. 7e).

Second, entry into S phase was monitored when synchronized cellswere exposed to inhibitor in G1 (Supplementary Fig. 9a). None of theconcentrations that affected G2/M had any effect on S phase (data notshown), even though G1 cells have lower levels of fusion protein(Supplementary Fig. 5). However, a dose-dependent delay in S-phaseonset was observed when 1–5mM NmPP1 was used (Fig. 2b andSupplementary Fig. 9b–e). The difference in inhibitor concentrationrequired to delay G1/S and G2/M supports the view that these transi-tions are associated with low and high kinase activities, respectively.

Table 1 | Characterization of cells operating with the minimal CDKmoduleGenotype Size at division

(mm)Generation time(min)

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Wild type 14 6 0.1 160 6 3 1.3 6 0.8cdc13-L-cdc2 D2D13 15.6 6 0.3 163 6 2 NDcdc13-L-cdc2 D2D13DCCP 15.9 6 0.2 162 6 6 0.2 6 0.2cdc13-L-cdc2as D2D13DCCP 14.7 6 0.2 158 6 6 NDcdc13-L-cdc2 D2D13DCCP Drum1* 15.9 6 0.1 155 6 3 NDcdc13-L-cdc2AF D2D13DCCP* 13.9 6 0.2 248 6 0 5.3 6 0.9cdc13-L-cdc2 D2D13DCCP Dwee1Dmik1

13.7 6 0 217 6 0 6.2 6 1.3

wee1-50ts{ 6.9 6 0.1 ND ND

Numbers are averages of three independent experiments with standard errors (n $ 50 for cell sizedetermination and $ 400 for dead cells). ND, not determined.*Similar sizes were obtained using NmPP1 sensitive strains.{Cell size at division was measured after 6 h at restrictive temperature (36 uC).

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Figure 2 | Oscillation of a single CDK activity between two thresholds.a, Percentage of binucleated cells (includes septated cells; n 5 400) insynchronized cdc13-L-cdc2as D2D13DCCP cultures incubated with NmPP1(added in early G2; T 5 0 in Supplementary Fig. 7a). Concentrations above300 nM also prevented mitosis (data not shown). b, DNA content analysis ofsynchronized cdc13-L-cdc2as D2D13DCCP cells treated with NmPP1 aftermitotic onset (T 5 0 in Supplementary Fig. 9a; flow cytometry profiles are inSupplementary Fig. 9c). Inhibitor-treated cells arrested in the next G2 andbecame elongated (data not shown). Block: 1mM NmPP1 for 2 h 45 min at32 uC. c, Inhibitor-mediated oscillation in activity using cdc13DDB-L-cdc2as

cdc2-33ts Dcig2 cells (see Supplementary Figs 10 and 11 for protocol anddescription of the entire experiment). DAPI/Blankophor staining (left panel)and DNA content analysis (right panel) at representative times during the firstartificial cycle. Scale bar, 10mm. The percentage of cells with a 1C DNA contentafter 40 min in 7.5mM NmPP1 is indicated.

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These data indicate that oscillation of qualitatively the same CDKactivity between two thresholds may be the sole requirement to drivethe minimal cell cycle. This interpretation predicts that substitutingthe oscillation in protein levels with an inhibitor-mediated oscillationin activity using a constantly present and non-degradable form of theCDK module should be sufficient to artificially drive the entire cycle.To test this, we deleted the Cdc13 destruction box within the fusionprotein (cdc13DDB-L-cdc2as) and drove its expression by the indu-cible urg1 regulatory elements33 (Fig. 2c and Supplementary Figs 10and 11). cdc13DDB-L-cdc2as cdc2-33ts Dcig2 cells (see Methods) main-tained at restrictive temperature entered mitosis when expression ofthe fusion cassette was induced. Impaired degradation of the proteinprevented mitotic exit, but addition of 7.5 mM NmPP1 allowed com-pletion of mitosis and cytokinesis with cells arresting in G1.Subsequent reduction to 1 mM inhibitor resulted in rapid DNA rep-lication. Finally, another cycle of inhibitor oscillation allowed cells toproceed through the next mitosis.

These results show that artificial modulation of the activity of asingle stable CDK module enables progression through S and M,supporting the idea of a quantitative CDK model of the cell cycle10,34,35.We propose that changes in protein levels and cyclin/CDK ratios arenot essential to cell cycle regulation and that a simple oscillation inactivity generated by a minimal control system is sufficient to drivethe mitotic cycle.

Resetting the cell cycleNext we asked whether the oscillator itself constitutes the primarysystem that sets the order and separation of cell cycle events. CDKactivity was manipulated in cdc13-L-cdc2as D2D13DCCP cells todetermine if this alone could change cell cycle architecture.

First, G2-arrested cells were released into medium with varyingconcentrations of inhibitor (Fig. 3a and Supplementary Fig. 12a–d).Cells released in dimethylsulphoxide (DMSO) resumed cycling,whereas 1 or 2.5 mM NmPP1 maintained the G2 block (data notshown). In contrast, treatments with 5 mM NmPP1 and more led toreplication without an intervening mitosis, after delays reflecting theconcentrations of inhibitor used and with increased amounts offusion protein. These data demonstrate that when CDK activity isreduced to a low level, G2 cells bypass mitosis and enter a G1/S-likeprogram. This is consistent with earlier studies showing that loss ofcdc13, overexpression of the CDK inhibitor Rum1 or chemical inhibi-tion of Cdc2 induces re-replication10,27,29,36,37. In these cases, however,Cig1 and Cig2 are required, indicating that the minimal CDK networkused here renders cells independent of additional regulation presentin wild-type cells. Finally, G2-arrested cdc13-L-cdc2as D2D13DCCPcells were subjected to a pulse of 10 mM NmPP1. Subsequent releaseinto 1mM inhibitor resulted in rapid entry into S phase without anintervening mitosis, showing that re-replication does not require per-sistence of low CDK activity (Supplementary Fig. 12e–g).

Next we investigated how G1 cells respond to an abrupt switch tohigh CDK activity. G2-arrested cdc13-L-cdc2as D2D13DCCP cellswere reset in G1 as in Supplementary Fig. 12e, bypassing mitoticdegradation of the fusion protein, but released into inhibitor-freemedium. An overlap between S and M was observed in most cells,resulting in aberrant nuclei and ‘cut’ phenotypes (G1 reset; Fig. 3b–d).Similar results were obtained when synchronized cells were main-tained in G1 to allow accumulation of the fusion protein and thenreleased into inhibitor-free medium (G1 arrest; Fig. 3b–d andSupplementary Fig. 13b). In this latter case, although bulk DNA syn-thesis appeared complete shortly after release, labelling of newly syn-thesized DNA showed that replication was still occurring whenmitotic phenotypes were apparent (Supplementary Fig. 13c).Moreover, the presence of aberrant nuclei reflected late stages of themitotic process. In both experiments, release into 1mM NmPP1 sup-pressed the mitotic phenotype but allowed DNA replication (data notshown), indicating that these effects are due to early induction of high

CDK activity which simultaneously brings about S and M.Furthermore, this shows that S phase can proceed with high CDKactivity as long as cells have previously experienced low activity.Finally, when S-phase progression was blocked using hydroxyureain these experiments, cells entered S and M simultaneously but failedto segregate distinct DNA masses (Fig. 4a and Supplementary Fig. 14),supporting the interpretation that the observed phenotype is a con-sequence of the mitotic machinery attempting to segregate incomple-tely replicated DNA. These data establish that cells with sufficientCDK activity can enter mitosis from inappropriate points in the cellcycle regardless of their previous state.

The apparent independency of S and M conflicts with models thatlink mitosis with completion of DNA replication through the S-phasecheckpoint. Surprisingly, we found that the checkpoint remained inac-tive (Fig. 4b and Supplementary Fig. 15a), indicating that it did notsense this overlap between S and M as a pathological situation.

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Figure 3 | Resetting the cell cycle. a, DNA content analysis of G2-arrestedcdc13-L-cdc2as D2D13DCCP cells released in various concentrations of NmPP1(T 5 0). Block: 1mM NmPP1 for 2 h 45 min at 32 uC. Black profiles showS-phase onset. Cells treated with inhibitor did not undergo mitosis(Supplementary Fig. 12a). b–d, G1 reset: cdc13-L-cdc2as D2D13DCCP cells weretreated as in Supplementary Fig. 12e but released into inhibitor-free medium(T 5 0). G1 arrest: Synchronized cdc13-L-cdc2as D2D13DCCP cells as inSupplementary Fig. 9a were arrested in G1 for 2 h with 10mM NmPP1 beforerelease (T 5 0). b, DAPI/Blankophor staining (see Supplementary Fig. 13a forthe entire time course). Scale bar, 10mm. c, Percentage of aberrant mitoticnuclei, including elongated, fragmented, asymmetrically divided and ‘cut’nuclei (n 5 400). d, DNA content analysis. Black profiles show first detection ofsignificant DNA synthesis.

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Furthermore, ectopic activation of the checkpoint using hydroxyureaonly prevented mitosis when entry into M phase was separated from theonset of S phase by temporary incubation in 1mM inhibitor beforerelease (Fig. 4c–f and Supplementary Fig. 15d). This demonstrates thatthe S-phase checkpoint can only have a role when the basic sequence ofcell cycle events is pre-established by proper kinetics of CDK oscillation.

We propose that the different phases of the mitotic cycle, defined byspecific CDK activity thresholds, can operate independently of eachother, establishing that the core cell cycle lacks inherent directionality.The oscillation of a single CDK activity can form the basic engine thatprovides directionality to this circuit and imposes the temporal orderof S and M (Fig. 4g).

CDK regulatory loops and size controlThe major systems directly regulating CDK activity in fission yeastinvolve the CDK inhibitor Rum122,36,38 and control of Cdc2 phosphor-ylation by Wee1, Mik1 and Cdc2539–42. We asked if these mechanismsare part of the core regulation or if they are only relevant in normalcells with a more complex CDK machinery.

Neither deregulation of Rum1 through deletion of cig1 and puc123,25,nor deletion of rum1 in cdc13-L-cdc2 D2D13DCCP cells had any effecton vegetative growth and checkpoint responses, but Drum1 cells didnot arrest in G1 after nitrogen starvation, consistent with previousobservations17 (Table 1 and Supplementary Figs 16 and 18).

Cdc2 phosphorylation has central roles in the control of cell size atdivision43, the S-phase and DNA-damage checkpoints44,45, and theresponse to changes in nutrient availability46,47. To determine theimportance of this regulation in the minimal cell cycle, Thr 14 (whichcan be phosphorylated by Wee148) and Tyr 15 (ref. 41) were altered(cdc13-L-cdc2AF cassette producing a Cdc13-L-Cdc2(T14A, Y15F)fusion protein; Supplementary Fig. 17). Whereas cells operating witha Cdc2(Y15F) protein showed poor viability41, cdc13-L-cdc2AFD2D13DCCP cells were surprisingly healthy, although with a longer

generation time (Fig. 5a and Table 1). Confirming these results, cdc13-L-cdc2 D2D13DCCP Dwee1 Dmik1 cells had similar characteristics(Fig. 5a, Table 1 and Supplementary Fig. 17), despite the co-lethalityof wee1 and mik1 in a normal background42. As expected, the S-phaseand DNA-damage checkpoints, which are operative in cdc13-L-cdc2D2D13DCCP cells, were impaired in the AF mutant (SupplementaryFig. 18). Although an imbalance in this regulatory loop affects thefunctioning of the wild-type fusion protein (Supplementary Fig. 19),these data show that simplifying the cell cycle network relieves cellsfrom tight regulation by Wee1, Mik1 and Cdc25.

In contrast to wee1 mutants, which divide at 50% of wild-type size,cdc13-L-cdc2AF D2D13DCCP cells divided only 15% smaller than cdc13-L-cdc2 D2D13DCCP cells (Table 1). Nevertheless, G1 was elongated as inwee1 cells43 (Fig. 5b). cdc13-L-cdc2AF D2D13DCCP cells also showed ahigher variability in size at division (Fig. 5c). However, the majority ofcells (67%) divided at a size within the range of variation observed for thecontrol strain. Furthermore, despite this increased heterogeneity, G2-arrested cdc13-L-cdc2AFas D2D13DCCP cells returned to their normalaverage size efficiently upon release (Fig. 5d and Supplementary Fig. 20).These data indicate that a Wee1-independent size control system isoperative in these cells and that Wee1 may integrate additional path-ways that render this control more accurate.

DiscussionWe have shown that a minimal control network based on a singlemonomolecular CDK module can autonomously drive the fissionyeast cell cycle, indicating that differential expression, degradationand subcellular localization of CDK subunits, attainment of specificratios of cyclin to CDK, and cyclin-mediated changes in substratespecificity are not essential for cell cycle progression. We demonstratethat the CDK oscillator provides timing and directionality to a simplecircuit of two activity thresholds that define independent cell cyclephases, and prevails over the S-phase checkpoint in organizing the

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Figure 4 | Timing and directionality of the minimal cell cycle. a, Percentageof aberrant mitotic nuclei in cells treated as in Fig. 3b–d but released in normalmedium or in 12 mM hydroxyurea (HU) (n 5 200). b, Western blot as inFig. 3b–d probed for the checkpoint effector Cds1. Controls are G2-arrestedcdc13-L-cdc2as D2D13DCCP cells (1mM NmPP1 for 2 h 45 min at 32 uC) thatwere released in 12 mM hydroxyurea, and proteins were extracted 2 h afterrelease. Phosphorylation of Cds1 upon checkpoint activation results in amobility shift (Methods). c, Western blot for the hydroxyurea-treated cells ina probed for Cds1. Despite the rapid activation of the S-phase checkpoint, S andM phases occurred simultaneously (a and Supplementary Fig. 14a). Controlsare as in b. d–f, Ectopic activation of the checkpoint using hydroxyureaprevents mitosis when the onsets of S and M are separated (modified G1 resetprotocol, Supplementary Fig. 15b). d, Percentage of aberrant mitotic nuclei

(n 5 200). Similar results were obtained using a G1 arrest-derived protocol(data not shown). e, DAPI/Blankophor staining. Scale bar, 10mm. f, Westernblots probed for Cds1, phospho-Cdc2(Y15), Cdc13 and tubulin. Controls are asin b. Cells incubated for 40 min with 1mM inhibitor entered S phase beforerelease (Supplementary Fig. 15c), resulting in checkpoint activation andinhibition of mitosis. Note that proper mitotic exit is set up in cells undergoingsimultaneous S and M as shown by the degradation of the fusion protein(10mM, T 5 30 min). g, The core cell cycle solely relies on changes inqualitatively the same CDK activity and lacks global timing and directionality(left panel). A temporal sequence is imposed on the critical independent cellcycle events by the characteristic oscillation of CDK activity between twothresholds (right panel: TS, S-phase threshold; TM, M-phase threshold). Theprecise kinetics of CDK activity accumulation are unknown (dashed line).

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mitotic cycle (Fig. 4g). We propose that this minimal architecturereveals the core control of the eukaryotic cell cycle. Although cell cycleregulation is more elaborate in multicellular eukaryotes, the redundancyobserved for metazoan CDK subunits11–13 indicates that our conclusionsmay also be relevant for more complex cells.

We show also that regulation by Wee1, Mik1 and Cdc25 is dispensablefor the minimal cell cycle. Interestingly, cdc13-L-cdc2AF D2D13DCCPcells divide at a length close to wild type, indicating that the Pom1gradient operating through Wee149,50 cannot be the only system thatprevents short cells from dividing. Cell-to-cell variation in size at divisionis higher in this strain. This could result from increased heterogeneity inthe timing and degree of CDK activation that may perturb the Wee1-independent size control. Feedback signalling through CDK phosphor-ylation may promote size homogeneity by reducing potential noise incore CDK expression, stability or activation. In other systems, CDKactivation shows Wee1/Cdc25-dependent stepwise and hysteretic prop-erties5–9, which provide sharp transitions and directionality to the cellcycle. In cdc13-L-cdc2AF D2D13DCCP cells, CDK activity may thereforerise more progressively, altering the kinetics of substrate phosphorylationand resulting in the observed phenotypes.

It is unclear how a single CDK activity can sequentially trigger DNAreplication and mitosis, as the possible overlap between S and M estab-lishes the simultaneous presence of G1/S and G2/M substrates. TheCDK may have higher affinity for G1/S substrates. Coupled withperiodic cyclin degradation, this would allow temporal separation ofS and M. Alternatively, activity-dependent changes in subcellularlocalization of the whole CDK machinery may provide substrate spe-cificity. It is also possible that specific phosphatases target G2/M sub-strates more readily. In G1, the significant CDK activity differential—from close to zero to a low level—coupled to a lower phosphorylationturnover would allow accumulation only of phosphorylated G1/S

substrates. G2/M substrate-specific phosphatases would establish afutile cycle with Cdc2, allowing the more modest differential in CDKactivity at the end of G2 to produce a significant increase in netphosphorylation of G2/M substrates.

The results presented here may have evolutionary implications. Asingle oscillating CDK module could be the way primitive eukaryotesregulated their cell cycle. Subsequent selection would have introducedother regulatory layers to improve and fine-tune the core system. Cellsmay have become dependent on these additional elements, renderingthem essential in modern cells and making it more difficult to fullyappreciate the core processes involved.

METHODS SUMMARYStandard methods for molecular biology, genetics and microscopy aredetailed in Methods. Strains are listed in Supplementary Table 1.Experiments were carried out in supplemented minimal medium at32 uC, except where otherwise noted, with various concentrations ofNmPP1 inhibitor (TRC). Cell size measurements were made fromimages of blankophor-stained cells. DNA was visualized in heat-fixedcells using 49,6-diamidino-2-phenylindole (DAPI) and in live cellsusing Hoechst. Western analyses were performed using total proteinextracts normalized by amounts of proteins except where otherwisenoted. DNA content was analysed using a BD FACSCalibur.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 23 April; accepted 28 September 2010.

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17. Martin-Castellanos, C., Labib, K. & Moreno, S. B-type cyclins regulate G1progression in fission yeast in opposition to the p25rum1 cdk inhibitor. EMBO J.15, 839–849 (1996).

18. Booher, R. & Beach, D. Involvement of cdc131 in mitotic control inSchizosaccharomyces pombe: possible interaction of the gene product withmicrotubules. EMBO J. 7, 2321–2327 (1988).

19. Booher, R. N., Alfa, C. E., Hyams, J. S. & Beach, D. H. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell58, 485–497 (1989).

20. Moreno, S., Hayles, J. & Nurse, P. Regulation of p34cdc2 protein kinase duringmitosis. Cell 58, 361–372 (1989).

21. Bueno, A., Richardson, H., Reed, S. I. & Russell, P. A fission yeast B-type cyclinfunctioning early in the cell cycle. Cell 66, 149–159 (1991).

22. Correa-Bordes, J., Gulli, M. P. & Nurse, P. p25rum1 promotes proteolysis of themitotic B-cyclin p56cdc13 during G1 of the fission yeast cell cycle. EMBO J. 16,4657–4664 (1997).

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Figure 5 | Role of Cdc2 T14 and Y15 phosphorylation. a–d, All strainscarried deletions of the endogenous copies of cdc2, cdc13, cig1, cig2 and puc1.a, b, Labels are indicated in box. a, Blankophor staining of exponentiallygrowing cells. Scale bar, 10mm. b, DNA content analysis. The percentages of 1Ccells are indicated. c, Distribution of cell size at division in exponentiallygrowing cultures presented as a percentage of the median size (n $ 150). d, G2-arrested cells (1mM NmPP1 for 3 h 30 min at 32 uC) were released and cell sizeat division determined at the peak of binucleated cells for the following threecycles (n $ 50; Supplementary Fig. 20). Box and whisker plot. Async,asynchronous cultures.

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23. Benito, J., Martın-Castellanos, C. & Moreno, S. Regulation of the G1 phase of the cellcycle by periodic stabilization and degradation of the p25rum1 CDK inhibitor.EMBO J. 17, 482–497 (1998).

24. Forsburg, S. L. & Nurse, P. Identification of a G1-type cyclin puc11 in the fissionyeast Schizosaccharomyces pombe. Nature 351, 245–248 (1991).

25. Martın-Castellanos, C., Blanco, M. A., de Prada, J. M. & Moreno, S. The puc1 cyclinregulates the G1 phase of the fission yeast cell cycle in response to cell size. Mol.Biol. Cell 11, 543–554 (2000).

26. Broek, D., Bartlett, R., Crawford, K. & Nurse, P. Involvement of p34cdc2 inestablishing the dependency of S phase on mitosis. Nature 349, 388–393 (1991).

27. Hayles, J., Fisher,D., Woollard,A.&Nurse, P. Temporal order ofSphaseandmitosisin fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex.Cell 78, 813–822 (1994).

28. Bishop, A. C. et al. A chemical switch for inhibitor-sensitive alleles of any proteinkinase. Nature 407, 395–401 (2000).

29. Dischinger, S., Krapp, A., Xie, L., Paulson, J. R. & Simanis, V. Chemical geneticanalysis of the regulatory role of Cdc2p in the S. pombe septation initiationnetwork. J. Cell Sci. 121, 843–853 (2008).

30. Decottignies, A., Zarzov, P. & Nurse, P. In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. J. CellSci. 114, 2627–2640 (2001).

31. Buck, V., Russell, P. & Millar, J. B. Identification of a cdk-activating kinase in fissionyeast. EMBO J. 14, 6173–6183 (1995).

32. Tanaka, K. & Okayama, H. A pcl-like cyclin activates the Res2p-Cdc10p cell cycle‘‘start’’ transcriptional factor complex in fission yeast. Mol. Biol. Cell 11,2845–2862 (2000).

33. Watt, S. et al. urg1: a uracil-regulatable promoter system for fission yeast with shortinduction and repression times. PLoS ONE 3, e1428 (2008).

34. Stern, B. & Nurse, P. A quantitative model for the cdc2 control of S phase andmitosis in fission yeast. Trends Genet. 12, 345–350 (1996).

35. Murray, A. W. Recycling the cell cycle: cyclins revisited. Cell 116, 221–234 (2004).36. Moreno, S. & Nurse, P. Regulation of progression through the G1 phase of the cell

cycle by the rum11 gene. Nature 367, 236–242 (1994).37. Snaith, H. A. & Forsburg, S. L. Rereplication phenomenon in fission yeast requires

MCM proteins and other S phase genes. Genetics 152, 839–851 (1999).38. Correa-Bordes, J. & Nurse, P. p25rum1 orders S phase and mitosis by acting as an

inhibitor of the p34cdc2 mitotic kinase. Cell 83, 1001–1009 (1995).39. Russell, P. & Nurse, P. cdc251 functions as an inducer in the mitotic control of

fission yeast. Cell 45, 145–153 (1986).40. Russell, P. & Nurse, P. Negative regulation of mitosis by wee11, a gene encoding a

protein kinase homolog. Cell 49, 559–567 (1987).

41. Gould, K. L. & Nurse, P. Tyrosine phosphorylation of the fission yeast cdc21 proteinkinase regulates entry into mitosis. Nature 342, 39–45 (1989).

42. Lundgren, K. et al. mik1 and wee1 cooperate in the inhibitory tyrosinephosphorylation of cdc2. Cell 64, 1111–1122 (1991).

43. Fantes, P. A. & Nurse, P. Control of the timing of cell division in fission yeast.Cell size mutants reveal a second control pathway. Exp. Cell Res. 115, 317–329(1978).

44. Rhind, N., Furnari, B. & Russell, P. Cdc2 tyrosine phosphorylation is required forthe DNA damage checkpoint in fission yeast. Genes Dev. 11, 504–511 (1997).

45. Rhind, N. & Russell, P. Tyrosine phosphorylation of cdc2 is required for thereplication checkpoint in Schizosaccharomyces pombe. Mol. Cell. Biol. 18,3782–3787 (1998).

46. Fantes, P. & Nurse, P. Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division. Exp. Cell Res. 107, 377–386 (1977).

47. Petersen, J. & Nurse, P. TOR signalling regulates mitotic commitment through thestress MAP kinase pathway and the Polo and Cdc2 kinases. Nature Cell Biol. 9,1263–1272 (2007).

48. Den Haese, G. J., Walworth, N., Carr, A. M. & Gould, K. L. The Wee1 protein kinaseregulatesT14phosphorylationoffissionyeastCdc2.Mol.Biol.Cell6,371–385(1995).

49. Martin, S. G. & Berthelot-Grosjean, M. Polar gradients of the DYRK-family kinasePom1 couple cell length with the cell cycle. Nature 459, 852–856 (2009).

50. Moseley, J. B., Mayeux, A., Paoletti, A. & Nurse, P. A spatial gradient coordinates cellsize and mitotic entry in fission yeast. Nature 459, 857–860 (2009).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank J. Hayles, P.-Y. Wu and F. Navarro for critically readingthe manuscript, and N. Rhind for the anti-Cds1 antibody. D.C. was supported bypost-doctoral fellowships from EMBO (ALTF 899-2007) and the Human FrontierScience Program (LT00623/2008) and P.N. by the Breast Cancer ResearchFoundation, The Rockefeller University and the Anderson Cancer Center Research atRockefeller University.

Author Contributions D.C. designed and performed the experiments and wrote themanuscript. Both authors discussed the experiments and edited the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should beaddressed to D.C. ([email protected]).

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METHODSStrains and growth conditions. Standard media and methods were used51,52.Strains used in this study are listed in Supplementary Table 1. All experimentswere carried out in minimal medium plus supplements (EMM4S) at 32 uC exceptwhere otherwise noted. The CDK module is a fusion of cdc13 and cdc2 openreading frames without introns. The fusion cassettes were cloned in a vectoradjacent to the ura41 cassette; flanking regions allowed a restriction fragmentto replace the leu1 gene by homologous recombination. The NmPP1 inhibitor(A603003; TRC) was dissolved in DMSO at a stock concentration of 10 mM andadded to the cultures at the indicated concentrations. The first 67 amino-terminalresidues of Cdc13 were deleted in the Cdc13DDB-L-Cdc2as protein53. Expressionof the cdc13DDB-L-cdc2as cassette was induced by addition of 250 mg l21 uracil tothe medium. For hydroxyurea treatments in Fig. 4 and Supplementary Figs 14and 15, hydroxyurea was added 10 min before release from the inhibitor. Thealteration in cdc13-117ts cells (Cdc13(C379Y)) was determined by sequencing.The cig1D::ura41, cig2D::ura41, puc1D::ura41, mik1D::leu21 deletions and thecdc2-33ts, wee1-50ts and cdc25-22ts alleles have been previously described14,21,54–58.The rad3D::ura1 deletion was a gift from R. Daga. The cdc2D::kanMX6,cdc13D::natMX6, rum1D::hphMX6 and cig2D::natMX6 deletions were exactreplacements of the open reading frames as described59,60.Cell size measurement and DNA staining. For size measurement, live cells werestained with Blankophor (MP Biochemicals). For DNA staining, cells were eitherheat-fixed on microscope slides and stained with DAPI (with 1:4 blankophorwhere indicated) or stained live with 50mg ml21 Hoechst DNA stain. Images wereacquired in Metamorph (MDS Analytical Technologies) using an Axioplan 2(Carl Zeiss) epifluorescence microscope and a CoolSNAP HQ camera (RoperScientific). Cell size was determined in ImageJ (National Institutes of Health)using the Pointpicker plug-in.Protein extracts and western blots. Western blots were performed on totalprotein extracts. Protein extracts in Fig. 1 and Supplementary Figs 6, 16 and 17were prepared using NaOH extraction61. In all other cases, cells were frozen inliquid nitrogen, broken with glass beads in the presence of protease and phos-phatase inhibitors (Roche) and resuspended in SDS buffer. Samples were normalizedby amounts of proteins except where otherwise noted. Antibodies used: Cdc13polyclonal (SP4 (ref. 20); 1:2,500), Cds1 polyclonal (1:3,000, a gift from N.Rhind62), phospho-Cdc2(Y15) polyclonal (Cell Signaling; 1:300) and tubulin mono-clonal (1:10,000, a gift from K. Gull63). Activation of the S-phase checkpoint wasmonitored by the phosphorylation-dependent shift in Cds1 mobility64 by 8% SDS–polyacrylamide gel electrophoresis.Flow cytometry profile interpretation. DNA content analysis was performed byflow cytometry using ethanol-fixed and propidium-iodide-stained cells (2 mgml21 propidium iodide in 50 mM sodium citrate) and a BD FACSCalibur. Thefission yeast cell cycle has a very short G1, and cells undergo DNA replicationbefore cytokinesis. As a result, fission yeast cells spend most of their cell cycle witha 2C DNA content. In synchronized cultures, a transient 4C peak appears as Sphase occurs in post-mitotic binucleated cells. This is resolved upon cytokinesis,producing mononucleated 2C cells. This phase only represents a small fraction ofthe population in an asynchronous culture; a larger 4C peak corresponds either tobinucleated cells that have completed S phase but show a cytokinesis defect or tomononucleated G2 cells that have undergone an additional round of DNA rep-lication without intervening mitosis. The appearance of a 1C peak reflects anelongation of G1 resulting in cytokinesis taking place before completion of DNAreplication. Intermediate, non-discrete profiles occur when cells divide althoughmitosis is not complete, resulting in the septum cutting through the DNA massand subsequent aberrant distribution of the DNA; this is referred to as ‘cut’ cells65.Finally, cell size has an effect on the position of the flow cytometry profiles as non-nuclear staining increases with size: profiles are shifted to the right in long cellsand to the left in newly divided cells, despite identical DNA contents66.Nuclear YFP quantification. Cells were imaged on agar pads under a coverslipand Z stacks were acquired using a DeltaVision RT microscope (AppliedPrecision). Quantification was performed on maximum projections usingImageJ (National Institutes of Health) as follows. Fluorescence intensity of equi-valent areas within the nucleus (N), the cytoplasm (C) and outside (B) of eachcdc13-L-cdc2-YFP D2D13DCCP cell was measured (strongly stained structuressuch as the spindle or spindle pole body were excluded) as well as cell size. Similarmeasurements were performed in cdc13-L-cdc2 D2D13DCCP cells as a control. C

was not significantly different than in the control cells and therefore was used as anormalization value. Using the cdc13-L-cdc2 D2D13DCCP cells, we estimated theaverage auto-fluorescence (A) in the nucleus as a constant percentage of B. Thevalue reflecting nuclear fluorescence for each cell was calculated as[N 2 B 2 (A 3 B)] / C. For binucleated and septated cells, N was calculated asthe average fluorescence of both nuclei. Values were then sorted by cell size. Therare cells showing a negative value were considered as negative for the YFP signal.Total YFP quantification by flow cytometry. The fluorescence (FL1-H) ofcdc13-L-cdc2-YFP D2D13DCCP and cdc13-L-cdc2 D2D13DCCP cells in minimalmedium at 25 uC was measured as a function of size (FSC-H) by flow cytometryusing a BD FACSCalibur. YFP measurements were averaged in size bins of 250cells. The equation of the linear regression obtained in the control strain was usedto subtract the auto-fluorescence background from the cdc13-L-cdc2-YFPD2D13DCCP measurements.Percentage of dead cells in liquid cultures. Liquid cultures were exponentiallygrown for 36 h at the appropriate temperatures in the presence of 25 mg l21 of thedye phloxin B (phloxin B stains dead cells in pink). The percentage of dead cellswas estimated by microscopy.Nitrogen starvation. Cells were exponentially grown at 28 uC in minimal mediumwith supplements, washed with water and inoculated at approximately 1.5 3 106

cells ml21 into non-supplemented minimal medium without nitrogen at 28 uC.EdU incorporation and detection. cdc13-L-cdc2as D2D13DCCP cells expressingthe human ENT1 transporter and herpes simplex virus thymidine kinase67 wereincubated with 2mM EdU (Invitrogen) for 5 min and fixed in 3.7% formaldehyde.After permeabilization (A. Kaykov, manuscript in preparation), EdU detectionwas performed according to manufacturer’s instructions (Invitrogen, Click-iTEdU Alexa Fluor 594 Imaging Kit) and imaged in Metamorph (MDS AnalyticalTechnologies) using an Axioplan 2 (Carl Zeiss) epifluorescence microscope and aCoolSNAP HQ camera (Roper Scientific).

51. Hayles, J. & Nurse, P. Genetics of the fission yeast Schizosaccharomyces pombe.Annu. Rev. Genet. 26, 373–402 (1992).

52. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeastSchizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

53. Yamano,H., Gannon, J. & Hunt, T. The role of proteolysis in cell cycle progression inSchizosaccharomyces pombe. EMBO J. 15, 5268–5279 (1996).

54. Obara-Ishihara, T. & Okayama, H. A B-type cyclin negatively regulates conjugationvia interacting with cell cycle ‘start’ genes in fission yeast. EMBO J. 13, 1863–1872(1994).

55. Forsburg,S. L.& Nurse, P.Analysis of theSchizosaccharomyces pombe cyclinpuc1:evidence for a role in cell cycle exit. J. Cell Sci. 107, 601–613 (1994).

56. Zarzov, P., Decottignies,A., Baldacci, G.&Nurse, P.G1/SCDK is inhibited to restrainmitotic onset when DNA replication is blocked in fission yeast. EMBO J. 21,3370–3376 (2002).

57. Nurse, P. Genetic control of cell size at cell division in yeast. Nature 256, 547–551(1975).

58. Thuriaux, P., Sipiczki, M. & Fantes, P. A. Genetical analysis of a sterile mutant byprotoplast fusion in the fissionyeastSchizosaccharomycespombe. J.Gen.Microbiol.116, 525–528 (1980).

59. Bahler, J. et al. Heterologous modules for efficient and versatile PCR-based genetargeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).

60. Hentges, P., Van Driessche, B., Tafforeau, L., Vandenhaute, J. & Carr, A. M. Threenovel antibiotic marker cassettes for gene disruption and marker switching inSchizosaccharomyces pombe. Yeast 22, 1013–1019 (2005).

61. Matsuo, Y., Asakawa, K., Toda, T. & Katayama, S. A rapid method for proteinextraction from fission yeast. Biosci. Biotechnol. Biochem. 70, 1992–1994 (2006).

62. Bhaumik, D. & Wang, T. S. Mutational effect of fission yeast pola on cell cycleevents. Mol. Biol. Cell 9, 2107–2123 (1998).

63. Woods, A. et al. Definition of individual components within the cytoskeleton ofTrypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93, 491–500(1989).

64. Lindsay, H. D. et al. S-phase-specific activation of Cds1 kinase defines asubpathway of the checkpoint response in Schizosaccharomyces pombe. GenesDev. 12, 382–395 (1998).

65. Uemura, T. & Yanagida, M. Isolation of type I and II DNA topoisomerase mutantsfrom fission yeast: single and double mutants show different phenotypes in cellgrowth and chromatin organization. EMBO J. 3, 1737–1744 (1984).

66. Sazer, S. & Sherwood, S. W. Mitochondrial growth and DNA synthesis occur in theabsenceofnuclearDNAreplication in fissionyeast. J. Cell Sci.97, 509–516 (1990).

67. Sivakumar, S., Porter-Goff, M., Patel, P. K., Benoit, K. & Rhind, N. In vivo labeling offission yeast DNA with thymidine and thymidine analogs. Methods 33, 213–219(2004).

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