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TRENDS in Genetics Vol.18 No.9 September 2002

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479Review

Ivan Rupeš

Dept of Biology, Queen’sUniversity, Kingston,Ontario, Canada K7L 3N6.e-mail: [email protected]

How does a cell control its size? Two schools ofthought exist: either a cell divides after it reaches acertain critical size, or cell growth and proliferationare regulated independently, with cell size emergingfrom a simple correlation of the two [1–5]. The mostimportant difference between the two hypotheses isthat cell size feeds back into the cell-cycle regulatorysystem in the former but not in the latter (Fig. 1a).Thus, the validity of the critical size theory dependson the existence of a ‘sizer’– a molecule or set ofmolecules whose activity correlates with cell size.Nevertheless, the sizer is only one component thatdetermines when cell division occurs; in addition, the extracellular environment influences the timingof the response to the changing activity of the sizer.

Two species of yeast, the budding yeastSaccharomyces cerevisiae and the fission yeastSchizosaccharomyces pombe, provide genetic modelsin which to study cell-cycle control. The evolutionarydivergence of these yeast is about the same as thatbetween each of them and human [6]. Both yeastshare cell-cycle characteristics with highereukaryotes, such as G1, S, G2 and M phases, cyclin-dependent kinases (CDKs) and checkpoint controls[7–9]. Owing to the uncertain supply of nutrients inthe wild, the yeast cell-division rate must becoordinated with widely variable rates of cell growth,otherwise cells would get progressively smaller orlarger. Although cell size is reduced in less favorable

growth conditions, the range of growth rates exceedsthe corresponding range of cell sizes for both yeast.Thus, a relationship between growth and division is afact of life for yeast cells. The early studies havepresented a compelling case for the existence of acritical size that is a prerequisite for progressionthrough the cell cycle. In budding yeast, cell divisionis asymmetrical and produces cells of unequal size. To compensate for this asymmetry, which becomesmore pronounced with increasing nutrient limitation,new daughter cells grow more before division than themother cells. This additional growth occurs almostentirely in G1, before the reference point known asStart. Once Start is passed, the rest of the cell cycle isrelatively constant in length [10] (Fig. 1b).

In fission yeast, G2–M is the primary cell-sizecontrol point [9]. The relative length of G2 variesgreatly with growth conditions (Fig. 1b). Nitrogenlimitation reduces cell size at division, and suddenshifts between different sources of nitrogen generaterapid acceleration or delay of mitosis in cells that areabove or below the new cell-size threshold (i.e. theminimum size required for initiation of mitosis) [9].Even during balanced growth, individual fission yeastcells can compensate for random fluctuations in theirsize at birth by adjusting their time spent in G2 [11].

Do fission yeast have a size control in G1, and dobudding yeast have a control in G2? Start is defined in fission yeast as in budding yeast, although infavorable growth conditions it occurs almostimmediately after exit from mitosis. A normallycryptic size-control point at Start is uncovered inmutants in which the G2–M size-control hascollapsed and cells enter mitosis prematurely. These cells have an extended G1, suggesting thatthey initiate S phase only after reaching a certainminimum size [12] (Fig. 1b). Although the two yeasthave traditionally been thought of as using differentsize-control strategies, the aim of this article is toshow that they use similar mechanisms. What isdifferent is their emphasis on the size-control points.Because these control mechanisms have beenconserved over such long evolutionary distances,

To remain viable, cells have to coordinate cell growth with cell division. In yeast,

this occurs at two control points: the boundaries between G1 and S phases,

also known as Start, and between G2 and M phases. Theoretically, coordination

can be achieved by independent regulation of growth and division, or by

participation of surveillance mechanisms in which cell size feeds back into

cell-cycle control. This article discusses recent advances in the identification of

sizing mechanisms in budding and in fission yeast, and how these mechanisms

integrate with environmental stimuli. A comparison of the G1–S and G2–M

size-control modules in the two species reveals a degree of conservation higher

than previously thought. This reinforces the notion that internal sizing could be

a conserved feature of cell-cycle control throughout eukaryotes.

Published online: 25 July 2002

Checking cell size in yeast

Ivan Rupeš

47 Zhaxybayeva, O. and Gogarten, J.P. (2002)Bootstrap, Bayesian probability and maximumlikelihood mapping: exploring new tools for comparative genome analyses. BMC Genomics 3, 4

48 Page, R.D. (2000) Extracting species trees fromcomplex gene trees: reconciled trees and vertebratephylogeny. Mol. Phylogenet. Evol. 14, 89–106

49 Bapteste, E. et al. (2002) The analysis of 100 genessupports the grouping of three highly divergentamoebae: Dictyostelium, Entamoeba, andMastigamoeba. Proc. Natl. Acad. Sci. U. S. A.99, 1414–1419

50 Woese, C.R. (2000) Interpreting the universalphylogenetic tree. Proc. Natl. Acad. Sci. U. S. A.97, 8392–8396

51 Burggraf, S. et al. (1991) Methanopyrus kandleri:an archaeal methanogen unrelated to all otherknown methanogens. Syst. Appl. Microbiol.14, 346–351

52 Slesarev, A.I. et al. (2002) The complete genome ofhyperthermophile Methanopyrus kandleriAV19and monophyly of archaeal methanogens.Proc. Natl. Acad. Sci. U. S. A. 99, 4644–4649

it might be worth considering whether analogoussystems operate in other eukaryotes.

Size control at Start: budding yeast

How does budding yeast measure its size, and howdoes this control Start? The G1–S size-control moduleis presented in Fig. 2. Its core consists of threeG1 cyclins (Cln1–3) and two B-type cyclins (Clb5 andClb6). These cyclins bind to a CDK1 homolog,Cdc28 (Table 1), and target its activity towards

specific sets of substrates. The level of Cln3 isimportant for timely expression of the CLN1 andCLN2 genes. The resulting increase in the activity ofCln1– and Cln2–Cdc28 complexes is responsible forinitiation of three major post-Start events: budding,spindle-pole duplication and, indirectly through


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TRENDS in Genetics

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Fig. 1. External conditions and internal sizing mechanismsco-determine cell size at division. (a) Integration map of the cell-sizecontrol modules. Cell size is a result of correlation between the rates ofcell-cycle progression (proliferation) and growth, but four differentgeneral processes modulate the two rates. (1) Cell-cycle machinerymonitors external conditions or activation of metabolic pathways.(2) Another class of mechanisms sets the growth rate depending onexternal conditions. (3) Growth rate can directly modulate cell-cycleprogression. (4) Cell size feeds back into cell-cycle control through aspecific size-surveillance mechanism (sizer). (b) Durations of individualcell-cycle phases in budding and fission yeast. Budding yeast: inwild-type conditions, smaller daughter cells undergo substantialgrowth before G1–S (Start), whereas larger mother cells rapidly initiateS phase. G1 is drastically reduced when Cln3 is overexpressed,resembling the wild-type cycle of fission yeast, whereas the S phaseremains virtually unchanged. G1 becomes relatively extended in poorgrowth conditions, and S, G2 and M are not affected. Fission yeast: cells initiate S phase rapidly after the completion of mitosis. Cellsdefective in wee1 enter mitosis prematurely. Their G1, however,becomes extended but their S phase remains unchanged, resemblingthe proportions in wild-type budding yeast. In poor conditions, G1 also becomes relatively extended in fission yeast.

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Fig. 2. The G1–S cell-size control module in budding yeast. A simplifiedminimum core consists of three G1 cyclins, Cln1–3, two B-type cyclins,Clb5 and Clb6, and a cyclin-dependent kinase (CDK) inhibitor Sic1. All the cyclins function in association with a CDK1 homolog, Cdc28.Cln3 is placed at the top of the cascade. In a dose-dependent manner,Cln3 induces expression of CLN1 and CLN2 through activation of atranscription activator complex that comprises two subunits: Swi6 andSwi4. This results in a sharp spike of Cln1– and Cln2–Cdc28 activity thatinitiates post-Start events, including budding and pre-mitoticspindle-pole duplication. The instability of Cln1 and Cln2 is increasedthrough the action of Grr1 (a protein that promotes degradation of Cln1and Cln2) in response to glucose. In addition, cAMP represses CLN1transcription. Cln1– and Cln2–Cdc28 phosphorylate and destabilizeSic1, a potent inhibitor of Clb5– and Clb6–Cdc28. Transcription of CLB5and CLB6 is also cell-cycle regulated through a complex consisting ofSwi6 and a Swi4 homolog, Mbp1. This regulation is not rate-limiting forStart. Activation of Clb5– and Clb6–Cdc28 leads to initiation of DNAreplication. Mitotic cyclins are also present in G1 but they are rapidlydegraded through the action of Cdh1, a homolog of Ste9 from fissionyeast. In contrast to Cdc13 in fission yeast, the budding yeast mitoticcyclins are unable to initiate DNA replication efficiently, and aretherefore omitted from this diagram. For simplicity, regulation of theindividual components at the transcriptional, post-transcriptional anddegradation levels is not distinguished in this diagram. Coloringreflects structural relationships as indicated.

release of the inhibition of Clb5– and Clb6–Cdc28 bythe CDK inhibitor Sic1, DNA replication [8,13].

Cln3 functions to coordinate Start with cell growthand the supply of nutrients. The level of Cln3 variesdramatically with the available carbon [14] or nitrogen[15] source. The glucose-response elements in theCLN3 promoter are a target for Azt1, a transcriptionfactor that stimulates CLN3 transcription in thepresence of glucose [16,17]. Cln3, therefore, isassociated with process 1 in Fig. 1a. CLN3 mRNAcontains a long 5′-untranslated region (UTR) thatharbors an upstream open reading frame (ORF) whosefunction is to slow down initiation of CLN3 translationwhen fewer ribosomes are available in the cytoplasm.Because the concentration of ribosomes correlates withgrowth rate, this mechanism provides a link betweengrowth rate and the rate of Cln3 synthesis [18]. Thus, Cln3 is also associated with process 3 in Fig. 1a.The translation efficiency of CLN3 is further controlledby the activity of the eukaryotic initiation factor (eIF)4F mRNA-cap-binding complex. This complex appearsto be a target for the target of rapamycin (TOR)phosphatidylinositol kinase pathway [19,20] andperhaps also for the cAMP-dependent protein kinasepathway [14]. Both these pathways are involved innutritional sensing and affect several metabolic andgrowth-related functions, thus coordinating processes1 and 2 (Fig. 1a). Finally, the Whi3 protein restrictsCln3 synthesis by localizing CLN3 mRNA to specificcytoplasmic loci with a possible function in nutritionalsensing (Ref. [21] and B. Futcher, pers. commun.).

Is Cln3 a sizer? Cln3 is a low-abundance,constitutively unstable protein [22,23] and, therefore,its level depends primarily on its rate of synthesis.According to one model [13], the rate of Cln3synthesis per cell increases with the volume ofcytoplasm. Because Cln3 localizes to the nucleus[24,25], assuming that nuclear volume depends onthe amount of DNA present, the effectiveconcentration of Cln3 in G1 rises with increasing cellsize. When a threshold level is reached, which takeslonger in smaller daughter cells than in mother cells,the cascade leading to Start is initiated [13]. Theshort answer to the above question is, therefore, yes,Cln3 is most likely a sizer. There is some uncertainty,however, about how exactly Cln3 might do the sizing.For one, it is not clear how a steady increase in Cln3levels in G1 could trigger the sharp rise in the levels ofCln1 and Cln2 with little apparent contribution froma positive amplification loop [22–27]. One couldimagine that the transcription factor complexSwi4–Swi6 (Fig. 2) operates as an ultra-sensitiveswitch that produces a steep activation curve once theCln3–Cdc28 level crosses the threshold value [28]. If so, one would expect excessive variation in cell sizecaused by minor random fluctuations of the Cln3levels in individual cells; this is not the case. The levelof CLN3 transcription oscillates modestly during thecell cycle, peaking in late M or G1 [29,30]. Recently,new evidence suggested that, in cooperation

with SWI4, this modest periodicity might, after all,contribute to more robust timing of Start. Theupstream-activating sequences of CLN3 and SWI4contain early cell-cycle box (ECB) elements that controlthe periodicity of the respective transcripts [29,30].Cells lacking functional ECBs in both CLN3 andSWI4 also lack the sharp peaks of CLN2transcription. They also exhibit additive delay andincreased size heterogeneity at Start [30], anindication that progression through Start has turnedinto a stochastic process. Importantly, the peaks ofCLN3 and SWI4 transcription seem to occur later indaughter cells than in mother cells, suggesting thattheir timing might be linked to cell size [30]. However,it is not yet clear whether the timing of the peaks iscrucial or whether the cln3ecb swi4ecb double-mutantcells delay Start simply because they fail to producesufficient levels of Cln3 and Swi4.

If cells can sense their size, at least in part,through Cln3, what sets the critical threshold of itsactivity? Clearly, the threshold varies in differentgrowth conditions. Part of the problem is that theexact link between Cln3 and Swi6–Swi4 is not known [31,32]. What is known, however, is that bothsynthesis and degradation of Cln1 and Cln2 aremodulated by several other pathways. Somewhatcounter-intuitively, the burst of CLN1 and CLN2transcription is delayed in rich media, resulting in alarger size at Start. This response is, at least in part,mediated by cAMP [33,34]. Addition of glucose orcAMP represses transcription of CLN1, and this issufficient to delay Start [35]. In addition, Grr1, a protein that promotes degradation of Cln1 and Cln2,is activated in response to glucose [36,37]. This further reduces the levels of Cln1 and Cln2 in cells.This type of regulation allows the response of Cln1and Cln2 to be sensitized or desensitized to Cln3 activity. Cln1 and Cln2 thus make a specificcontribution to the integration of environmentalsignals within the G1–S module (Fig. 1a, process 1)and, therefore, to the setting of a critical size.

Size control at Start: fission yeast

In fission yeast, Start is initiated by a CDK1 homolog,Cdc2, in association with a G1 cyclin, Puc1, and threeB-type cyclins, Cig1, Cig2 and Cdc13. Two inhibitorymechanisms prevent premature initiation of S phase:one involves a CDK inhibitor, Rum1, and the otherinvolves a component of the ubiquitin proteolysismachinery, Ste9. The wiring diagram reveals strikingsimilarity between fission yeast and budding yeast(Fig. 3, Table 1) [9]. As already mentioned, the fissionyeast G1–S size control is cryptic when cells grow infavorable conditions. Under limiting conditions,however, G1 is extended to a varying degree, with nosimple correlation to G2–M regulation. This suggeststhat, as in budding yeast, there might be a minimumsize requirement for progression through Start thatvaries with external conditions [38]. How cell sizeimpinges on the G1–S module in fission yeast is still



481Review

largely a mystery. Puc1 alone is insufficient to initiateS phase, indicating that it functions upstream of theB-cyclins. Puc1–Cdc2 phosphorylates anddestabilizes Rum1, an inhibitor of Cdc13– andCig2–Cdc2, paralleling the action of Cln1– andCln2–Cdc28 on Sic1 in budding yeast [39]. Similar toCLN3 mRNA, puc1 mRNA contains a long 5′-UTRand upstream ORFs, suggesting tight regulation atthe translational level [40]. These properties and thelow abundance and high instability of puc1 mRNAportray Puc1 as a counterpart of Cln3 in fission yeast.However, in contrast to Cln3, Puc1 seems completelydispensable for the timing of Start in rich medium. By contrast, in poor nitrogen sources, Puc1 becomesrate-limiting for Start [39]. Puc1 might thus functionas a G1–S sizer in fission yeast, but this function is

probably shared with additional components. Anequivalent of Cln1 and Cln2 is conspicuously missingdownstream of Puc1, but so is the extra requirementto coordinate budding with the initiation of S phasefor which Cln1 and Cln2 are responsible. Puc1 adoptsthe role of Cln1 and Cln2 in relieving the Rum1 (Sic1)-mediated CDK inhibition, but there appears to be nointermediate Cdc2-dependent transcription step inthis cascade [41], and Cig2 activity occurs in a sharpspike that is reminiscent of those of Cln1 and Cln2[42,43]. The two Clns participate in the modulation ofcritical size in response to the environment.One might, therefore, expect a similar function forCig2. Indeed, cig2 mRNA contains long UTRs andtranslational regulation reduces the levels of Cig2 inresponse to nitrogen limitation (Fig. 1a, process 1),delaying progression through Start [44]. Althoughpuc1 mRNA is induced by nitrogen starvation [40],the protein levels do not change dramatically(S. Moreno, pers. commun.). Even so, with the relativedrop in Cig2 levels, this could contribute to theincreased impact of puc1 deletion on cell size at Startthat is observed under nutrient limitation [39].

Size control at G2–M: fission yeast

The wee1 gene was isolated as the prototype cell-sizemutant that enters mitosis precociously and loses thewild-type ability to modify cell size at mitosis inresponse to the availability of nitrogen [9]. As cellsprogress through S and G2, Cdc13 slowlyaccumulates, but the associated Cdc2 activity remainsinhibited by Wee1-dependent tyrosinephosphorylation (Fig. 4a). A Wee1 inhibitory kinase,Cdr1, is probably a component of the nutrition-sensingmodule (Fig. 1a, process 1) as cdr1-deficient mutantsare unable to adjust cell size over a range of nitrogenconcentrations [9,45]. This property is shared with arelated kinase, Cdr2, which also phosphorylates Wee1in vitro [45–47]. The exact relationship between Cdr1and Cdr2 and their possible upstream regulators isnot known. Cdr1 localizes predominantly to thecytoplasm, whereas Wee1 is present mainly in thenucleus. Controlled accessibility of the two partners,therefore, is one possible mechanism by which Cdr1could exert control over the setting of cell size [48].Independent of Cdr1 and Cdr2, Wee1 is upregulatedby inhibition of protein synthesis [49].

To initiate mitosis, cells must reverse Cdc2tyrosine phosphorylation. The essential phosphatasefor this is Cdc25 [9] (Fig. 4a). Switch-like activation ofCdc13–Cdc2 is ensured by a positive feedback loopbetween Cdc2 and Cdc25 [50] and probably also by anegative loop between Cdc2 and Wee1, the latter ofwhich is related to the Cdc2–Wee1 loop identified inother eukaryotes [51]. The level of Cdc25, as well asthat of Cdc13, is correlated with general syntheticactivity through translational regulation involvingthe 5′-UTRs of the cdc25 and cdc13 mRNAs,providing a potential link between growth rate andtiming of mitosis [52] (Fig. 1a, process 3).



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Fig. 3. The G1–S cell-size control module in fission yeast. In thesimplified version, there is one G1 cyclin homolog, Puc1, and threeB-type cyclins, Cig1, Cig2 and Cdc13, active in G1–S in association witha cyclin-dependent kinase (CDK)-1 homolog, Cdc2. Two inhibitoryactivities are crucial for preventing premature initiation of S phase: a CDK inhibitor, Rum1 (a homolog of Sic1 in budding yeast), and Ste9, a protein involved in targeting its substrates for degradation. Puc1 functions upstream of the B-cyclin–Cdc2 complexes. In G1, Rum1inhibits Cdc13–Cdc2 and, to a lesser extent, Cig2–Cdc2. In addition, the levels of Cdc13 and Cig1 are kept low by the action of Ste9. At Start,Puc1 and Cig1 phosphorylate and destabilize Rum1, possibly withincreasing contribution from Cig1 as the level of Ste9 decreases. Cdc13phosphorylates Ste9, resulting in Ste9 degradation. This creates anegative loop that contributes to gradual accumulation of Cdc13 duringS phase. Transcription of cig2 is induced by a heteromeric complex thatcontains a Swi6 homolog, Cdc10, and two Swi4/Mbp1 homologs, Res1 and Res2. This transcription is not rate-limiting for Start but ispresent in this diagram as a feature conserved between the two yeast.Coloring reflects structural relationships as indicated.

So, which components qualify for thesize-dependent process (Fig. 1a, process 4)? Onepossibility is that gradual accumulation of Cdc13,which destabilizes the ‘off ’ state of the switch,represents a sizing mechanism [53,54]. However,there seems to be little experimental evidencesupporting this because accumulation of Cdc13 in thenucleus appears to level off long before cells entermitosis [55]. The same seems to be true for thepre-mitotic nuclear accumulation of Cdc25 [56]. Wee1 is also an unlikely candidate as Cdc2-tyrosinephosphorylation persists even after Wee1 and arelated kinase, Mik1, have been inactivated in aconditional mutant [57,58]. That leaves us with a direct activation of the Cdc25–Cdc2 loop. Indeed, the sizing in G2–M has recently been shown to bemediated by Cdc25. Cells that have stopped growingbecause of interference with the actin cytoskeletonfail to initiate mitosis if their size is below thethreshold, but do progress into mitosis unperturbed ifthey are oversized [58]. The inhibition is lost in cellsin which Cdc25 has been replaced with a constitutiveCdc2-tyrosine phosphatase activity. Conversely, cells in which the critical size has been reduced by anutritional down-shift accelerate their entry intomitosis even if their further growth has beeninterrupted by actin depolymerization. These cellssuddenly became oversized relative to the new sizethreshold [58]. The size-related upstream regulatorsof Cdc25 (or Cdc13–Cdc2, as the presence of thepositive loop makes it difficult to distinguish betweendirect activation of Cdc13–Cdc2 and Cdc25) remain tobe identified. However, these results demonstratethat cell size impinges on cell-cycle regulation as acell-cycle checkpoint. The identification of thecheckpoint has an important consequence: it justifiesthe integration model of cell-size control by closingthe loop between cell size and cell-cycle progression(Fig. 1a, process 4). It shows that the activity of thesizer does not simply coincide with cell growth but is

directly determined by cell size. The checkpointfunction cannot be executed without previous Cdc2-tyrosine phosphorylation. Consistently, wee1-deficientmutants are unable to control their size in G2–M.Furthermore, simultaneous collapse of the G1–Smodule in the wee1 background, owing to deletion ofrum1 or ste9, results in a loss of viability [59–61].



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Table 1. Conserved components of the G1–S and G2–M size-control modulesa,b

Budding yeast Fission yeast Protein function

Cln1, Cln2, Cln3 Puc1 G1 cyclinClb1, Clb2, Clb5, Clb6 Cig1, Cig2, Cdc13 B-type cyclinCdc28 Cdc2 CDK1Sic1 Rum1 CDK inhibitorCdh1 Ste9 APC activatorSwi6 Cdc10 Transcription factorSwi4, Mbp1 Res1, Res2 Transcription factorSwe1 Wee1 Protein tyrosine kinaseMih1 Cdc25 Protein tyrosine phosphataseHsl1, Kcc4, Gin4 Cdr1, Cdr2 Protein kinase

aAbbreviations: APC, anaphase-promoting complex (part of the ubiquitin proteolytic machinery);CDK, cyclin-dependent kinase.bCdc (cell-division cycle) genes were first identified as mutants that cause cell-cycle arrest inbudding and fission yeast. The conserved components display a significant degree of structuralhomology and, mostly, they also have conserved interacting partners, upstream regulators ortargets, suggesting that they constitute conserved regulatory modules. The context in whichthese modules operate, however, can differ in the two yeast, sometimes leading to differentphenotypic manifestations in the respective mutants.

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Fig. 4. The G2–M size-control module. (a) Fission yeast: the essentialB-cyclin–CDK (cyclin-dependent kinase) complex, Cdc13–Cdc2, is inhibited by phosphorylation of Tyr15. The phosphorylation ismediated by Wee1 and reversed by Cdc25. Two feedback loops areengaged, resulting in a switch-like activation of Cdc2 kinase. Cdr1 andCdr2 are upstream regulators of Wee1 that are involved in nutritionalsignaling. (b) Budding yeast: the Clb1– and Clb2–Cdc28 complexesshare the mitosis-promoting CDK activity, Clb2 being the dominant ofthe pair (the interactions of Clb1 parallel those of Clb2 and, therefore,are omitted from this diagram). Swe1 and Mih1, the respectivehomologs of Wee1 and Cdc25, modulate phosphorylation of Cdc28Tyr19, the equivalent of Cdc2 Tyr15. Hsl1 is a homolog of Cdr1 and Cdr2,and Hsl7 is its interacting partner.

Size control in G2–M: budding yeast

In G2, Cdc28 is phosphorylated by the Wee1 homologSwe1, and dephosphorylated by the Cdc25 homologMih1 [8] (Fig. 4b, Table 1). Unlike their counterpartsin fission yeast, however, neither of these componentsare essential in budding yeast. It has been proposedthat instead of size control, Clb2–Cdc28 tyrosinephosphorylation evolved a specialized function inexecution of a morphogenesis checkpoint. Thecheckpoint is activated when cells fail to form a bud orwhen the integrity of the actin cytoskeleton is perturbed[62]. Part of this signal is believed to be mediated byinhibition of Hsl1, one of the three Cdr1 and Cdr2homologs in budding yeast (Table 1), and its interactingpartner, Hsl7. Inhibition of Hsl1 and Hsl7 is thought toresult in stabilization of Swe1 and inhibition of mitosis[63]. However, a more sensitive assay revealed thatSwe1 is, in fact, normally present in cells until the endof mitosis, just as in other eukaryotes [64]. This revivesthe question as to whether other functions of Wee1 andCdc25 homologs, such as size control, might beconserved in budding yeast. Overexpression of SWE1results in pre-mitotic arrest, and deletion of MIH1increases cell size [65,66]. Conversely, deletion of SWE1causes subtle but consistent reduction of cell size(D. Kellogg, pers. commun.). This alone does not tell uswhether Swe1 and Mih1 actively participate in sizecontrol, but indirect evidence strongly suggests thatG2–M size-control is, indeed, present in buddingyeast, although it normally remains cryptic. Reversingthe situation seen in fission yeast, budding yeast cellsthat have their G1–S size-control compromised, suchas Cln3 overproducers, are small, but their generationtime (i.e. time between two subsequent cell divisions)remains largely unaffected, suggesting that the secondcontrol-point becomes active and prevents catastrophicregression of cell size [67,68] (Fig. 1b). At the molecularlevel, Clb2–Cdc28 and Swe1 are engaged in a strongnegative feedback loop, suggesting the presence of thesame switch-like mechanism of Cdc28 activation thatis conserved throughout eukaryotic cells (D. Kellogg,pers. commun.). In light of these results, the concept ofa morphogenesis checkpoint in budding yeast requiresreexamination to separate the specific response to failedbud formation from a simple manifestation of cell-sizecontrol. The effect of cell size has not been directlyaddressed in this context. It seems, however, that theinability of cells to reach a sufficient size for G2–Mmight explain the pre-mitotic arrest that is caused byperturbation of actin integrity, as occurs in fission

yeast [58]. It would be premature to speculate aboutthe identity of the G2–M sizer in budding yeast, but itwould be intriguing to test whether some of the Cdr1and Cdr2 homologs (Table 1) can mediate nutritionalsignals. If the G2–M module is conserved, whatmakes Mih1 non-essential? One explanation is that,in the absence of Mih1, Cdc28 inhibition is eventuallyoverturned by continued accumulation of Clb2,activating more and more free Cdc28 molecules [23]until the switch is flipped.

Concluding remarks

What is ‘cell size’? Entirely in accord with Pringle andHartwell [10], I use this deliberately vague expressioninstead of more specific terms simply because we arestill far from fully understanding what represents cellsize within cells. Production of Cln3 is clearlydependent on the total number of ribosomes in thecytoplasm, which increases as cells grow, but otherpossible sizers do not offer such clear-cut solutions.Protein mass, the number of ribosomes and cell volumeare obvious candidates, and one or another blend thatincludes these, and perhaps other structurally baseddeterminants, might constitute an integratedmeasure of size. So, why did cells bother to evolve sucha complicated system of size monitoring and feedbackcontrols if, intuitively, a simple correlation of growthand proliferation could suffice? One possible answer isthat cells need it to boost robustness of the cell-cycleregulatory network. There is always variation in thesize at which cell-cycle transitions occur. If unchecked,the variation would increase from generation togeneration until the cells at the tails of the distributionwould enter territory that is incompatible with survival.The cell-size homeostasis phenomenon that preventsthis from happening has been demonstrated elegantlyin fission yeast [11]. Stochastic processes, to whichlow-abundance constituents are especially vulnerable,are inherent to biological systems. Even in eukaryoticcells, gene expression can occur in erratic burstsrather than smoothly [69]. Eroding components of theregulatory network causes increased heterogeneity incell size and unpredictable behavior [30,70]. This leadsto the argument that cell-size feedback mechanisms orcell-size checkpoints are a conserved solution to thenoise problem. They might underlie more complexmechanisms that have evolved from preexisingbuilding blocks such as the ones that constituteprocesses 1–3 in Fig. 1a to ensure proper developmentand differentiation in multicellular organisms.



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Acknowledgements

I thank E. Boye,L. Breeden, B. Futcher,D. Kellogg, D. Lew,S. Moreno and A. Sveiczerfor fruitful discussionsand for sharing their databefore publication. I also thank P. Young,J. Karagiannis andD. Kellogg for criticalreading of themanuscript.

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