RATE OF CELL CYCLE INITIATION OF YEAST CELLS WHEN CELL SIZE IS NOT A RATE-DETERMINING FACTOR

J. Cell Sci. 59, 183-21 (1983) 183 Printed in Great Britain Cmpany f Bilgists Limited 1983 RATE OF CELL CYCLE INITIATION OF YEAST CELLS WHEN CELL SIZE IS NOT A RATE-ETERMINING FACTOR P. G. LOR* AN A. E. WHEALS Micrbilgy Grup, Schl f Bilgical Sciences, University f Bath, Bath, U.K. SUMMARY The cntrl f cell prliferatin under steady-state cnditins in the budding yeast, Saccharmyces cerevisiae, is well described by either the tandem r slppy size cntrl mdels, bth f which suggest that differences in cycle time between individual cells r between parents and daughters is largely due t differences in birth size. These mdels have been investigated further under cnditins in which cell size has nt been a rate-determining factr fr cell cycle initiatin. Tw appraches have been used. The first invlves the grwth f cells in lw cncentratins f hydrxyurea (HU), which has the effect f prlnging the duratin f NA synthesis. This leads t a lengthening f the budded perid, which in turn leads t daughter cells being larger at divisin than the nrmal cell cycle initiatin size f daughters in steady-state ppulatins. The secnd apprach invlves the accumulatin f cells at the key cntrl pint f the cycle, called start, using the phermne a-iactr. Since grwth is unaffected, all cells eventually becme larger than the vlume at which they wuld nrmally initiate the cell cycle. The kinetics f prliferatin were fllwed after release frm a-factr arrest. The results frm bth appraches were bradly cnsistent with the predictins f bth mdels. Hwever, ablitin f birth-size differences between parents and daughters in the presence f HU did nt lead t a cmplete disappearance f differences in either cycle time r prliferatin kinetics. Furthermre, fllwing release frm a-factr arrest, the rate f cell cycle initiatin f parent cells was slwer than in steady-state culture and the daughters' cells behaved as if cmprising tw separate ppulatins. These discrepancies suggest that besides a size difference, there are additinal physilgical differences between parent and daughter cells. INTROUCTION Start is defined as the stage, in the G\ perid f the cell cycle f Saccharmyces cerevisiae, the cmpletin f which cmmits a cell t a sequence f events (the cell divisin sequence) culminating in a mittic divisin (Hartwell, 1974). All the knwn events f the cell cycle are dependent n the prir ccurrence f start (Pringle & Hartwell, 1981), which thus represents the majr pint f cntrl in the cell cycle f this yeast. Jhnstn, Pringle & Hartwell (1977) prduced evidence that the attainment f a critical cell size is an imprtant prerequisite fr the cmpletin f start. An imprtant implicatin f a size cntrl is that much f the duratin f the cell cycle is determined by the birth-size f the cell. In the case f yeast cells this means that it is the prtin f G\ between cell divisin and start whse duratin is determined by birth-size. Since 5. cerevisiae cells divide asymmetrically int large parents and smaller daughters (Jhnsn & Gibsn, 1966; Hartwell & Unger, 1977) and since, at divisin, parent cells are already abve the critical size, it is nly the duratin f the G\ perid f daughter cells that is subject t the size cntrl (Hartwell & Unger, 1977).

184 P. G. Lrd and A. E. Wheats Supprt fr the view that much f the G\ perid is due t the fulfilment f a grwth requirement was given by Singer & Jhnstn (1981). They shwed that when the 5 phase f yeast cells is lengthened, by incrprating a lw cncentratin f hydrxyurea (HU) in the grwth medium, the G\ perid is shrtened (particularly that part f G\ between cytkinesis and start). They als reprted that daughter cells tended t prduce buds at the same time as their sibling parent cells, in the presence f HU. Their frmal explanatin fr these results was that daughter cells are brn at a larger size due t the extended budded perid (which is due t the lengthened S phase) and, therefre, need less time t attain a critical size fr traversing start than daughter cells grwn in the same medium but withut HU. The imprtance f cell size in triggering start was questined by Shil, Shil & Simchen (1976). They released yeast cells frm start arrest and shwed that these cells traversed start at a similar rate t expnentially grwing cells, bth shwing first-rder kinetics. They prpsed that start is triggered in the manner suggested in the Transitin Prbability hypthesis f Smith & Martin (1973). That is, that cells initiate the cell divisin sequence with cnstant prbability per unit time. Hwever, t accunt fr the difference between parent and daughter cycle times, Shil, Shil & Simchen (1977) accepted the ntin that cells must attain a minimum cell size befre they can traverse start. We termed the prpsal f Shil et al. (1976, 1977) the Tandem mdel (Lrd & Wheals, 1981) since it implies that there are tw mechanisms, which perate in tandem t trigger start (i.e. a size mechanism fllwed by a prbabilistic mechanism). We shwed that this mdel adequately describes the kinetics f yeast cell prliferatin as lng as it is further assumed that there is cnsiderable variatin in critical cell size (Lrd & Wheals, 1981; Wheals, 1982). We als shwed that anther mdel f cell prliferatin cntrl, which cmbines the ideas f a size cntrl and a prbabilistic traverse f start, prvides a gd descriptin f yeast cell cycle kinetics. In this slppy size cntrl (SSC) mdel (Wheals, 1982), the prbability f traverse f start increases smthly with increasing cell size. We have used the methds f Singer & Jhnstn (1981) and f Shil et al. (1976) as further means f testing the Tandem and the SSC mdels by analysing their effect n individual cells. Using time-lapse cinephtmicrgraphy we have measured the size and cycle times f yeast cells grwing in steady state in lw cncentratins f HU. Under these cnditins daughter cells shuld be large enugh at birth t prevent size being ratedetermining fr start. Bth the Tandem and the SSC mdels predict that in lw cncentratins f HU: (1) the mean daughter cycle time shuld apprach the mean parent cycle time; and (2) the rate f traverse f start shuld be the same fr parent and daughter cells. a-factr prevents the traverse f start in a strains f 5. cerevisiae withut inhibiting grwth (Herefrd & Hartwell, 1974; Thrm & untze, 197). We have used a-factr t accumulate cells at start and released them after sufficient time fr daughter cells t attain a size at which they shuld be as cmpetent as parent cells t traverse start. We have cmpared the kinetics f the traverse f start fllwing release, f parent and daughter cells, by fllwing the kinetics f bud emergence using time-lapse cinephtmicrgraphy. Shil et al. (1976) shwed that the rate f bud

Cell cycle initiatin in yeast 185 emergence prvides a gd estimate f the rate f emergence frm start. The Tandem and SSC mdels predict that after release frm start arrest, parent and daughter cells shuld traverse start with the same kinetics. Althugh the difference between parent and daughter cycle times in expnentially grwing ppulatins f S. cerevisiae can be bradly explained by the asymmetrical mde f divisin and the presence f a size cntrl ver start (Hartwell & Unger, 1977), we recently fund indirect evidence that part f the duratin f the unbudded perid f daughter cells is nt determined by their size (Lrd & Wheals, 1981). The experiments reprted here prvide mre direct evidence fr this perid. MATERIALS AN METHOS Organism A haplid strain f S. cerevisiae, A364A (Hartwell, 1967) btained frm L. H. Hartwell was used thrughut. Media YEP-galactse-PVP medium cnsisted f 15g yeast extract, 3g bacterilgical peptne, 3g galactse, 28 g plyvinylpyrrlidne (PVP-4, Sigma) and 5 \vg adenine in 11 distilled water. This was sterilized by autclaving. Hydrxyurea was btained frm Sigma. Synthetic a-factr was btained frm the Peptide Institute, Osaka, Japan. Culture chamber A Pwell chamber (Pwell, 1956) was used. In this, cells are grwn n a piece f cellphane and liquid medium is passed underneath. The chamber was set up in the fllwing manner. The cellphane (Cuprphan 15PM, 11-5/im thick, Medicell Internatinal Ltd) and its supprting PVC washers were sterilized by saking them fr lomin in 7% ethanl and then fr 5 min in sterile distilled water. The cellphane was placed in psitin between the washers n the chamber, the tp was screwed n and the vacuum (prvided by an electric vacuum pump) applied. Sterile distilled water was pumped (using a LKB Vari-perpex pump) thrugh slwly fr apprx. 3 min t leach ut plasticizers in the cellphane. After that, medium was pumped thrugh at the maximum flw rate. A small drp f cell suspensin was placed n the cellphane surface, which was then made cncave by tightly squeezing the utlet tube, and a cverslip was placed n tp f the cellphane. The pressure n the utlet tube was slwly released. The latter peratins ensured a firm even cntact between the cellphane and the cverslip. Once the cvershp was in place the flw rate was reduced t 4ml/h. Cells were grwn fr several generatins t ensure that they were grwing expnentially, then redistributed prir t filming. Redistributin was accmplished by intrducing a small amunt f liquid medium (nt cntaining PVP) underneath the cverslip and then twice raising and lwering the cverslip. The latter was achieved by setting the flw rate t maximum, applying and then releasing pressure (by squeezing) n the utlet tube. After redistributing the cells, the flw rate was reduced t 4ml/h. Filming equipment The micrscpe was a Wild M2 fitted with a lng-wrking-distance phase cndenser. A 1 X eyepiece and 2 X phase bjective were used thrughut. The camera was a Blex H165BM cntrlled by a Blex/ Wild Varitimer timing system. An electrmagnetic shutter, perated by the timer unit, was fitted beneath the cndenser s that cells were nt cntinuusly expsed t light. Films were taken at a rate f 1 frame/min. n Eastman Ektachrme Cmmercial 7252 16 mm film. All peratins were carried ut at 3 C in a temperature-cntrlled rm. Unless therwise stated the films were analysed as previusly (Lrd & Wheals, 1981). 13 CEL 59

186 P. G. Lrd and A. E. Wheals RESULTS Kinetics f cell prliferatin in lw cncentratins f hydrxyurea Time-lapse cine films were made f A364A cells grwing n YEP-galactse PVP medium with and withut HU, in a Pwell chamber at 3 C. This medium was chsen because there is a large difference between the cycle times f parent and daughter cells grwing n this medium (Lrd & Wheals, 1981). Three films were analysed: f cells grwing in the absence f HU (1), and in the presence f (2) 1-5 mg/ml and (3) 2-5 mg/ml HU. In each case the cells were judged t be in expnential grwth by sme r all f the criteria f Lrd & Wheals (1981). The values f the ppulatin dubling time (T) and f the ppulatin vlume dubling time (T V ) are given in Table 1. As in previus kinetic analyses f this strain T<TV (Lrd & Wheals, 1981). The mean duratins f the cell cycle and its cnstituent perids fr parent and daughter cells are listed in Table 2. Cells grwn in the presence f HU have lnger budded perids than cells grwn in the absence f HU. The perids frm nuclear migratin t cell separatin shwed little increase in duratin with increasing cncentratins f HU. These results are cnsistent with 5 phase being lengthened by the additin f HU t the medium (Singer & Jhnstn, 1981). Nuclear divisin, and cnsequently cytkinesis and cell separatin, are thus delayed since nuclear divisin is dependent n the cmpletin f NA synthesis (Hartwell, 1974). Nuclear migratin was als delayed s there may be sme dependency fr this event n the cmpletin f NA synthesis. The parent cycle time increased with increasing cncentratin f HU due t the expansin f the budded perid. The parental unbudded perid was unaffected by the presence f HU. The increased variance f the parent cycle times in the presence f HU is due t the increased variance f the budded perid. The mean parent cycle time was less than the mean daughter cycle time in the presence r in the absence f HU, althugh the difference between the means was less fr cells grwn in the presence f HU. In the absence f HU and in the presence f 1-5 mg/ ml HU, all daughter cells had a lnger unbudded perid than their sibling parent cells, whereas in the presence f 2-5 mg/ml HU all except six daughter cells had lnger unbudded perids than their sibling parent cells (data nt shwn). The mean daughter cycle time in the presence f l p 5 mg/ml HU was slightly less than that in Table 1. Values fx* and x v in different cncentratin f hydrxyurea Cncentratin f hydrxyurea (mg/ml) r (min) r, (min) 13-4 135-2 1-5 118-141-3 2-5 138-1 NMf * r, ppulatin dubling time in number; x^., ppulatin dubling time in vlume. fnm, nt measured.

Cell cycle initiatin in yeast 187 Table 2. Mean duratin f the cell cycle and cnstituent perids fr parent and daughter cells in each cncentratin f hydrxyurea Cncentratin f A 1-5 HU (mg/ml) 2-5 Perid P* * P P Cycle time 78-2 (9-l)f 133-2 (21-2) 17-1 (18-3) 128-8 (21-6) 122-1 (17-7) 147-3 (24-2) Unbudded perid 8- (4-6) 59-1 (18-5) 6-6 (7-2) 26-8 (14-1) 6-2 (S-6) 17-9 (12-3) Budded perid 7-2 (7-7) 74-2 (6-7) 1-5 (13-9) 12-1 (12-6) 115-9 (16-9) 128-5 (17-5) Bud emergence t nuclear migratin 44-7 (7-1) 49-3 (6-2) 71-1 (11-8) 73-3 (11-) 81- (15-9) 9-7 (16-5) Nuclear migratin t nuclear divisin 8-1 (1-5) 7-8 (1-2) 11-2 (4-3) 1-9 (3-3) 13-2 (4-8) 16-8 (6-1) Nuclear divisin t cytkinesis 8-3 (2-2) 8-2 (2-3) 9-4 (2-3) 9 (2-3) 9-7 (3-4) 12- (4-3) Cytkinesis t cell separatin 9-1 (2-) 8-9 (1-6) 8-9 (1-6) 8-9 (1-6) 12 (3-8) 1- (2-5) All values are in minutes; 4 parent and 4 daughter cycles were mnitred in each case. * P, parent cells;, daughter cells. Values in parentheses are standard deviatins. ata pled as previusly (Lrd & Wheals, 1981). the absence f HU. There was a marked increase in the mean length f the daughter cycle time in the presence f 2-5 mg/ml HU. The mean duratin f the unbudded perid f daughter cells decreased with increasing cncentratins f HU. The variability f the unbudded perid cntributed mre t the variability f the daughter cycle time with increasing cncentratins f HU. The ratinale behind these experiments was t increase the size f daughter cells at birth s that they were brn at a size either abve a 'critical size' (in terms f the Tandem mdel) r with a transitin prbability as high as parent cells (in terms f the SSC mdel). It can be seen frm Table 3 that the mean birth size f daughter cells grwing in the presence f 1-5 mg/ml HU is larger than the mean daughter cell size at bud emergence in the absence f HU. It is als evident that in the presence f HU the mean increase in cell vlume f daughter cells frm birth t bud emergence was less than that in the absence f HU. The distributins f daughter cell size at birth and at bud emergence in the absence and in the presence f HU are presented in Fig. 1. The majrity f daughter cells in the presence f HU were brn at a size greater

188 P. G. Lrd and A. E. \Wieals Table 3. Mean size f daughter cells, at three stages in the cell cycle, in the absence and in the presence f hydrxyurea Birth Mean size (j*m 3 ) at: Bud emergence Cell separatin Withut HU 26-8 (7-)» 37-6 (8-3) 38-2 (7-7) With l-5mg/mlhu 46-1 (11-3) 54-2 (1-2) 55-8 (9-5) Values in parenthesis are standard deviatins. than the mean daughter cell size at bud emergence in the absence f HU. The difference in the timing f start between parent and daughter cells, which is due t a size cntrl, shuld therefre be negligible at the cncentratin f HU used. Cell cycle kinetics f parent and daughter cells are best cmpared by pltting the 15-1 - n 5-2 4 6 8 Vlume 15 - B c a 1-5 " - 2 4 6 8 Vlume I Fig. 1. The distributin f the sizes f daughter cells at tw stages f the cell cycle, in the absence and in the presence f hydrxyurea. Histgrams shw the sizes f daughter cells at birth (slid line) and at bud emergence (brken line), A. Cells grwing in the absence f HU; B, cells grwing in the presence f 1-5 mg/ml HU.

Cell cycle initiatin in yeast 189 distributins f cycle times as a plts (Lrd & Wheals, 1981). An a plt is the percentage f cells with cycle times greater r equal t / pltted (n a lgarithmic scale) against / (Smith & Martin, 1973). a plts f the duratin f the unbudded perid give a mre accurate indicatin f the kinetics f initiatin f cell cycle events (i.e. f traverse f start) since the pre-start perid frms part f the unbudded perid and since variatin in the duratin f the budded perid can have a prnunced effect n the slpe f the a plt f cycle times, particularly when the unbudded perid is shrt. In this study it is especially imprtant t cmpare the a plts f the duratin f the unbudded perid since the budded perid was mre directly affected by HU. It is clear frm Table 2 that HU increased bth the length and the variability f the budded perid and, cnsequently, with increasing cncentratins f HU the distributins f the lengths f the budded perid had an increasing effect n the shape f the a plts f cycle times. The distributins f the lengths f the unbudded perids f parent and daughter cells in the absence and in the presence f HU are presented as a plts in Fig. 2. The a plt f parent unbudded perids were unaffected by the presence f HU. HU had a cnsiderable effect n the shape f the a plt f daughter unbudded perids. In the absence f HU the a plt had a prnunced initial dwnward curvature, which included abut 5% f the data, befre becming apprximately linear. This initial curvature was greatly reduced in the presence f HU. All the daughter unbudded perids were shrter in the presence than in the absence f HU, with the a curves being shifted mre t they-axis as the cncentratin f HU was increased. There was little difference in the slpes f the (apprximately) linear prtins f the three curves f the daughter unbudded perids, but in each case the slpe was less steep than the slpe f the crrespnding parent a curve. The relatinship between cell size at cell separatin and the length f the subsequent unbudded perid is shwn in Fig. 3 fr cells grwn in the absence and in the presence f 1-5 mg/ml HU. In bth cases there was n crrelatin between the size at cell separatin f parent cells and their subsequent unbudded perid. There was a crrelatin between the birth size f daughter cells and the length f their unbudded perid (r = - 66, which is significantly different frm at the -1 % level) in the absence f HU. In the presence f 1*5 mg/ml HU there was less crrelatin between these parameters (r= -42, which is nt significantly different frm ). Size, therefre, plays less f a rle in determining the length f the unbudded perid fr cells grwn with HU. The data pints fr HU-grwn daughter cells merge int the data pints f HU-grwn parent cells because f the brader distributin f birth sizes and the shrter unbudded perids. Release frm a-factr arrest Fr this experiment cells f A364A were filmed grwing n YEP-galactse-PVP medium at 3 C in the Pwell chamber. At t = 2 min a-factr was added. This was achieved by replacing the medium in the reservir with YEP-galactse-PVP medium cntaining a-factr at a cncentratin f 5 fig/ ml at t = 198 min and adjusting the flw rate t maximum. After 5 min the flw rate was returned t the riginal setting. This

19 P. G. Lrd and A. E. Wheals 1 A \ A n A O Q O A A A m A O AO A\ O 1 A? 1 - a? AO AO A * A m A I 5 f (min) I 1 Fig. 2. a Plts f the unbudded perids f parent and daughter cells, grwing in the absence and in the presence f hydrxyurea. The percentage f cells with unbudded perids f duratin, f u b, greater than r equal t /, is pltted against /. ata are cells grwing: in the absence f HU (O, ); in the presence f l-5mg/ml HU (A, ); and f 2-5mg/ml HU (, ). Open symbls, parent cells; clsed symbls, daughter cells.

Cell cycle inttiattn in yeast 191 p A 1 5-8 O» cb f < c s - 5 1 1 {$ E 5»cc» V. 5 1 Unbudded perid (min) Fig. 3. Cell size at cell separatin versus the duratin f the subsequent unbudded perid in different cncentratins f HU. A. Cells grwing in the absence f HU; B, cells grwing in the presence f 1-5 mg/ml HU; (O) parent cells; () daughter cells. The crrelatin cefficients f daughter cell size at cell separatin versus the duratin f the unbudded perid were: A, '66; B, -42.

192 P. G. Lrd and A. E. Wheats prcedure ensured that the a-factr-cntaining medium came int cntact with the cells at t = 2 min and that all the medium withut a-factr was fully replaced in the chamber. a-factr was remved at t = 395 min by repeating the abve prcedure in reverse. The cells were expsed t a-factr fr a time at least as lng as the lngest daughter cycle time, s that n release mst r all daughter cells shuld be as large r larger than daughter cells at bud emergence in medium withut a-factr. Filming was stpped when it was judged that all cells had prduced a bud fllwing a-factr release. In the analysis f the film all cells f each clne in fcus were scred. The cells in even the largest clne was easily identified and fllwed, since after release the cells prduced buds rientated away frm the centre f the clne in a radial array. Cmplete cessatin f divisin ccurred 1 min after additin f a-factr. The ppulatin dubling time was calculated t be 113 min. The mean duratins f the measured perids in the cell cycle f cells prir t a- factr arrest and f cells after a-factr release are cmpared in Table 4. It shuld be nted that the unbudded perid f cells fllwing a-factr release refers t the perid f time frm a-factr release (i.e. t = 395 min) t bud emergence. The mean duratin f the perids between nuclear migratin and cell separatin were equivalent fr cells befre a-factr arrest and after a-factr release. The budded perid f cells after a- factr release was, hwever, abut 1 min shrter than that f cells befre a-factr Table 4. Mean duratin f cnstituent perids f the cell cycle prir t and fllwing release frm a-factr arrest Perid Cycle time Unbudded peridf Budded perid Bud emergence t nuclear migratin Nuclear migratin t nuclear divisin Nuclear divisin t cytkinesis Cytkinesis t cell separatin Befre a-factr arrest t ^ # 9-8(9-9)f 129-6(28-6) 9-2(6-4) 45-3(25-9) 81-6(7-9) 84-1(8-5) 51-6(6-7) 55-6(7-3) 9-4(2-) 9-4(3-) 11-3(3-4) 8-5(1-9) 9-5(2-5) 1-9(2-) After release frm a-factr arrest t P 81-2(24-7) 73-2(5-1) 44-1(4-4) 9-(2-3) 9-6(2-8) 1-6(2-7) 115-9(34-3) 71-8(7-3) 43-8(5-9) 8-2(1-7) 9-6(2-4) 9-9(3-) A4I values are-in minutes; 55 parent and 38 daughter cycles were mnitred befre a-factr arrest. After release frm a-factr arrest, the unbudded perids f 64 parent and 78 daughter cells were measured and f these, 56 parent and 34 daughter cells were mnitred thrugh t cell separatin. P, parent cells;, daughter cells. f Standard deviatins in parentheses. \ In the case f cells released frm a-factr arrest the unbudded perid refers t the time frm remval f a-factr frm the medium t bud emergence.

Cell cycle initiatin in yeast 193 arrest. Since the perid between bud emergence and nuclear migratin is shrter fr cells after a-factr release, either bud emergence may be delayed r thse events that ccur during this perid may be cmpleted mre quickly. The mst interesting feature f these data was that parent cells had a shrter unbudded perid than daughter cells bth befre a-factr arrest and after a-factr release, the mean differences being 36-1 and 34-7 min, respectively. The mean size f daughter cells at the time f a-factr release was 58-3 /xm 3, which is larger than the mean size f daughter cells at bud emergence (47-7 /xm ) befre a- factr arrest, althugh the distributins f the tw sizes verlap cmpletely (Fig. 4). Since there may be a substantial lag perid after a-factr release during which cells are unable t traverse start (Samkhin et al. 1981; and Fig. 5), it is likely that all daughter cells will be abve a critical size (Tandem mdel) r will be in the hightransitin-prbability size range (SSC mdel). After a-factr release, therefre, daughter cell size shuld nt be a determining factr fr traverse f start. Indeed there is n significant crrelatin between the size f daughter cells at a-factr release and the time frm a-factr release t bud emergence (r= --38). 2 - "I s 1 2-4 8 r Vlume 12 B jency» 1 - L - 4 8 Vlume Ln q n Fig. 4. The distributins f daughter cell size at tw stages f the cell cycle and at the time f a-factr release, A. Histgrams f the sizes f daughter cells at birth (slid line) and at bud emergence (brken line) prir t a-factr arrest, B. Histgram f the sizes f daughter cells at f = 395 min (i.e. the time f remval f a-factr frm the medium). 12

194 P. G. Lrd anda.e. Wheals Assuming that the lag perid after a-factr release is apprximately the same fr each cell (i.e. the lag perid has little variability), the perid f time frm a-factr release t bud emergence is cmparable t the time frm start t bud emergence, since the duratin f each is determined mainly by the rate f cmpletin f start. Bth the Tandem and the SSC mdel prvide the same clear predictin abut the shape f the a plts f the distributins f the length f time frm a-factr release t bud emergence fr parent and daughter cells; namely, that after a lag perid, apprximately cnstant fr all cells, the a plts f this perid shuld becme linear and shuld be the same fr parent and daughter cells, and the linear prtins f these a plts shuld be apprximately parallel t the a plt f the lengths f the unbudded perids f parent cells prir t a-factr arrest. These a plts are presented in Fig. 5. The fllwing features are evident frm Fig. 5. (1) The a plt f the parent unbudded perids is apprximately linear. (2) The a plt f daughter unbudded perids is nt linear. (3) There is a lag perid, f abut 5 min fr parent cells and f abut 6 min fr daughter cells, after a-factr release befre bud emergence ccurs. (4) After the initial lag and an initial dwnward curvature the a plt fr parent cells, after a-factr release, becmes apprximately linear but the slpe is much less steep than the slpe f the a plt f parent unbudded perids. (5) The a plt f the time frm a-factr release t bud emergence f daughter cells is nt linear. The distributins f the perids f time frm a-factr release t bud emergence, fr parent and daughter cells, are presented mre cnventinally as histgrams in Fig. 6. The distributin fr parent cells is skewed as was expected since the a plt was mainly linear. In cntrast, the distributin fr daughter cells is bimdal, suggesting that the daughter cells are nt a hmgeneus ppulatin but are cmpsed f at least tw subsets. The rigin f the bimdality is nt apparent frm any f these data. Neither cell size at the time f a-factr release nr the length f time that daughter cells, after birth, were expsed t a-factr determined the length f time frm a-factr release t bud emergence (data nt shwn). There is slight evidence (data nt shwn) that, after a-factr release, daughter cells frm the same clne prduce buds after a time that falls in the same half f the distributin in Fig. 6B. Hwever, any explanatin f clnal variatin cannt satisfactrily explain why the data fr parent cells appear hmgeneus (Fig. 6A). These results seem t be incnsistent with the results f Shil et al. (1977). They shwed that the kinetics f bud emergence in an expnentially grwing culture and in a cell ppulatin fllwing a-factr release were similar. They did nt, hwever, distinguish between parent and daughter cells. Fig. 7 is equivalent t fig. 1 f Shil et al. (1977). The a plt f the lengths f the unbudded perids f cells (bth parent and daughter cells) prir t a-factr arrest is equivalent t the a plt f the % unbudded cells against the time after plating expnentially grwing cells. The a plt f the lengths f the perid frm a-factr release t bud emergence is equivalent t the a plt f the % unbudded cells against time after a-factr release. As shwn in Fig. 7, by pling the data fr parent and daughter cells as was, in effect, dne by Shil et al. (1977), the kinetics f bud emergence f expnentially grwing cells d appear t be similar t the kinetics f bud emergence fllwing a-factr release.

Cell cycle initiatin in yeast 195 1 - V 5-1 5 1 15 f (min) Fig. 5. a Plts f the unbudded perids f parent and daughter cells prir t a-factr arrest and fllwing release frm a-factr arrest. The percentage f cells with unbudded perids f duratin, / u b, greater than r equal t t versus t, prir t a-factr arrest (O, ); and the percentage f cells with t, (time frm remval f a-factr t bud emergence) greater than r equal t t versus t (A, A) are shwn. Open symbls, parent cells; clsed symbls, daughter cells. ISCUSSION As expected, lw cncentratins f HU in the grwth medium increased the length f the budded perid and increased the size f daughter cells at birth. Whereas in the similar experiments f Singer & Jhnstn (1981) the ppulatin dubling time (r) was unaltered by the presence f HU, in these experiments r increased as the cncentratin f HU in the medium increased. The ppulatin vlume dubling time (r v ) in the presence f 1-5 mg/ml HU was apprximately the same as in the absence f HU, which suggests that this cncentratin f HU des nt alter the (vlume) grwth rate.

196 P. G. Lrd and A. E. Wheats There are tw pssible reasns fr the increase in x. (1) If the sle effect f HU is t expand S phase and if the sum f the lengths f G\, Gz and M phases cannt be decreased belw a minimum value, then beynd a threshld cncentratin f HU when the sum f G\, G 2 andm phases becmes minimal, X wuld increase due t the expansin f S phase with increasing cncentratins f HU. (2) HU als slws dwn synthesis f RNA and prtein, althugh t a much smaller extent than it slws NA synthesis and, cnsequently, culd have slwed dwn ther prcesses. The latter explanatin is unlikely since the cncentratins f HU used were lw (-2 M and -3 M) and Slater (1973) fund that these cncentratins f HU had little effect n RNA and prtein synthesis. Apart frm increasing the length f the budded perid (between bud emergence and nuclear migratin) the presence f HU has little effect n the duratin f the ther perids in the parent cycle (Table 2). The delayed ccurrence f nuclear divisin, cytkinesis and cell separatin in the presence f HU (Table 2) was expected, since 2-1 5 1 15 2 f (min) 15 H B & 1 - c I CJ a> * 5 H n_ j 5 1 15 f (min) 2 Fig. 6. The distributins f the times between release frm a-factr arrest and bud emergence f parent and daughter cells, A. Histgram f the duratin f the perid between remval f a-factr and bud emergence f parent cells. B. Histgram f the duratin f the perid between remval f a-factr and bud emergence f daughter cells.

1 a Cell cycle initiatin in yeast m^ 197 \ a a a <h a \ 1 - " \ a a 1 5 15 1 r(min) Fig. 7. a Plts f the unbudded perids f cells prir t a-factr arrest and fllwing remval f cr-factr. The data f parent and daughter cells were pled fr these plts. () The percentage f cells with unbudded perids f a duratin greater than r equal t t versus t, prir t cr-factr arrest. () The percentage f cells with t T (time frm remval f a-f actr t bud emergence) greater than r equal t t versw t. these events are dependent n cmpletin f NA synthesis (Hartwell, 1974). Althugh it has been prpsed that nuclear migratin is independent f NA synthetic events (Hartwell, Cultti, Pringle & Reid, 1974), the delay in the ccurrence f nuclear migratin in the presence f HU (Table 2) suggests that this event is dependent n cmpletin f NA synthesis. The prpsal f Hartwell et al. (1974) was based n the bservatins f Hartwell (1973) and Slater (1973) that nuclear migratin takes place when cmpletin f NA synthesis is prevented. Hwever, their bservatins were n fixed, Giemsa-stained cells and it is pssible that the fixatin prcedure affected the psitin f the nucleus. This pssibility culd be tested by repeating their

198 P. G. Lrd and A. E. Wheats experiments n unfixed cells using time-lapse cinephtmicrgraphy and immersin refractmetry. The rate f bud emergence (i.e. the rate f exit frm the unbudded perid; Fig. 2) f parent cells is als little affected by HU. This bservatin and Table 2 suggest that, even in the absence f HU, the length f G\ is minimal and the rate f traverse f start is maximal in parent cells. It is clear that the unbudded perid is shrter fr daughter cells in the presence f HU (Table 2). It is nt clear whether the rate f traverse f start f daughter cells (rate f exit frm the unbudded perids; Fig. 2) is altered by the presence f HU, since the shape f the a plt f the lengths f the unbudded perid f daughter cells in the absence f HU is determined t sme extent by the variatin in the birth size f daughter cells. The results are cnsistent with there being a size cntrl early in the cell cycle, since the difference between the cycle times (and in particular, between the lengths f the unbudded perids) f parent and daughter cells is reduced when the birth size f daughter cells is increased. Hwever, there is still a difference between parent and daughter cells in the duratins f their unbudded perids in the presence f HU. The cause f this is the difference in rate f exit frm the unbudded perid and, by inference, the rate f traverse f start, fr parent and daughter cells. This bservatin is incnsistent with bth the Tandem and the SSC mdels. These mdels predict that the length f the unbudded perid and the rate f traverse f start shuld be the same fr parent and daughter cells in the presence f HU (prvided that the daughter cells are brn large enugh, which they are; see Table 3 and Fig. 1). Either mdel can be mdified t accunt fr the results. The inclusin f the assumptin that there is an additinal perid in the daughter cycle prir t bud emergence is ne adequate way f mdifying bth mdels. This perid wuld have t be f variable duratin t accunt fr the a plts in Fig. 2, but the shape f the distributin and the exact tempral lcatin f this perid cannt be deduced frm the data. As shwn by Fig. 7 the results f the t-factr-release experiment are nt t dissimilar frm the results f Shil et al. (1977). Hwever, we have lked at the kinetics f a-factr release in mre detail by distinguishing between parent and daughter cells, and the cnclusins frm these results are quite different frm the cnclusins f Shil et al. (1976, 1977). The rate f bud emergence f parent cells fllws apprximately first-rder kinetics bth befre a-factr arrest and fllwing release frm the arrest, althugh the rate f bud emergence is nt the same. Samkhin et al. (1981) have shwn that the rate f bud emergence fllwing cmplete arrest is decreased when cells are transferred t medium cntaining lw cncentratins f a- factr. This suggests the pssibility that a-factr was nt cmpletely washed ut f the Pwell chamber. Since a cells actively degrade a-factr (Ciejek & Thrner, 1979), and since any residual a-factr within the chamber will have been cntinuusly diluted by a-factr-free medium, the cncentratin f residual a-factr in the chamber shuld have decreased with time. Even if the decrease in the cncentratin f a- factr was slw, the a plt f the time frm release t bud emergence f parent cells (Fig. 5) wuld nt be linear (after the initial plateau) as was bserved. Instead, it

Cell cycle initiatin in yeast 199 wuld be a curve with increasing (negative) slpe. It is perhaps mre likely that the traverse f start fllwing release frm a-factr arrest des nt ccur at the same rate as in steady-state cnditins, wing t sme prperty intrinsic t the mde f actin f a-factr. The questin then arises as t what the cells are ding during the lag perid fllwing remval f a-factr frm the medium, a lag perid very similar in length t that bserved by Shil et al. (1977) and Samkhin et al. (1981). T cmplicate the issue further, the kinetics f bud emergence f daughter cells fllwing a-factr release are strikingly different frm the kinetics f bud emergence f: (1) daughter cells prir t a-factr arrest; (2) parent cells prir t arrest; and (3) parent cells after release (Fig. 5). The distributin f the times frm a-factr release t bud emergence f daughter cells is bimdal (Fig. 6B), which suggests that there is a difference in the kinetics f release frm a-factr arrest nt nly between parent and daughter cells, but als between at least tw subsets within the daughter cell ppulatin. The basis f the hetergeneity f the daughter cell ppulatin fllwing a-factr release remains a mystery, althugh it is pssible that a prprtin f the daughters enter a G -like state, as can ccur in slw-grwing r statinary-phase cultures (B. Carter, persnal cmmunicatin). The duratin f the budded perid fllwing a-factr release is equivalent fr parent and daughter cells, but is sme lomin shrter than fr cells in a steady state. This des nt necessarily mean that the time between cmpletin f start and cell separatin is shrter in cells fllwing a-factr release, althugh it is a pssible reasn fr the shrter budded perid. A secnd pssibility is that events specific t the emergence f the bud (e.g. micrfilament-ring frmatin) are executed at a slwer rate because f the changes in the cell wall prduced by the actin f a-factr (Lipke, Taylr & Ballu, 1976). This is plausible since the bud is frmed, in mst cases, at the 'shming tip' and the Calcflur-stainable ring at the base f the bud has a larger diameter n 'shms' than n steady-state cells (unpublished bservatin). These experiments were designed t reveal hw much influence 'pre-start' cell size has n the timing f start. Under the cnditins f the experiments the pre-start cell size f all cells was large enugh in thery t essentially remve the effect f cell size in determining the timing f start. In the case f the timing f start in parent cells in balanced grwth, size has little r n effect. Cell size is an imprtant determining factr fr the timing f start in daughter cells in balanced grwth. The experiments with HU cnfirm this and reveal that an additinal factr influences the timing f start in daughter cells but nt in parent cells. The experiments with HU als supprt the interpretatin f the results presented previusly (Lrd & Wheals, 1981), that the daughter cell cycle cntains an additinal perid, called G w, whse duratin is nt influenced by cell size. C w is unique t daughter cells and may be due t an event (r events) distinct frm, but a prerequisite fr, start events. If this were true then, during a-factr arrest, nt nly will the effect f cell size be reduced (by cntinued grwth) but the effect due t this pre-start event will als be reduced, assuming that a-factr blck start events and nt the hypthetical pre-start event. In view f the difference in the kinetics f a-factr release between parent and daughter cells, a pre-start event unique t daughter cells is unlikely.

2 P. G. Lrd and A. E. Wheals This leaves tw further pssibilities fr G w - It may be due t an event that lies between start and bud emergence r it may lie within the cmplex f start events (Nurse, 1982; Pringle & Hartwell, 1981). In the frmer case the rate f traverse f the start cmplex wuld be the same fr parent and daughter cells, but the rate f bud emergence fllwing start wuld be different fr parent and daughter cells. In the latter case the rate f traverse f start wuld be different, but the rate f bud emergence after start wuld be the same fr parent and daughter cells. The rate f traverse f start was nt directly mnitred in the a-factr arrest experiment and, apart frm this, there is a prnunced qualitative difference in the kinetics f bud emergence after a-factr release between parent and daughter cells, which is difficult t interpret. The a-factr release experiment, therefre, des nt prvide evidence t discriminate between these tw pssibilities. It is held that the difference between the mean cycle times f parent and daughter cells f S. cerevisiae is due t the asymmetrical mde f divisin and the presence f a 'size cntrl' (Hartwell & Unger, 1977; Carter & Jagadish, 1978). Furthermre, it is held that the difference is due t the difference in the mean pre-start perid f parent and daughter cells (Hartwell & Unger, 1977; Singer & Jhnstn, 1981). The results presented here imply that, whilst the difference in cell size at divisin is the majr cause f the difference in the mean pre-start cycle time f parent and daughter cells, it is nt the sle cause. The difference in the mean pre-start perid f parent and daughter cells des appear, in the light f this evidence, t be caused slely by the difference in cell size at divisin. Hwever, an additinal surce f the mean cycle time difference is apparent in daughter cells that, if nt within the start cmplex, lie immediately after start. These results further emphasize the need t treat ppulatins f 5. cerevisiae cells as cmprising tw distinct sub-ppulatins. Treating them as hmgeneus ppulatins in cell cycle experiments can lead t misleading, if nt errneus, cnclusins. We thank the SRC frfinancialsupprt. REFERENCES CARTER, B. L. A. & JAGAISH, M. N. (1978). The relatinship between cell size and cell divisin in the yeast Saccharmyces cerevisiae. Expl Cell Res. 112, 15-24. CIEJEK, E. & THORNER, J. (1979). Recvery f Saccharmyces cerevisiae a cells frm Gl arrest by a factr phermne requires endpeptidase actin. Cell 18, 623 635. HARTWELL, L. H. (1967). Macrmlecular synthesis in temperature-sensitive mutants f yeast. J. Bad. 93, 1662-167. HARTWELL, L. H. (1973). Three additinal genes required fr NA synthesis in 5. cerevisiae. J. Bact. 115, 966-974. HARTWELL, L. H. (1974). Saccharmyces cerevisiae cell cycle. Bact. Rev. 38, 164 198. HARWELL, L. H., CULOTTI, J., PRINGLE, J. R. & REI, B. J. (1974). Genetic cntrl f the cell divisin cycle in yeast. Science, N.Y. 183, 46-51. HARTWELL, L. H. & UNGER, M. W. (1977). Unequal divisin in Saccharmyces cerevisiae and its implicatins fr the cntrl f cell divisin.,?. Cell Bil. 75, 422-435. HEREFOR, L. M. & HARTWELL, L. H. (1974). Sequential gene functin in the initiatin f Saccharmyces cerevisiae NA synthesis..7. mlec. Bil. 84, 445-461.

Cell cycle initiatin in yeast 21 JOHNSON, B. F. & GIBSON, E. J. (1966). Autradigraphic analysis f reginal cell wall grwth f yeasts. III. Saccharmyces cerevisiae. Expl Cell Res. 41, 58-591. JOHNSTON, G. C, PRINGLE, J. R. & HARTWELL, L. H. (1977). C-rdinatin f grwth with cell divisin in the yeast Saccharmyces cerevisiae. Expl Cell Res. 15, 79-98. LIPKE, P. N., TAYLOR, A. & BALLOU, C. E. (1976). Mrphgenic effects f a factr n Saccharmyces cerevisiae a cells. J. Bact. 127, 61-618. LOR, P. G. & WHEALS, A. E. (1981). Variability in individual cell cycles f Saccharmyces cerevisiae. jf. Cell Sci. 5, 361-376. NURSE, P. M. (1981). Genetic cntrl f the yeast cell cycle: a reappraisal f start. In The Fungal Nucleus (ed. K. Gull & S. Oliver), pp. 331-345. Cambridge University Press. POWELL, E. O. (1956). An imprved culture chamber fr the study f living bacteria.7'^- micrsc. Sc. 75, 235-243. PRINGLE, J. R. & HARTWELL, L. H. (1981). The Saccharmyces cerevisiae cell cycle. In The Mlecular Bilgy f the Yeast Saccharmyces (ed. J. N. Strathern, E. W. Jnes& J. R. Brach), pp. 97-142. New Yrk: Cld Spring Harbr Labratry. SAMOKHIN, G. P., LIZLOVA, L. V., BESPALOVA, J.., TITOV, M. I. & SMIRNOV, V. N. (1981). The effect f a-factr n the rate f cell cycle initiatin in Saccharmyces cerevisiae. Expl Cell Res. 131, 267-275. SHILO, B., SHILO, V. & SIMCHEN, G. (1976). Cell cycle initiatin in yeast fllws first rder kinetics. Nature, Lnd. 264, 767-769. SHILO, B., SHILO, V. & SIMCHEN, G. (1977). Transitin prbability and cell cycle initiatin in yeast. Nature, Lnd. 267, 648-649. SINGER, R. A. & JOHNSTON, G. C. (1981). Nature f the Gl phase f the yeast Saccharmyces cerevisiae. Prc. natn. Acad. Sci. U.SA. 78, 33-333. SLATER, M. L. (1973). Effect f reversible inhibitin f NA synthesis n the yeast cell cycle. J. Bact. 113, 263-27. SMITH, J. A. & MARTIN, L. (1973). cells cycle? Prc. natn. Acad. Sci. U.SA. 7, 1263-1267. THROM, E. & UNTZE, W. (197). Mating type dependent inhibitin f NA synthesis in Saccharmyces cerevisiae. jf. Bact. 14, 1388-139. WHEALS, A. E. (1982). Size cntrl mdels f Saccharmyces cerevisiae cell prliferatin. Mlec. CellBil. 2, 361-368. (Received 2 July 1982) CEL59