Prinred in Sweden CopyrQhr N@)1977 by Academic Press, Inc. in any form reserved A// righrs of reproduction ISSN WI44827
Experimental Cell Research 105 (1977) 79-98
COORDINATION OF GROWTH WITH CELL DIVISION IN THE YEAST SACCHAROMYCES CEREVZSZAE G. C. JOHNSTON,’ Department
J. R. PRINGLE,* and L. H. HARTWELL
of Genetics, University
of Washington,
Seattle, WA 98195, USA
SUMMARY Yeast cell growth and cell division are normally coordinated. The mechanism of this coordination can be examined by attempting to dissociate the two processes. We have arrested division (with the use of temperature-sensitive cell division cycle mutants) and observed the effect of this arrest on growth, and we have limited growth (by nutritional deprivation) and observed the effect of this limitation on division. Mutants blocked at various stages of the cell cycle were able to continue growth, as evidenced by increases in cell volume, mass, and protein content. Cells that had initiated cell cycles were able to complete their cycles and arrest in Gl even when growth was very severely restricted. Under these conditions the daughter cells produced were abnormally small. Such abnormally small cells did not initiate new cell cycles (i.e., did not bud or complete any of the three known gene-controlled steps (cdc28, cdc4, cdc7) in Gl) after nutrients were restored until growth to a critical size had occurred. We have also prepared abnormally large cells (by arresting division temporarily with the appropriate mating pheromone); when such cells were allowed to bud, the buds produced were much smaller than the mother cells when cytokinesis occurred. We propose that the normal coordination of cell growth with cell division is a consequence of the following two relationships. (1) Growth, rather than progress through the DNAdivision cycle, is normally rate-limiting for cell proliferation. (2) A specific early event in G 1, at or before the event controlled by the cdc28 gene product, cannot be completed until a critical size is attained.
Cells of any particular type display a characteristic and rather narrow range of sizes. Moreover, a population of cells recovers its normal size distribution after the removal of an insult that had perturbed this distribution ([l], p. 248). Thus it seems reasonable to suppose that there exist mechanisms that coordinate the overall growth of the cell with the process of cell division, and there’ Present address: Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. ’ Present address: Division of Biological Sciences, The University of Michigan, Ann Arbor, MI 48109, USA. 6-771814
by ensure that cells of a particular type are the appropriate size. Although the relation between growth and division has been discussed for many years [2-S], the nature of the coordinating mechanisms remains obscure. A particularly thoughtful consideration of the problem was provided by Swann [4]. He defined “growth” as a “not too precise shorthand word for those synthetic processes that do not appear, as yet, to be immediately connected with division and which provide the bulk of the new cytoplasm”. Swann pointed out that growth and division must be relatively independent pro&p
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cesses, since a variety of insults, such as the presence of inhibitors or the absence of nutrients, could result in changes in cell size of several-fold. More recent observations have strengthened this concept, and have been formalized by Mitchison in a model that portrays the cell cycle as composed of two loosely coupled cycles, the “growth cycle” and the “DNA-division cycle” (see [1], pp. 244-249). For the bacterial cell, events in the DNA-division cycle include DNA replication, nuclear body segregation, and septation; the growth cycle consists of macromolecule synthesis and the production of division proteins [l, 61. For the yeast cell, events in the DNA-division cycle would include the duplication and segregation of the nuclear plaque, bud emergence, DNA replication, nuclear migration and division, cytokinesis, and cell wall separation [ 1,7-91; the growth cycle would include the synthesis of most macromolecules and the bulk of the cell wall. The coordinating mechanisms that couple the growth cycle to the DNA-division cycle must prevent two types of imbalance between the two cycles. (1) It is necessary to prevent cells from becoming too small; (2) it is necessary to prevent cells from becoming too large. Abnormally small cells would result from continued division in the absence of a normal amount of growth. This problem could be avoided if the completion of some event(s) in the DNA-division cycle were dependent on growth beyond some minimum size. In the two most obvious possibilities, either the initiation of cell cycles or division itself (i.e., mitosis, cytokinesis, and cell separation) would be dependent on the attainment of some minimum cell size. The existence of a control mechanism of this type would be revealed by the observation that cells limited for Exp CdlRes I05 (1977)
growth would arrest at some specific point or points in the DNA-division cycle. Such a control mechanism would be sufficient to coordinate growth and division if growth, rather than progress through the DNAdivision cycle, were always rate-limiting for cell proliferation. (That is, if under various environmental conditions the individual cells could always traverse DNA-division cycles more rapidly than they could double their masses, protein contents, etc.). The problem of becoming too large would not arise, and the control mechanism would prevent cells from becoming too small. Abnormally large cells would result from continued growth in the absence of division. This problem could be avoided if growth beyond some maximum size required the completion of some specific event(s) in the DNA-division cycle. The existence of such a control mechanism would be revealed by the observation that cells arrested at some step(s) in the DNAdivision cycle would cease growth. A control mechanism of this type would be sufficient to coordinate growth and division if progress through the DNA-division cycle, rather than growth, were always rate-limiting for cell proliferation. (That is, if under various environmental conditions the individual cells could always double their masses, protein contents, etc., more rapidly than they could traverse DNA-division cycles.) The problem of becoming too small would not arise, and the control mechanism would prevent cells from becoming too large. The two types of control mechanism mentioned are of course not mutually exclusive, and a combination would prevent cells from becoming either too large or too small. In the work reported here, we have attempted to test for the existence of these
Coordination of growth with cell division two types of control mechanism in cells of the yeast Saccharomyces cerevisiae. The possibility that growth is dependent upon events in the DNA-division cycle has been examined by arresting the DNA-division cycle at various specific steps with the aid of temperature-sensitive cell division cycle (cdc) mutants [7, 8, lo] and then determining the effect of this arrest upon growth. The possibility that events of the DNAdivision cycle are dependent upon growth has been tested by limiting growth through nutritional deprivation and examining the effect of this limitation upon the completion of events in the DNA-division cycle. In addition, we have addressed the question of whether growth or progress through the DNA-division cycle is rate-limiting for cell proliferation in yeast. It is obviously necessary in this analysis to distinguish events of the DNA-division cycle from those of the growth cycle. In addition to the events enumerated above, we tentatively attribute the steps mediated by the temperature-sensitive cdc gene products to the yeast DNA-division cycle. Cdc mutants are identified by the criterion that upon a shift to the restrictive temperature an initially asynchronous population of cells arrests synchronously within the sequence of morphologically and biochemically defined events that comprises the DNA-division cycle [8, 10, 111. The definition of the events that comprise the growth cycle is little better today than at the time of Swann’s review [4]; they remain more or less by default those events that do not comprise the DNA-division cycle. In experiments with mass populations of cells, we have monitored dry mass, protein content, and volume as measures of growth; these three parameters appear to be coupled more closely with each other than they are with the events of the DNA-division
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cycle. In some experiments it has been desirable to monitor the growth responses of individual cells. In these cases we have been limited, for technical reasons, to using volume alone as a measure of growth. EXPERIMENTAL
PROCEDURE
Strains The prototrophic diploid and haploid strains C276 ala and X2180-IALI have been described oreviouslv r12. 131,as have haploid strain A364Aa [li] and the ternperature-sensitive cell division cycle (cdc) mutants derived from it r7, 8, 10. 111.The temperature-sensitive haploid photoirophs GJ298-8 (cd&),‘GJ299-16 (cdc 7), GJ296-3 (c&25) and GJ297-1 (c&28) were constructed from crdsses Between EM-63 (OL,?zis2), kindly provided by G. Fink, and the appropriate cdc mutant.
Media YEPD solid medium and the rich liquid medium YM-1 have been described previously r141. However. our YM-I contained 20 g/l glucose. %o types of synthetic liquid medium, both based on the formulation of Wickerham [15], were used. The first (MIN) contained, per liter of medium: 1 g (NH&SO,, 0.5 g MgS0,x7Hz0, 0.87 g KH,PO,, 0.12 g K2HP04, 0.1 g NaCI, 0.1 g CaCl,xZH,O, 10 pg HaOar IO pg KI, 10 pg FeCl,x6H,O, 10 pg CuCl,x2H,O, 10 +g ZnCl,. 4 mg inositol, 0.8 mg calcium pantothenate, 0.8 mg pyridoxine hydrochloride, 0.8 mg thiamine hydrochloride, 4 pg biotin, 8.3 g succinic acid, 5 g NaOH, and 20 g glucose. To make nitrogen-free medium (MIN-N), the (NH&SO, was omitted, and an additional 1.8 g/l of MgSO&7Hz0 were added. To make phosphorous-free medium (MIN-P), the KH,PO, and K,HPO, were replaced by 0.59 g/l of KCl. To make sulfur-free medium (MIN-S), the (NH&SO4 and MgSO,x7H,O were replaced by 0.81 g/l NH,CI and 0.41 all MeCl,x6H,O. The second tvne of svnthetic liquicmed&(Yfi) contained, per i;ter of hedium: 1.6 g Difco Yeast Nitrogen Base w/o amino acids and ammonium sulfate, 1 g (NH4)$OI, 10 g succinic acid, 6.7 g NaOH, and 20 g glucose. To make nitrogen-free medium (YNB-N), the (NH&SO, was omitted. Synthetic media were supplemented with the appropriate nutrients when auxotrophic strains were used.
Culture conditions All cultures were incubated in Erlenmever flasks with vigorous rotary shaking either at room temperature (23°C or 2S”C) or in a water bath held at 36°C or 38°C.
Measurement of cellular parameters Procedures for determinations of numbers of cells and of proportions of unbudded cells [16, 171,for measurements of dry weights by filtration [17], and for timeErp Cell Re.\ 105 (IY77)
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lapse photography [l l] have been described previously. The volumes of individual cells were calculated from photographs by assuming the cells to be prolate spheroids [l&24]. The major axis (1) and minor axis (w; taken as the maximum width perpendicular to the major axis) were measured, and the volume (V) was taken to be V=(~/6)lwZ. To obtain absolute cell volumes, styrene beads of uniform volume (22.26 pm;; obtained from Coulter Electronics. Hialeah. Fla) were photographed and measured at the’same magnitication used for the cells. Distributions of cell volumes were also obtained by using an automatic particle size distribution analyzer (Coulter channelyzer, Coulter Electronics). The analyzer was calibrated using the 22.26 pm3 styrene beads.
Determination of protein contents Two methods were employed for the estimation of protein contents. In the first, cultures were uniformlv labeled by growth of the cells in the presence of a required W-labeled amino acid for several generations prior to the start of the experimental period. The labeling was then continued, and at appropriate times 0.5 ml samples of culture were removed to tubes containing 0.5 ml of 10% trichloroacetic acid (TCA). The precipitates were collected on glass fiber filters and washed 4 times with 15 ml aliquots of 5 % TCA. TCA solutions contained unlabeled amino acid at a concentration 10X that of the culture. Filters were dried and counted in a liquid scintillation counter with Omnifluor (New England Nuclear); the ratio of the total radioactivities of two samples.was taken as equal to the ratio of their total protein contents. Protein contents were also determined by the Lowry method, using bovine serum albumin as a standard. Samples containing approx. lo’-lOa cells were treated with 5 % TCA for 30 min at 0°C. and then centrifueed. The pellets were resuspended’in 95% ethanol and again centrifuged; the resulting pellets were resuspended in 0.5 ml of 1 N NaOH and incubated at 37°C for 12 h. (Standards were treated identically.) Cell debris was then removed by centrifugation and 0.1 ml of sample was tested for protein. The Lowry reagents were made up without NaOH, and the proportions were adjusted to give the correct final concentration of NaOH.
Isolation of small unbudded cells and of cells with small buds Cells growing exponentially in YNB medium at 23°C were collected on Millipore filters (0.45 pm pore size) and washed briefly with YNB-N medium. For the isolation of a population enriched in cells with small buds, the washed population was immediately subjected to Ludox density gradient centrifugation by the method of Shulman et al. [25]. For the isolation of small unbudded cells, the washed population was suspended in YNB-N medium and incubated for 24 h at 23aC, then subjected to Ludox density gradient centrifugation. In both cases the desired cells were the Exp CdIRes 105 (1977)
densest cells in the gradient, and were collected by puncturing the side of the tube and removing the cells with a 1 ml syringe.
Preparation and use of cxfactor The mating pheromone a factor was prepared as described by Bucking-Throm et al. [26] by Russell Chan, and was added to culture medium at a concentration sufficient to arrest budding for 6 h.
RESULTS Growth in cells arrested in the DNA-division cycle As a test of the degree to which growth is dependent on continued progress through the DNA-division cycle, cdc mutants that arrest at various points within the DNAdivision cycle [7, 8, lo] were shifted to the restrictive temperature, and growth was then monitored as increases in the mean volume, dry weight, and protein content/ cell. Data obtained with a cdc7 mutant, defective in the initiation of DNA synthesis, are typical (fig. 1; table 1). When an asynchronous, exponential culture was shifted to the restrictive temperature, cell number increased by approximately 80% (fig. 1A), as expected from the execution point of this mutant [lo, 27, 281. Volume, dry weight, and protein content continued to increase after cell number increase had ceased. After 8 h at the restrictive temperature, the average cell in the population had a volume approx. 2.6-fold greater (fig. IB), a dry weight approx. 2.6-fold greater (fig. lC), and a protein content approx. 3.5fold greater (fig. 1D) than the average cell in the population at the permissive temperature. A control experiment with strain A364A (table 1) established that the volume and dry weight/ cell did not change significantly when a wild-type strain was shifted to 36°C. The other mutants examined (table 1) were blocked in spindle plaque duplication (cdc25, cdc28, cdc33), in spindle plaque
Coordination of growth with cell division
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Table 1. Growth in cells arrested in the DNA-division cycle’
Strain
Mutant gene
Growth temp., (“C)
Ratio of final to initial volume/cell
Ratio of final to initial dry wt/cell
Ratio of final to initial protein content/cell*
::i
1.2 2.9
:.0
19041 I H131:l:l
cdc2
23 36
cdc4
H201: 14:4
cdc7
13052
cdc8
23 36 23 36 23 36 23 36 23 36 23 36 23 36 23 38 23 36 23 36 38 23 (a factor)e
428
cdcl3
7041
cdcll
5OllD6
cdc24
BR205.2A
cdc25
H185:3:4
cdc28
El7
cdc33
A364A
1.0 4.0 1.0 2.6 ::!i 1.0 2.3 1.0 3.6 1.0 5.2 1.0 1.0 1.0 3.7 1.0 1.1 1.0 1.0 1.0 2.0
0.9 3.5 1.0 2.6 1.3 2.3 1.15 2.8 1.2 2.7 0.9 3.5 1.2 1.3
::i 0.75 1.0 1.2 1.1 I.0 2.1
1.1’ 3.6c l.lC 3.5” ::i 1.1 2.8 ::t 0.75d 2.3d 1.1 1.2 0.8 3.0 0.8 1.0
o Experiments were performed as that of fig. 1. The ratios given are based on the values obtained 8 h after the shifts to 36°C. Changes in the ratios after 8 h were small (as in fig. I), and complicated by cell lysis (see text). Note that A364A had generation times of 3.6 h at 23”C, 2.5 hat 36”C, and 3.2 hat 38°C. The various mutants had generation times of 3.54.0 h at 23°C. * Except where noted, values are based on the incorporation of radioactivity into acid-precipitable material (see Experimental Procedure). ’ Protein contents were determined both by the incorporation of radioactivity and by the Lowry method. d Protein contents were determined by the Lowry method only. e One half of a culture growing exponentially in YM-1 medium was exposed to (Yfactor beginning at time 0. The ratios given were obtained 2.5 h after adding the a! factor. See fig. 2 and [30] for additional data.
separation (cd&), in nuclear DNA synthesis (cdc8), in bud emergence (cdc24), in medial nuclear division (cdc2 and cdcl3), and in late nuclear division (cdcll). With the exception of the cdc25 and cdc33 strains, all cdc mutants tested continued to grow after the arrest of cell division at the restrictive temperature. The volume, dry weight, and protein content of the average
cell attained values 2- to 4-fold greater than those of the average cell at the permissive temperature. Although the various mutants (cdd, cdcl0 and cdcll) blocked in cytokinesis are not included in table 1, it seems clear from data presented elsewhere [29] that they undergo even more extensive increases in size at the restrictive temperature. ExpCdRes
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Fig. 1. Abscissa: time after temperature shift (hours); ordinate: (A) cells/mix 10m6;(B) volume/cell (pm3); (C) dry wt/106 cells (pg); (D) protein/lOBcells (pg). O-O, 23°C culture. O-O. 36°C culture. Volume, dry weight, and protein content/cell of a cdc7 strain grown at the nermissive and restrictive temperatures. At time 0, one-half of a culture growing exuonentiallv in YNE3 medium at 23°C was shifted to the restricti;e temperature (36°C). At intervals, samples were taken from both the 23°C and 36°C cultures for the determination of cell number, the distribution of cell volumes using the Coulter channelyzer, dry weight, and protein content by the Lowry method. At 7 h after the shift to 36”C, the cells in both cultures were diluted four-fold with fresh medium, to ensure that nutrients were not limiting for growth. Experimental points are corrected by the dilution factor. Shown as functions of time are (A) cell number; (B) the volume/cell at the peak of the volume distribution curve; (C) mean dry wt/cell; (0) mean protein content/cell. (Note that because the volume distribution curves approximated normal curves for these samples, the volume/cell at the peak of the distribution curve is not grossly different from the mean volume/cell.)
asynchronous population is greater (by a factor of approx. 1.3) than the size of a cell at the time of the initiation of DNA synthesis, and our measurements of the increases in mean cell size are underestimates (by a factor of approx. 1.3) of the increase that a typical mutant cell undergoes after encountering a block in the initiation of DNA synthesis. (2) Most of the mutants display some cell lysis, especially after about 8 h at the restrictive temperature. This lysis clearly reduces the measured values of all the parameters, particularly at later times, but no attempt was made to evaluate it quantitatively. (3) Despite the effects of cell lysis most mutants did show some further increase in the measured size parameters when held at the restrictive temperature longer than 8 h. These results suggest that continued growth is not dependent on passage through any of the events of the NDA-division cycle with the possible exception of some of the early Gl events (as represented by cdc25 and cdc33). Sizes of the daughter cells produced by abnormally large mother cells
Since growth can continue when progress through the DNA-division cycle is blocked (fig. 1, table l), abnormally large cells can For three reasons these factors of in- be produced. It is important to know whethcrease are in most cases underestimates of er the progeny produced by these abnorthe increases undergone by individual mu- mally large cells are also abnormallly large. tant cells after their progress through the A temporary arrest of the DNA-division DNA-division cycle has stopped. (1) The cycle by exposure to mating pheromone initial asynchronous populations contain provides a good approach to this question, cells with buds of all sizes, whereas many since the arrested cells become exceptionof the steps in the DNA-division cycle that ally large (table 1, fig. 2; see also [30]), yet are blocked in the mutants occur in cells remain viable. The results presented in fig. that have not yet budded or possess only 2 show that the daughter cells derived from small buds [8, 10, 27, 281. For example, the the buds of these abnormally large mother mean cell size (by any parameter) in an cells were for the most part substantially Exp Cdl Kc.\ 105 (1977)
Coordination of growth with cell division
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Fig. 2. Abscissa:
cell volume (pm3); ordinate: rel. no. of cells. Volume distributions for (Y factor-treated cells and for the daughter cells produced by them. A population of strain X2180-1A growing exponentially in YM-1 medium at 23°C was exposed to (Ymating pheromone in a concentration sufftcient to prevent the initiation of new DNA-division cvcles. After about 2 h in the presence of LY factor,- cell number increase had ceased, but the Gl-arrested cells continued to grow. After 6 h in the presence of QLfactor, the cells were washed free of o factor by centrifugation in fresh YM-1
and spotted on YEPD plates at 23°C. Each cell was photographed immediately after spotting and at 20 min intervals thereafter. The volume of each large cell was determined from the photograph taken immediately after spotting; these volumes remained nearly constant during the subsequent cycle. The first bud produced by each large cell was followed until it had itself initiated a bud; it was then scored as an independent daughter cell, and its volume was determined. 0, Volume distribution for 85 OLfactor-arrested cells immediately after spotting; n , volume distribution for the 85 independent daughter cells.
larger than normal cells. (Measurements on cells of this strain growing exponentially in several different types of media have given mean volumes of about 30 pm3 for cells with small buds, with very few such cells having volumes greater than 40 pm3; cf also fig. 7.) Nevertheless, these large daughter cells were much smaller than the mother cells that produced them. A continuation of this trend would lead to the restoration of the normal size distribution within a few generations.
the DNA-division cycle, as evidenced by the presence of S-60% budded cells with buds of various sizes (fig. 3A; see, i.a. [7-9, 16, 24, 31-341 for information on the relationship between bud size and position in the DNA-division cycle). The unbudded cells present were rather homogeneous in size, and similar in size to the mother cell portions of the budded cells (fig. 3A, fig. 4). In contrast, virtually all of the cells in the starved cultures were arrested at the beginning of the DNA-division cycle, as evidenced by the presence of less than 2% budded cells (fig. 3B). Moreover, the unbudded cells were very heterogeneous in size (fig. 3B; fig. 4); in particular, nearly half of the cells from the starved cultures were smaller than virtually all of the cells from the unstarved cultures. (The small number of particles with volumes of about 10 pm3 observed with the Coulter channelyzer in the exponentially growing culture (fig. 4A) may not have been yeast cells; note that no comparable cells were ob-
Division under growth limitation As a test of the degree to which progress through the DNA-division cycle is dependent on continued growth, the ability of cells to divide was examined in nutrient-limited cultures. Typical samples of cells from exponentially growing and nitrogen-starved cultures are shown in fig. 3, and volume distributions for such cultures are given in fig. 4. The exponentially growing cultures of course contained cells at all positions in
Erp
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Fig. 3. Photomicrographs of typical samples of cells
from exponentially growing and nitrogen-starved populations. (A) Cells from a culture of strain C276 growing exponentially in MIN medium at 25°C; (B)
cells from the same population 10 h after an abrupt shift (by filtration, washing, and resuspension) to MIN-N medium. Note that all of the objects visible here seem to be viable yeast cells (cf fig. SA).
served microscopically in a similar growing culture (fig. 4B).) Similarly, sulfur-starvation and phosphorus-starvation (shifts of populations growing exponentially in MIN to MIN-S and MIN-P, respectively), or simply allowing a culture to grow to stationary phase in YM-1 medium (see fig. 5A), also yielded populations that were uniformly unbudded, and that contained large numbers of cells smaller than any of the cells present in the cultures prior to starvation. The production of abnormally small unbudded cells under starvation conditions suggests that at least some of the yeast DNA-division cycle can be completed without the normal amount of growth; cytokinesis occurs even though the bud has not reached normal size. In an effort to determine how much of the DNA-division cycle can be traversed, and how much growth occurs, under conditions of nutrient limitation, a population of cells near the beginning of the DNA-division cycle was isolated and subjected to nitrogen starvation. The isopycnic banding method (see Experimental Procedure) was used to obtain a population enriched in cells with small buds (25% unbudded cells, 65% cells with buds
whose long axes were less than f the length of the long axes of the mother cells, 10% cells with larger buds). The presumption that this population was enriched in cells near the beginning of the DNA-division cycle was confirmed by placing a sample of cells on a YEPD plate containing 0.3 M Time-lapse observations hydroxyurea. showed that approx. 63 % of the cells arrested within the fist cell cycle. Since hydroxyurea blocks DNA synthesis specifically [35], at least 63% of the tested cells were within or prior to the S phase of the cell cycle. In contrast, about 35% of the cells in the exponentially growing population were within or prior to the S phase by this criterion. Portions of the isolated population were resuspended in media with and without a nitrogen source (table 2). The population resuspended in YNB medium resumed its exponential increases in protein content and (after a short lag) in cell number. The population resuspended in YNB-N medium increased in cell number by approx. 2-fold, although the total protein content increased by less than 10% (table 2). Thus much of the DNA-division cycle can be completed
Coordination of growth with cell division
Fig. 4. Abscissa: (A) cell volume (pm3); (B) rel. cell volume (arbitrary units); ordinate: rel no. of cells. ---, Exponentially growing cultures; -, nitrogenstarved cultures. Volume distributions of cells from exponentially growing and nitrogen-starved cultures. (A) The Coulter channelyzer was used to obtain volume distributions for cells of strain X2180-1A in a culture growing exponentially in YNB medium at 23°C (---), and in a portion of this culture 24 h after an abrupt shift (by filtration, washing, and resuspension) to YNB-N medium (-). Note that the volume distribution for the growing culture includes both budded and unbudded cells; (B) individual unbudded cells from the populations pictured in fig. 3 were measured and the volume distributions plotted. The distribution for the growing population (---) is based on 69 cells, while that for the starved population (-) is based on 240 cells.
starved culture, these data do not necessarily imply that all events in the DNA-division cycle are independent of growth. Indeed, experiments in which size-heterogeneous stationary-phase populations were exposed to fresh medium suggest that the initiation of DNA-division cycles is coupled to the attainment of a critical cell size. When both the initial volume and the length of the lag period (defined here as the time until emergence of the first bud) were determined for a number of individual cells from a stationary phase population in YM-1, a striking correlation was observed (fig. 5A). The cells that were initially smaller required more time in fresh medium before they initiated DNA-division cycles (as judged by the emergence of buds; see further comments in the next section). Although phosphorous-starved, nitrogen-starved, and sulfur-starved populations were not analysed in detail in this type of experi-
Table 2. Zncreases in cell number and in protein content of populations in media with and without a nitrogen sourcea YNB medium
with scarcely any net increase in protein content, although the detailed relationship (e.g., whether there exists a point in the DNA-division cycle beyond which IZOnet increase in protein is necessary for the successful completion of the cycle) cannot be determined from these data. Growth of small cells prior to bud emergence Although the preceding data suggest that most events in the DNA-division cycle are independent of the growth that would normally occur during the cell cycle in an un-
87
YNB-N medium
Protein Time (hours) Cells/ml (pg/ml)
Protein Cells/ml @g/ml)
0 3 6*
1.5~10~ 4.00f0.16 2.3x lo6 4.25k0.23 3.2x lo6 4.1OkO.60
1.5x 106 4.OOtO.16 2.3x lo6 8.00f0.95 4.8x lo6 12.50f0.80
a A population enriched in cells with small buds was isolated from an exponentially growing population of strain X2180-1A by isopycnic banding as described in Experimental Procedure. At time 0, portions of the isolated population were resuspended in YNB and in YNB-N at 23°C and the numbers of cells and total protein contents (by the Lowry method) were then determined as functions of time. Protein values are based on 6 replicate samples for each of the 0 h points, and 3 replicate samples for each of the other points; mean va1ueskS.D. are shown. * Both cell number and protein content continued to increase exponentially in the YNB culture for at least another 8 h; neither parameter showed any further increase in the YNB-N culture. Exp Cd Res 105 (1977)
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Johnston, Pringle and Hartwell
Fig. 5. Abscissa: time until first bud appeared (mm); ordinate: (A) initial cell volume (arbitrary units);
(B) length of first cell cycle (min). Length of the lag period and of the first cell cycle in relation to the initial cell volume when stationarv phase cells were exposed to fresh medium. Strain C276 was inoculated at low cell density (less than IO” cells/ ml) into YM-I medium and allowed to grow into stationary phase at 23°C. At the time of this experiment the culture contained 99.5% unbudded cells and had been in stationary phase less than 6 h (i.e., cell number increase had ceased less than 6 h earlier). A sample of cells was diluted with water and spotted on the surface of a YEPD plate at 23°C. Four fields, containing 104 cells, were photographed immediately after spotting and at intervals of approx. 8 min thereafter. The relative volume of each cell was calculated from the initial photographs, and the time of emergence of each cell’s first bud was noted. For most of the cells the time of emergence of the cell’s second bud was also determined. (A) Initial cell volume plotted against the length of the lag period (i.e., the time until emergence of the first bud); (B) length of the first cell cycle (i.e., interval between the time of emergence of the first bud and the time of emergence of the-second bud) plotted against the length of the Ian period. (Note that in this plot each cell simply retains the position along the abscissa that it had in (A).)
ment, limited observations suggest that they behave similarly. It seems likely that the smaller cells require longer lag periods because they must grow back into the normal size range before they can initiate new DNA-division cycles. In contrast to their long lag periods, the initially small cells displayed first cell cycles (including the subsequent GI period) that were normal in duration (fig. 5B). E.rp Cdl Rr., 105 (IY77)
In order to obtain more direct evidence that the small cells present in a stationary phase population grow to a critical size prior to bud emergence in fresh medium, a population enriched in small cells was isolated from a nitrogen-starved culture and exposed to fresh medium. When the volume distribution of this population was then determined as a function of time (fig. 6), it was clear that the smaller cells initially present increased substantially in volume prior to bud emergence. (Note that even at 6 h about 15% of the cells-presumably the smallest cells that had been present initially-had still not produced first buds.) At 5.5 h, when about 70% of the cells had pro-
cell volume (pm3); ordinate: rel. no. of cells. Volume distributions for cells of strain X2180-IA at various times after the exposure of nitrogen-starved cells to fresh medium. A population enriched in small unbudded cells was isolated from a nitrogen-starved culture of strain X2180-IA as described in Experimental Procedure. The isolated population was incubated in YNS medium at 23”C, and the Coulter channelvzer was used to determine volume distributions immediately after resuspension in YNB (-; at this point the population contained IO8cells/ml and about 1-2 % budded cells), after 4 h of incubation (. . .; at this point the population contained IO6 cells/ml and about 5% budded cells), and after 6 h of incubation (---; at this point the population contained I .25x IO” cells/ml and-about 60% budded cells). Note that the distributions are plotted so that they have the same peak heights, but do not enclose the same areas.
Fig. 6. Abscissa:
Coordination of growth with cell division duced a first bud, the volumes of unbudded cells and of the mother cell portions of budded cells were determined photographically. The results in fig. 7 show that the mother cells carrying small buds (i.e., the cells that had just initiated DNA-division cycles) were more homogeneous in volume than was the population as a whole, and were significantly larger than most of the unbudded cells that had been present in the stationary-phase population. Taken together, the data of figs 6 and 7 indicate that the small cells present in a nitrogen-starved population of strain X2180-1A must, upon exposure to fresh medium, grow to a minimum volume of roughly 25 pm3 before bud emergence occurs. This volume is similar to that displayed by cells that have just undergone bud emergence in cultures of this strain growing exponentially in YNB medium. (A value of 33.6k8.2 pm3 was obtained for the exponentially growing population. In comparing these values, recall that the population of figs 6 and 7 was a selected population of the smallest cells present initially; this may account for the slightly lower mean volume at bud emergence.) Thus it seems likely that either bud emergence itself, or some still earlier event in the DNA-division cycle, is dependent upon the cell reaching a specific “initiation size”.
89
100 -
80-
60 -
20 -
10
20
30
40
50
60
70
Fig. 7. Abscissa: cell volume (pm3); ordinate: rel. no. of cells. Volume distribution of cells with small buds after nitrogen-starved cells were exposed to fresh medium. A sample of cells was taken from the population described in fig. 6 after 5.5 h of incubation in YNE3, and the volumes of unbudded cells and of the mother cell portions of budded cells were calculated from photographs. Shown are the volume distributions for the total population (entire histogram; based on measurements of 280 cells) and for the subpopulation of cells with buds whose long axes were less than f the lengths of the long axes of
Experimental Cell Research 105 (1977) 79-98
COORDINATION OF GROWTH WITH CELL DIVISION IN THE YEAST SACCHAROMYCES CEREVZSZAE G. C. JOHNSTON,’ Department
J. R. PRINGLE,* and L. H. HARTWELL
of Genetics, University
of Washington,
Seattle, WA 98195, USA
SUMMARY Yeast cell growth and cell division are normally coordinated. The mechanism of this coordination can be examined by attempting to dissociate the two processes. We have arrested division (with the use of temperature-sensitive cell division cycle mutants) and observed the effect of this arrest on growth, and we have limited growth (by nutritional deprivation) and observed the effect of this limitation on division. Mutants blocked at various stages of the cell cycle were able to continue growth, as evidenced by increases in cell volume, mass, and protein content. Cells that had initiated cell cycles were able to complete their cycles and arrest in Gl even when growth was very severely restricted. Under these conditions the daughter cells produced were abnormally small. Such abnormally small cells did not initiate new cell cycles (i.e., did not bud or complete any of the three known gene-controlled steps (cdc28, cdc4, cdc7) in Gl) after nutrients were restored until growth to a critical size had occurred. We have also prepared abnormally large cells (by arresting division temporarily with the appropriate mating pheromone); when such cells were allowed to bud, the buds produced were much smaller than the mother cells when cytokinesis occurred. We propose that the normal coordination of cell growth with cell division is a consequence of the following two relationships. (1) Growth, rather than progress through the DNAdivision cycle, is normally rate-limiting for cell proliferation. (2) A specific early event in G 1, at or before the event controlled by the cdc28 gene product, cannot be completed until a critical size is attained.
Cells of any particular type display a characteristic and rather narrow range of sizes. Moreover, a population of cells recovers its normal size distribution after the removal of an insult that had perturbed this distribution ([l], p. 248). Thus it seems reasonable to suppose that there exist mechanisms that coordinate the overall growth of the cell with the process of cell division, and there’ Present address: Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. ’ Present address: Division of Biological Sciences, The University of Michigan, Ann Arbor, MI 48109, USA. 6-771814
by ensure that cells of a particular type are the appropriate size. Although the relation between growth and division has been discussed for many years [2-S], the nature of the coordinating mechanisms remains obscure. A particularly thoughtful consideration of the problem was provided by Swann [4]. He defined “growth” as a “not too precise shorthand word for those synthetic processes that do not appear, as yet, to be immediately connected with division and which provide the bulk of the new cytoplasm”. Swann pointed out that growth and division must be relatively independent pro&p
Cell
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10.5 (1977)
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Johnston, Pringle and Hartwell
cesses, since a variety of insults, such as the presence of inhibitors or the absence of nutrients, could result in changes in cell size of several-fold. More recent observations have strengthened this concept, and have been formalized by Mitchison in a model that portrays the cell cycle as composed of two loosely coupled cycles, the “growth cycle” and the “DNA-division cycle” (see [1], pp. 244-249). For the bacterial cell, events in the DNA-division cycle include DNA replication, nuclear body segregation, and septation; the growth cycle consists of macromolecule synthesis and the production of division proteins [l, 61. For the yeast cell, events in the DNA-division cycle would include the duplication and segregation of the nuclear plaque, bud emergence, DNA replication, nuclear migration and division, cytokinesis, and cell wall separation [ 1,7-91; the growth cycle would include the synthesis of most macromolecules and the bulk of the cell wall. The coordinating mechanisms that couple the growth cycle to the DNA-division cycle must prevent two types of imbalance between the two cycles. (1) It is necessary to prevent cells from becoming too small; (2) it is necessary to prevent cells from becoming too large. Abnormally small cells would result from continued division in the absence of a normal amount of growth. This problem could be avoided if the completion of some event(s) in the DNA-division cycle were dependent on growth beyond some minimum size. In the two most obvious possibilities, either the initiation of cell cycles or division itself (i.e., mitosis, cytokinesis, and cell separation) would be dependent on the attainment of some minimum cell size. The existence of a control mechanism of this type would be revealed by the observation that cells limited for Exp CdlRes I05 (1977)
growth would arrest at some specific point or points in the DNA-division cycle. Such a control mechanism would be sufficient to coordinate growth and division if growth, rather than progress through the DNAdivision cycle, were always rate-limiting for cell proliferation. (That is, if under various environmental conditions the individual cells could always traverse DNA-division cycles more rapidly than they could double their masses, protein contents, etc.). The problem of becoming too large would not arise, and the control mechanism would prevent cells from becoming too small. Abnormally large cells would result from continued growth in the absence of division. This problem could be avoided if growth beyond some maximum size required the completion of some specific event(s) in the DNA-division cycle. The existence of such a control mechanism would be revealed by the observation that cells arrested at some step(s) in the DNAdivision cycle would cease growth. A control mechanism of this type would be sufficient to coordinate growth and division if progress through the DNA-division cycle, rather than growth, were always rate-limiting for cell proliferation. (That is, if under various environmental conditions the individual cells could always double their masses, protein contents, etc., more rapidly than they could traverse DNA-division cycles.) The problem of becoming too small would not arise, and the control mechanism would prevent cells from becoming too large. The two types of control mechanism mentioned are of course not mutually exclusive, and a combination would prevent cells from becoming either too large or too small. In the work reported here, we have attempted to test for the existence of these
Coordination of growth with cell division two types of control mechanism in cells of the yeast Saccharomyces cerevisiae. The possibility that growth is dependent upon events in the DNA-division cycle has been examined by arresting the DNA-division cycle at various specific steps with the aid of temperature-sensitive cell division cycle (cdc) mutants [7, 8, lo] and then determining the effect of this arrest upon growth. The possibility that events of the DNAdivision cycle are dependent upon growth has been tested by limiting growth through nutritional deprivation and examining the effect of this limitation upon the completion of events in the DNA-division cycle. In addition, we have addressed the question of whether growth or progress through the DNA-division cycle is rate-limiting for cell proliferation in yeast. It is obviously necessary in this analysis to distinguish events of the DNA-division cycle from those of the growth cycle. In addition to the events enumerated above, we tentatively attribute the steps mediated by the temperature-sensitive cdc gene products to the yeast DNA-division cycle. Cdc mutants are identified by the criterion that upon a shift to the restrictive temperature an initially asynchronous population of cells arrests synchronously within the sequence of morphologically and biochemically defined events that comprises the DNA-division cycle [8, 10, 111. The definition of the events that comprise the growth cycle is little better today than at the time of Swann’s review [4]; they remain more or less by default those events that do not comprise the DNA-division cycle. In experiments with mass populations of cells, we have monitored dry mass, protein content, and volume as measures of growth; these three parameters appear to be coupled more closely with each other than they are with the events of the DNA-division
81
cycle. In some experiments it has been desirable to monitor the growth responses of individual cells. In these cases we have been limited, for technical reasons, to using volume alone as a measure of growth. EXPERIMENTAL
PROCEDURE
Strains The prototrophic diploid and haploid strains C276 ala and X2180-IALI have been described oreviouslv r12. 131,as have haploid strain A364Aa [li] and the ternperature-sensitive cell division cycle (cdc) mutants derived from it r7, 8, 10. 111.The temperature-sensitive haploid photoirophs GJ298-8 (cd&),‘GJ299-16 (cdc 7), GJ296-3 (c&25) and GJ297-1 (c&28) were constructed from crdsses Between EM-63 (OL,?zis2), kindly provided by G. Fink, and the appropriate cdc mutant.
Media YEPD solid medium and the rich liquid medium YM-1 have been described previously r141. However. our YM-I contained 20 g/l glucose. %o types of synthetic liquid medium, both based on the formulation of Wickerham [15], were used. The first (MIN) contained, per liter of medium: 1 g (NH&SO,, 0.5 g MgS0,x7Hz0, 0.87 g KH,PO,, 0.12 g K2HP04, 0.1 g NaCI, 0.1 g CaCl,xZH,O, 10 pg HaOar IO pg KI, 10 pg FeCl,x6H,O, 10 pg CuCl,x2H,O, 10 +g ZnCl,. 4 mg inositol, 0.8 mg calcium pantothenate, 0.8 mg pyridoxine hydrochloride, 0.8 mg thiamine hydrochloride, 4 pg biotin, 8.3 g succinic acid, 5 g NaOH, and 20 g glucose. To make nitrogen-free medium (MIN-N), the (NH&SO, was omitted, and an additional 1.8 g/l of MgSO&7Hz0 were added. To make phosphorous-free medium (MIN-P), the KH,PO, and K,HPO, were replaced by 0.59 g/l of KCl. To make sulfur-free medium (MIN-S), the (NH&SO4 and MgSO,x7H,O were replaced by 0.81 g/l NH,CI and 0.41 all MeCl,x6H,O. The second tvne of svnthetic liquicmed&(Yfi) contained, per i;ter of hedium: 1.6 g Difco Yeast Nitrogen Base w/o amino acids and ammonium sulfate, 1 g (NH4)$OI, 10 g succinic acid, 6.7 g NaOH, and 20 g glucose. To make nitrogen-free medium (YNB-N), the (NH&SO, was omitted. Synthetic media were supplemented with the appropriate nutrients when auxotrophic strains were used.
Culture conditions All cultures were incubated in Erlenmever flasks with vigorous rotary shaking either at room temperature (23°C or 2S”C) or in a water bath held at 36°C or 38°C.
Measurement of cellular parameters Procedures for determinations of numbers of cells and of proportions of unbudded cells [16, 171,for measurements of dry weights by filtration [17], and for timeErp Cell Re.\ 105 (IY77)
82
Johnston, Pringle and Hartwell
lapse photography [l l] have been described previously. The volumes of individual cells were calculated from photographs by assuming the cells to be prolate spheroids [l&24]. The major axis (1) and minor axis (w; taken as the maximum width perpendicular to the major axis) were measured, and the volume (V) was taken to be V=(~/6)lwZ. To obtain absolute cell volumes, styrene beads of uniform volume (22.26 pm;; obtained from Coulter Electronics. Hialeah. Fla) were photographed and measured at the’same magnitication used for the cells. Distributions of cell volumes were also obtained by using an automatic particle size distribution analyzer (Coulter channelyzer, Coulter Electronics). The analyzer was calibrated using the 22.26 pm3 styrene beads.
Determination of protein contents Two methods were employed for the estimation of protein contents. In the first, cultures were uniformlv labeled by growth of the cells in the presence of a required W-labeled amino acid for several generations prior to the start of the experimental period. The labeling was then continued, and at appropriate times 0.5 ml samples of culture were removed to tubes containing 0.5 ml of 10% trichloroacetic acid (TCA). The precipitates were collected on glass fiber filters and washed 4 times with 15 ml aliquots of 5 % TCA. TCA solutions contained unlabeled amino acid at a concentration 10X that of the culture. Filters were dried and counted in a liquid scintillation counter with Omnifluor (New England Nuclear); the ratio of the total radioactivities of two samples.was taken as equal to the ratio of their total protein contents. Protein contents were also determined by the Lowry method, using bovine serum albumin as a standard. Samples containing approx. lo’-lOa cells were treated with 5 % TCA for 30 min at 0°C. and then centrifueed. The pellets were resuspended’in 95% ethanol and again centrifuged; the resulting pellets were resuspended in 0.5 ml of 1 N NaOH and incubated at 37°C for 12 h. (Standards were treated identically.) Cell debris was then removed by centrifugation and 0.1 ml of sample was tested for protein. The Lowry reagents were made up without NaOH, and the proportions were adjusted to give the correct final concentration of NaOH.
Isolation of small unbudded cells and of cells with small buds Cells growing exponentially in YNB medium at 23°C were collected on Millipore filters (0.45 pm pore size) and washed briefly with YNB-N medium. For the isolation of a population enriched in cells with small buds, the washed population was immediately subjected to Ludox density gradient centrifugation by the method of Shulman et al. [25]. For the isolation of small unbudded cells, the washed population was suspended in YNB-N medium and incubated for 24 h at 23aC, then subjected to Ludox density gradient centrifugation. In both cases the desired cells were the Exp CdIRes 105 (1977)
densest cells in the gradient, and were collected by puncturing the side of the tube and removing the cells with a 1 ml syringe.
Preparation and use of cxfactor The mating pheromone a factor was prepared as described by Bucking-Throm et al. [26] by Russell Chan, and was added to culture medium at a concentration sufficient to arrest budding for 6 h.
RESULTS Growth in cells arrested in the DNA-division cycle As a test of the degree to which growth is dependent on continued progress through the DNA-division cycle, cdc mutants that arrest at various points within the DNAdivision cycle [7, 8, lo] were shifted to the restrictive temperature, and growth was then monitored as increases in the mean volume, dry weight, and protein content/ cell. Data obtained with a cdc7 mutant, defective in the initiation of DNA synthesis, are typical (fig. 1; table 1). When an asynchronous, exponential culture was shifted to the restrictive temperature, cell number increased by approximately 80% (fig. 1A), as expected from the execution point of this mutant [lo, 27, 281. Volume, dry weight, and protein content continued to increase after cell number increase had ceased. After 8 h at the restrictive temperature, the average cell in the population had a volume approx. 2.6-fold greater (fig. IB), a dry weight approx. 2.6-fold greater (fig. lC), and a protein content approx. 3.5fold greater (fig. 1D) than the average cell in the population at the permissive temperature. A control experiment with strain A364A (table 1) established that the volume and dry weight/ cell did not change significantly when a wild-type strain was shifted to 36°C. The other mutants examined (table 1) were blocked in spindle plaque duplication (cdc25, cdc28, cdc33), in spindle plaque
Coordination of growth with cell division
83
Table 1. Growth in cells arrested in the DNA-division cycle’
Strain
Mutant gene
Growth temp., (“C)
Ratio of final to initial volume/cell
Ratio of final to initial dry wt/cell
Ratio of final to initial protein content/cell*
::i
1.2 2.9
:.0
19041 I H131:l:l
cdc2
23 36
cdc4
H201: 14:4
cdc7
13052
cdc8
23 36 23 36 23 36 23 36 23 36 23 36 23 36 23 38 23 36 23 36 38 23 (a factor)e
428
cdcl3
7041
cdcll
5OllD6
cdc24
BR205.2A
cdc25
H185:3:4
cdc28
El7
cdc33
A364A
1.0 4.0 1.0 2.6 ::!i 1.0 2.3 1.0 3.6 1.0 5.2 1.0 1.0 1.0 3.7 1.0 1.1 1.0 1.0 1.0 2.0
0.9 3.5 1.0 2.6 1.3 2.3 1.15 2.8 1.2 2.7 0.9 3.5 1.2 1.3
::i 0.75 1.0 1.2 1.1 I.0 2.1
1.1’ 3.6c l.lC 3.5” ::i 1.1 2.8 ::t 0.75d 2.3d 1.1 1.2 0.8 3.0 0.8 1.0
o Experiments were performed as that of fig. 1. The ratios given are based on the values obtained 8 h after the shifts to 36°C. Changes in the ratios after 8 h were small (as in fig. I), and complicated by cell lysis (see text). Note that A364A had generation times of 3.6 h at 23”C, 2.5 hat 36”C, and 3.2 hat 38°C. The various mutants had generation times of 3.54.0 h at 23°C. * Except where noted, values are based on the incorporation of radioactivity into acid-precipitable material (see Experimental Procedure). ’ Protein contents were determined both by the incorporation of radioactivity and by the Lowry method. d Protein contents were determined by the Lowry method only. e One half of a culture growing exponentially in YM-1 medium was exposed to (Yfactor beginning at time 0. The ratios given were obtained 2.5 h after adding the a! factor. See fig. 2 and [30] for additional data.
separation (cd&), in nuclear DNA synthesis (cdc8), in bud emergence (cdc24), in medial nuclear division (cdc2 and cdcl3), and in late nuclear division (cdcll). With the exception of the cdc25 and cdc33 strains, all cdc mutants tested continued to grow after the arrest of cell division at the restrictive temperature. The volume, dry weight, and protein content of the average
cell attained values 2- to 4-fold greater than those of the average cell at the permissive temperature. Although the various mutants (cdd, cdcl0 and cdcll) blocked in cytokinesis are not included in table 1, it seems clear from data presented elsewhere [29] that they undergo even more extensive increases in size at the restrictive temperature. ExpCdRes
IO5 (1977)
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Johnston, Pringle and Hartwell
Fig. 1. Abscissa: time after temperature shift (hours); ordinate: (A) cells/mix 10m6;(B) volume/cell (pm3); (C) dry wt/106 cells (pg); (D) protein/lOBcells (pg). O-O, 23°C culture. O-O. 36°C culture. Volume, dry weight, and protein content/cell of a cdc7 strain grown at the nermissive and restrictive temperatures. At time 0, one-half of a culture growing exuonentiallv in YNE3 medium at 23°C was shifted to the restricti;e temperature (36°C). At intervals, samples were taken from both the 23°C and 36°C cultures for the determination of cell number, the distribution of cell volumes using the Coulter channelyzer, dry weight, and protein content by the Lowry method. At 7 h after the shift to 36”C, the cells in both cultures were diluted four-fold with fresh medium, to ensure that nutrients were not limiting for growth. Experimental points are corrected by the dilution factor. Shown as functions of time are (A) cell number; (B) the volume/cell at the peak of the volume distribution curve; (C) mean dry wt/cell; (0) mean protein content/cell. (Note that because the volume distribution curves approximated normal curves for these samples, the volume/cell at the peak of the distribution curve is not grossly different from the mean volume/cell.)
asynchronous population is greater (by a factor of approx. 1.3) than the size of a cell at the time of the initiation of DNA synthesis, and our measurements of the increases in mean cell size are underestimates (by a factor of approx. 1.3) of the increase that a typical mutant cell undergoes after encountering a block in the initiation of DNA synthesis. (2) Most of the mutants display some cell lysis, especially after about 8 h at the restrictive temperature. This lysis clearly reduces the measured values of all the parameters, particularly at later times, but no attempt was made to evaluate it quantitatively. (3) Despite the effects of cell lysis most mutants did show some further increase in the measured size parameters when held at the restrictive temperature longer than 8 h. These results suggest that continued growth is not dependent on passage through any of the events of the NDA-division cycle with the possible exception of some of the early Gl events (as represented by cdc25 and cdc33). Sizes of the daughter cells produced by abnormally large mother cells
Since growth can continue when progress through the DNA-division cycle is blocked (fig. 1, table l), abnormally large cells can For three reasons these factors of in- be produced. It is important to know whethcrease are in most cases underestimates of er the progeny produced by these abnorthe increases undergone by individual mu- mally large cells are also abnormallly large. tant cells after their progress through the A temporary arrest of the DNA-division DNA-division cycle has stopped. (1) The cycle by exposure to mating pheromone initial asynchronous populations contain provides a good approach to this question, cells with buds of all sizes, whereas many since the arrested cells become exceptionof the steps in the DNA-division cycle that ally large (table 1, fig. 2; see also [30]), yet are blocked in the mutants occur in cells remain viable. The results presented in fig. that have not yet budded or possess only 2 show that the daughter cells derived from small buds [8, 10, 27, 281. For example, the the buds of these abnormally large mother mean cell size (by any parameter) in an cells were for the most part substantially Exp Cdl Kc.\ 105 (1977)
Coordination of growth with cell division
85
Fig. 2. Abscissa:
cell volume (pm3); ordinate: rel. no. of cells. Volume distributions for (Y factor-treated cells and for the daughter cells produced by them. A population of strain X2180-1A growing exponentially in YM-1 medium at 23°C was exposed to (Ymating pheromone in a concentration sufftcient to prevent the initiation of new DNA-division cvcles. After about 2 h in the presence of LY factor,- cell number increase had ceased, but the Gl-arrested cells continued to grow. After 6 h in the presence of QLfactor, the cells were washed free of o factor by centrifugation in fresh YM-1
and spotted on YEPD plates at 23°C. Each cell was photographed immediately after spotting and at 20 min intervals thereafter. The volume of each large cell was determined from the photograph taken immediately after spotting; these volumes remained nearly constant during the subsequent cycle. The first bud produced by each large cell was followed until it had itself initiated a bud; it was then scored as an independent daughter cell, and its volume was determined. 0, Volume distribution for 85 OLfactor-arrested cells immediately after spotting; n , volume distribution for the 85 independent daughter cells.
larger than normal cells. (Measurements on cells of this strain growing exponentially in several different types of media have given mean volumes of about 30 pm3 for cells with small buds, with very few such cells having volumes greater than 40 pm3; cf also fig. 7.) Nevertheless, these large daughter cells were much smaller than the mother cells that produced them. A continuation of this trend would lead to the restoration of the normal size distribution within a few generations.
the DNA-division cycle, as evidenced by the presence of S-60% budded cells with buds of various sizes (fig. 3A; see, i.a. [7-9, 16, 24, 31-341 for information on the relationship between bud size and position in the DNA-division cycle). The unbudded cells present were rather homogeneous in size, and similar in size to the mother cell portions of the budded cells (fig. 3A, fig. 4). In contrast, virtually all of the cells in the starved cultures were arrested at the beginning of the DNA-division cycle, as evidenced by the presence of less than 2% budded cells (fig. 3B). Moreover, the unbudded cells were very heterogeneous in size (fig. 3B; fig. 4); in particular, nearly half of the cells from the starved cultures were smaller than virtually all of the cells from the unstarved cultures. (The small number of particles with volumes of about 10 pm3 observed with the Coulter channelyzer in the exponentially growing culture (fig. 4A) may not have been yeast cells; note that no comparable cells were ob-
Division under growth limitation As a test of the degree to which progress through the DNA-division cycle is dependent on continued growth, the ability of cells to divide was examined in nutrient-limited cultures. Typical samples of cells from exponentially growing and nitrogen-starved cultures are shown in fig. 3, and volume distributions for such cultures are given in fig. 4. The exponentially growing cultures of course contained cells at all positions in
Erp
Cd
Rrs
IO.5 (I 977)
Fig. 3. Photomicrographs of typical samples of cells
from exponentially growing and nitrogen-starved populations. (A) Cells from a culture of strain C276 growing exponentially in MIN medium at 25°C; (B)
cells from the same population 10 h after an abrupt shift (by filtration, washing, and resuspension) to MIN-N medium. Note that all of the objects visible here seem to be viable yeast cells (cf fig. SA).
served microscopically in a similar growing culture (fig. 4B).) Similarly, sulfur-starvation and phosphorus-starvation (shifts of populations growing exponentially in MIN to MIN-S and MIN-P, respectively), or simply allowing a culture to grow to stationary phase in YM-1 medium (see fig. 5A), also yielded populations that were uniformly unbudded, and that contained large numbers of cells smaller than any of the cells present in the cultures prior to starvation. The production of abnormally small unbudded cells under starvation conditions suggests that at least some of the yeast DNA-division cycle can be completed without the normal amount of growth; cytokinesis occurs even though the bud has not reached normal size. In an effort to determine how much of the DNA-division cycle can be traversed, and how much growth occurs, under conditions of nutrient limitation, a population of cells near the beginning of the DNA-division cycle was isolated and subjected to nitrogen starvation. The isopycnic banding method (see Experimental Procedure) was used to obtain a population enriched in cells with small buds (25% unbudded cells, 65% cells with buds
whose long axes were less than f the length of the long axes of the mother cells, 10% cells with larger buds). The presumption that this population was enriched in cells near the beginning of the DNA-division cycle was confirmed by placing a sample of cells on a YEPD plate containing 0.3 M Time-lapse observations hydroxyurea. showed that approx. 63 % of the cells arrested within the fist cell cycle. Since hydroxyurea blocks DNA synthesis specifically [35], at least 63% of the tested cells were within or prior to the S phase of the cell cycle. In contrast, about 35% of the cells in the exponentially growing population were within or prior to the S phase by this criterion. Portions of the isolated population were resuspended in media with and without a nitrogen source (table 2). The population resuspended in YNB medium resumed its exponential increases in protein content and (after a short lag) in cell number. The population resuspended in YNB-N medium increased in cell number by approx. 2-fold, although the total protein content increased by less than 10% (table 2). Thus much of the DNA-division cycle can be completed
Coordination of growth with cell division
Fig. 4. Abscissa: (A) cell volume (pm3); (B) rel. cell volume (arbitrary units); ordinate: rel no. of cells. ---, Exponentially growing cultures; -, nitrogenstarved cultures. Volume distributions of cells from exponentially growing and nitrogen-starved cultures. (A) The Coulter channelyzer was used to obtain volume distributions for cells of strain X2180-1A in a culture growing exponentially in YNB medium at 23°C (---), and in a portion of this culture 24 h after an abrupt shift (by filtration, washing, and resuspension) to YNB-N medium (-). Note that the volume distribution for the growing culture includes both budded and unbudded cells; (B) individual unbudded cells from the populations pictured in fig. 3 were measured and the volume distributions plotted. The distribution for the growing population (---) is based on 69 cells, while that for the starved population (-) is based on 240 cells.
starved culture, these data do not necessarily imply that all events in the DNA-division cycle are independent of growth. Indeed, experiments in which size-heterogeneous stationary-phase populations were exposed to fresh medium suggest that the initiation of DNA-division cycles is coupled to the attainment of a critical cell size. When both the initial volume and the length of the lag period (defined here as the time until emergence of the first bud) were determined for a number of individual cells from a stationary phase population in YM-1, a striking correlation was observed (fig. 5A). The cells that were initially smaller required more time in fresh medium before they initiated DNA-division cycles (as judged by the emergence of buds; see further comments in the next section). Although phosphorous-starved, nitrogen-starved, and sulfur-starved populations were not analysed in detail in this type of experi-
Table 2. Zncreases in cell number and in protein content of populations in media with and without a nitrogen sourcea YNB medium
with scarcely any net increase in protein content, although the detailed relationship (e.g., whether there exists a point in the DNA-division cycle beyond which IZOnet increase in protein is necessary for the successful completion of the cycle) cannot be determined from these data. Growth of small cells prior to bud emergence Although the preceding data suggest that most events in the DNA-division cycle are independent of the growth that would normally occur during the cell cycle in an un-
87
YNB-N medium
Protein Time (hours) Cells/ml (pg/ml)
Protein Cells/ml @g/ml)
0 3 6*
1.5~10~ 4.00f0.16 2.3x lo6 4.25k0.23 3.2x lo6 4.1OkO.60
1.5x 106 4.OOtO.16 2.3x lo6 8.00f0.95 4.8x lo6 12.50f0.80
a A population enriched in cells with small buds was isolated from an exponentially growing population of strain X2180-1A by isopycnic banding as described in Experimental Procedure. At time 0, portions of the isolated population were resuspended in YNB and in YNB-N at 23°C and the numbers of cells and total protein contents (by the Lowry method) were then determined as functions of time. Protein values are based on 6 replicate samples for each of the 0 h points, and 3 replicate samples for each of the other points; mean va1ueskS.D. are shown. * Both cell number and protein content continued to increase exponentially in the YNB culture for at least another 8 h; neither parameter showed any further increase in the YNB-N culture. Exp Cd Res 105 (1977)
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Fig. 5. Abscissa: time until first bud appeared (mm); ordinate: (A) initial cell volume (arbitrary units);
(B) length of first cell cycle (min). Length of the lag period and of the first cell cycle in relation to the initial cell volume when stationarv phase cells were exposed to fresh medium. Strain C276 was inoculated at low cell density (less than IO” cells/ ml) into YM-I medium and allowed to grow into stationary phase at 23°C. At the time of this experiment the culture contained 99.5% unbudded cells and had been in stationary phase less than 6 h (i.e., cell number increase had ceased less than 6 h earlier). A sample of cells was diluted with water and spotted on the surface of a YEPD plate at 23°C. Four fields, containing 104 cells, were photographed immediately after spotting and at intervals of approx. 8 min thereafter. The relative volume of each cell was calculated from the initial photographs, and the time of emergence of each cell’s first bud was noted. For most of the cells the time of emergence of the cell’s second bud was also determined. (A) Initial cell volume plotted against the length of the lag period (i.e., the time until emergence of the first bud); (B) length of the first cell cycle (i.e., interval between the time of emergence of the first bud and the time of emergence of the-second bud) plotted against the length of the Ian period. (Note that in this plot each cell simply retains the position along the abscissa that it had in (A).)
ment, limited observations suggest that they behave similarly. It seems likely that the smaller cells require longer lag periods because they must grow back into the normal size range before they can initiate new DNA-division cycles. In contrast to their long lag periods, the initially small cells displayed first cell cycles (including the subsequent GI period) that were normal in duration (fig. 5B). E.rp Cdl Rr., 105 (IY77)
In order to obtain more direct evidence that the small cells present in a stationary phase population grow to a critical size prior to bud emergence in fresh medium, a population enriched in small cells was isolated from a nitrogen-starved culture and exposed to fresh medium. When the volume distribution of this population was then determined as a function of time (fig. 6), it was clear that the smaller cells initially present increased substantially in volume prior to bud emergence. (Note that even at 6 h about 15% of the cells-presumably the smallest cells that had been present initially-had still not produced first buds.) At 5.5 h, when about 70% of the cells had pro-
cell volume (pm3); ordinate: rel. no. of cells. Volume distributions for cells of strain X2180-IA at various times after the exposure of nitrogen-starved cells to fresh medium. A population enriched in small unbudded cells was isolated from a nitrogen-starved culture of strain X2180-IA as described in Experimental Procedure. The isolated population was incubated in YNS medium at 23”C, and the Coulter channelvzer was used to determine volume distributions immediately after resuspension in YNB (-; at this point the population contained IO8cells/ml and about 1-2 % budded cells), after 4 h of incubation (. . .; at this point the population contained IO6 cells/ml and about 5% budded cells), and after 6 h of incubation (---; at this point the population contained I .25x IO” cells/ml and-about 60% budded cells). Note that the distributions are plotted so that they have the same peak heights, but do not enclose the same areas.
Fig. 6. Abscissa:
Coordination of growth with cell division duced a first bud, the volumes of unbudded cells and of the mother cell portions of budded cells were determined photographically. The results in fig. 7 show that the mother cells carrying small buds (i.e., the cells that had just initiated DNA-division cycles) were more homogeneous in volume than was the population as a whole, and were significantly larger than most of the unbudded cells that had been present in the stationary-phase population. Taken together, the data of figs 6 and 7 indicate that the small cells present in a nitrogen-starved population of strain X2180-1A must, upon exposure to fresh medium, grow to a minimum volume of roughly 25 pm3 before bud emergence occurs. This volume is similar to that displayed by cells that have just undergone bud emergence in cultures of this strain growing exponentially in YNB medium. (A value of 33.6k8.2 pm3 was obtained for the exponentially growing population. In comparing these values, recall that the population of figs 6 and 7 was a selected population of the smallest cells present initially; this may account for the slightly lower mean volume at bud emergence.) Thus it seems likely that either bud emergence itself, or some still earlier event in the DNA-division cycle, is dependent upon the cell reaching a specific “initiation size”.
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Fig. 7. Abscissa: cell volume (pm3); ordinate: rel. no. of cells. Volume distribution of cells with small buds after nitrogen-starved cells were exposed to fresh medium. A sample of cells was taken from the population described in fig. 6 after 5.5 h of incubation in YNE3, and the volumes of unbudded cells and of the mother cell portions of budded cells were calculated from photographs. Shown are the volume distributions for the total population (entire histogram; based on measurements of 280 cells) and for the subpopulation of cells with buds whose long axes were less than f the lengths of the long axes of
