The effect of CO2 on the timing of cell cycle events in fission yeast
Schizosaccharomyces pombe
B. NOVAK, J. HALBAUER and E. LASZL6
Department of Agricultural Chemical Technology, Technical University of Budapest, 1521 Budapest Gellert ter 4, Hungary
Summary
The effect of CO2 removal on the cell cycle phases
of Schizosaccharomyces pombe has been examined in minimal, aspartate-containing and complete medium. The removal of CO2 shortened the
G2 phase of the cell cycle and arrested the cells in
Gi phase in minimal medium. The Gx block
caused by C0 2 deprivation was demonstrated by
transition-point and flow-cytometry analyses. The
slow-down of anapleurotic CO2 fixation might be
responsible for this effect, as aspartic acid could
abolish the Gi block. The shortening of G2 phase
in the wild-type cells was observed in every
medium irrespective of whether the growth rate
was changed or not. The experiments in which
growth rate was not changed by CO2 shift-down
suggest that this CO2 effect can be independent
from its action on CO2-fixing steps in metabolism.
Introduction
There are basically two different ways of looking at the
cell cycle (Mitchison, 1971; Kirschner et al. 1985).
The first sees it as a dependent sequence of events
leading to DNA synthesis and mitosis and the size
controls over them (Fantes & Nurse, 1981). The other
sees it as a master oscillator which can be described as a
limit cycle type oscillation (Kauffman & Wille, 1975).
There is a substantial amount of evidences that cell size
controls the cycle time of growing systems (see e.g.
Fantes, 1977), therefore size control models are more
preferable (Mitchison, 1977). However there are some
clear indications for persistent periodicities after the
cell division cycle has been blocked, which fits into the
clock model (Mitchison, 1984; Novak & Mitchison,
1986, 1987).
We have tried to resolve this discrepancy between the
models in a limit cycle type oscillation model, which is
based on the tricarboxylic acid (TCA) cycle completed
Journal of Cell Science 89, 433-439 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Therefore we propose that CO2 inhibits mitosis in
fission yeast and we explain the proportionality
between growth rate and cell size at mitosis found
by Fantes & Nurse by this CO2 inhibition. The
larger CO2 production in fast-growing cells leads
to a higher CO2 concentration, which could exert
a stronger inhibition of mitosis. A wee mutant,
which has lost its mitotic size control, also shows
the Gi block after CO2 deprivation, but its mitosis
is insensitive to CO2. Comparing the respiration
of wee and wild-type cells we conclude that CO2
inhibits the citric acid cycle in the wild type. The
consequence of these results in the regulation of
fission yeast cell cycle is discussed.
Key words: CO2, fission yeast, Schizosaccharomyces
pombe, cell cycle, wee mutant, size control.
by the CC>2-fixing anapleurotic reactions (Novdk &
Liszl6, 1986). In this model the cell size influences the
period of the oscillator through the intracellular CO 2
concentration in a negative manner (the larger cells
have shorter cycle times). A very simple consequence
of this model is that the reduction of intracellular CO 2
concentration switches off the oscillator and stops the
cell cycle. Therefore we have examined the effect of
CO2 removal on the proliferation of Schizosaccharomyces pombe. The fission yeast is a very attractive
organism for studying the effect of CO 2 because it does
not have the two characteristic enzymes of the glyoxalate shunt (Flury, 1973), which is an alternative way for
filling up the TCA cycle, e.g. in budding yeast (Beck &
von Meyenburg, 1968).
The regulation of the fission yeast cell cycle can be
described by two size controls (Nurse & Fantes, 1981;
Fantes, 1984). One acts just before DNA synthesis, at
the start of the cycle, and it is cryptic in fast-growing
cells (Nurse & Thuriaux, 1977; Nasmyth et al. 1979).
433
The cells also have to attain a critical size before mitosis
but the actual size is a function of nutritional status or
growth rate (Fantes & Nurse, 1977). Our results
support these findings, but they also show that the
relation between the critical cell size for mitosis and
growth rate could be indirect, because the growth rate
could influence mitosis through the intracellular CO2
concentration.
Materials and methods
Strains and nutrients
The wild-type and the wee 1.6 mutant of Scizosaccharomyces
pombe was a gift from James Creanor (Department of
Zoology, University of Edinburgh). The wild-type strain
972 h~ was originally obtained from Professor U. Leupold
(Bern). The mutant weel.6 divides at about half the size of
the wild-type cell (Thuriaux et al. 1978).
Edinburgh Minimal Medium (EMM3) was used in most of
the experiments (Creanor & Mitchison, 1982). In some
experiments the NH4CI in EMM3 was replaced by aspartic
acid at 5 g l " ' and the pH adjusted to 5-4 (EMM+ASP). In
others, the medium was supplemented with 5 g l ~ ' yeast
extract (EMM + YE).
Strategy of experiments
Cultures were grown overnight in a fermentor at 35°C to
mid-exponential phase (to 5X 10 5 -lXl0 6 cellsml" 1 ). The
fermentor containing about 0 5 1 yeast suspension was stirred
and aerated, and the change in dissolved O2 and CO2
concentration was measured as described below. The exponential growth was followed for about one generation time,
which seemed to be enough to determine the specific growth
rate of the culture. The dissolved O2 concentration was held
constant at 80 % of saturation value by changing the aeration
intensity. After this time the CO2 was flushed out by aflowof
N2 (flow rate ~4001 h" 1 ) through the medium. The dissolved
O2 concentration was not changed during the experiment so
some air was mixed with the N2 flow.
Elimination of the CO2 in the medium reduces the
intracellular CO2 concentration by increasing the diffusion
flux between the cell and its environment.
Measurements of cell number and cell size
distribution
Cell number was measured after mild sonication with a
Laborscale (Medicor, Hungary) electronic particle counter
with a 70-^m aperture. The mean cell volume (V) was
estimated from the cell volume distribution obtained with the
PSA-1 channel-analyser (Medicor, Hungary) by the following equation:
V =
'-^-L
where iV, is the number of cells and A", the cell volume in the /
channel (0 < / < 64): A, = 3-265/.
DNA flow cytometry
Up to now the DNA content of fission yeast has only been
analysed by flow cytometry in one laboratory (Beach et al.
434
B. Novak et al.
1985; Costello et al. 1986): our sample preparation procedure
is basically the same as described in these papers. Samples of
cells (~10 7 cells) were mildly sonicated, collected by centrifugation, washed free of growth medium and suspended in
40 % ethanol. The cells were fixed in 70 % ethanol for at least
24 h at 4°C. Thereafter the cells were washed with lml
005 M-phosphate buffer (pH 7-5) and resuspended in 1 ml of
the same buffer. They were treated with preboiled RNase
(Reanal, Hungary) at 1 mgrnl" concentration for 90min at
37°C. After centrifugation the cells were stained at 4°C for at
least 15min in 2ml solution containing: l g l ~ ' sodium
citrate, 0-58gl~' NaCl and 50mgl~' ethidium bromide.
Total fluorescence intensities were measured in an FC
200/4800A-50 instrument (Biophysics Inc) equipped with a
5W argon-ion laser giving 120 mW at 488 nm.
Light to the fluorescence amplifier was filtered using a
600 nm filter and the analyses were based on the accumulation
of 10s cells. The mean DNA content of the cells was
calculated from the distribution with a similar equation used
for calculating the mean cell size.
In some experiments the digestion by RNase was omitted
on a portion of the sample to obtain the total ethidium
bromide fluorescence of nucleic acids. The comparison of
two samples showed that fluorescence from DNA is negligible compared to the RNA. It is not surprising since the
DNA/RNA ratio is 1/100 in fission yeast (Mitchison & Lark,
1962).
Other techniques
The dissolved O2 and CO2 concentration was determined
with electrodes sensitive to these gases (Radelkis, Hungary).
The aeration of the culture was disrupted for a short time
(~10min) and the rate of respiration was determined from
the slope of O2 concentration decrease. It is impossible to
measure the respiration rate during the intensive aeration
period.
The optical density (o.D.) of the culture was measured at
595 nm on a Specord M40 photometer (Zeiss, Jena).
Results
Fig. 1 shows the reaction of wild-type yeast to the
removal of CO2 after some time in exponential growth
in three independent experiments. The downward
pointing arrow indicates the start of flushing out and
the upward pointing arrow marks the time at which the
N2 flow was switched off. The CO2 concentration and
the mean cell size curves belong with the lower cell
number curve.
The reduction of CO2 concentration causes two
dramatic changes in the cell density curves. The rate of
cell division accelerates and the cell number remains
constant after it has reached about double the value it
had at the start of the CO2 reduction. Thus all the cells
divide once again in the absence of CO2 with a rate
higher than normal. In parallel the mean cell size
decreases from the 90 fim value characteristic of
balanced growth to 60 /im 3 .
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Fig. 1. The growth of wild-type cells in EMM3 at 35 °C in
the presence and in the absence of CO^. The N2 flow was
switched on at the downward pointing arrows and switched
off at upward pointing arrows. Curves A-C, cell number;
1 arbitrary unit = 10s cells ml" 1 . Curve D, specific
respiration rate; 1 arbitrary unit = 10~8^l C^celF 1 min" 1 .
Curve E, mean cell volume. Curve F, partial pressure of
CO2 in the medium.
After switching off the N 2 flow, the CO 2 starts to
increase together with the cell size, but the cell number
remains constant for about a cycle time (~2-5h).
Curve D in Fig. 1 shows the specific respiration of
wild-type before CO2 deprivation and after the CO2
shift up.
The stage in the cell cycle at which a cell cycle block
arrests the cells can be estimated from the residual cell
number increase after the block, based on a theoretical
age distribution (Cook & James, 1964). Cells that at the
time of shift are before this 'transition' point cannot
divide, but cells that are over this stage can complete
their cycle and divide (Nurse et al. 1976). The mean
residual cell number increase after CO 2 reduction from
eight independent experiments gives a —0-18 transition
point, which is a good estimate for a Gi block.
This conclusion is strengthened by DNA flowcytometry analysis of the population. Fig. 2 shows the
change in the cell number and in the mean DNA
content of the cells calculated from flow-cytometry data
in another experiment. In fast-growing fission yeast
populations, almost all the cells have a 2C (G 2 ) DNA
content, because the time of DNA synthesis coincides
with cell division (Mitchison & Creanor, 1971). Therefore the decrease of the cellular DNA content to nearly
half of its original value is a clear indication that most of
the cells have changed to a Gi DNA content. Fig. 3
shows the cellular DNA distribution of the exponentially growing and the CO2-deprived cultures.
The bottom curve on Fig. 2 shows the fraction of the
cells in the population carrying a septum (Septal
Index, SI). This septum is formed shortly after
mitosis, therefore the SI gives an estimate similar to the
mitotic index. The rapid increase in SI after the
reduction of CO 2 indicates that the cells accelerate into
mitosis. It means that the faster division rate is due to
the shortened G 2 phase rather than to the shortened
period between mitosis and cell division. After switching off the N 2 flow, the mean DNA content and the SI
rise transiently above the value characteristic for the
balanced growth, because the culture shows some
degree of synchrony.
The top curve in Fig. 2 is the change in the optical
density, which gives an estimate proportional to cell
number and cell size. After the CO 2 shift-down the
optical density reaches a plateau after a transient and
slight increase in its slope. During the transient increase in slope the cell number and the cell size change
in the opposite way (see Fig. 1), therefore the optical
density curve does not give a real estimate for the
specific growth rate. However the lack of increase in
optical density indicates that the absence of CO 2
inhibits cell growth and reduces the specific growth
rate to zero or very close to it. A plausible explanation
for this is that the reduced level of CO 2 limits the
Effect of CO2 on cell cycle events in S. pombe
435
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Fig. 2. The growth of wild-type cells in EMM3 at 35°C in
the presence and in the absence of CO2. Curve A, optical
density; 1 arbitrary unit = 2X 10~3A. Curve B, cell
number; 1 arbitrary unit = 105 cells nil"'. Curve C, mean
DNA content. Curve D, septal index.
growth rate through CO2-fixing reactions. The acceleration into the mitosis could be a consequence of the
reduced growth rate, according to the model of Fantes
& Nurse (1977).
The experiments with EMM + ASP medium were
done to test the hypothesis that anapleurotic CO2
fixation is the rate-limiting step at the reduced CO2
level. Aspartic acid can be transaminated to oxaloacetate and so it can fill up the TCA cycle independently of the CO2 fixating anapleurotic reactions.
Fig. 4 shows the effect of CO2 on proliferation of wild
type in EMM + ASP. The reduction of CO2 makes little
difference to the slope of the optical density and does
not stop the cell number increase. Thus the presence of
aspartate abolished the G| block caused by CO2
436
B. Novak et al.
10
Fig. 4. The growth of wild-type cells in EMM + ASP
medium at 35 °C in the presence and in the absence of
CO2. Curve A, optical density; 1 arbitrary unit = 10~ A.
Curve B, cell number; 1 arbitrary unit =
8 5 X 10s cells ml"'. Curve C, mean cell volume.
Curve D, septal index.
16
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Fig. 5. The growth of wild-type cells in EMM+YE
medium at 35 CC in the presence and in the absence of
CO2. Curve A, optical density; 1 arbitrary unit = \0~zA.
Curve B, cell number; 1 arbitrary unit = 5x 105 cells ml" 1 .
Curve C, mean cell volume. Curve D, septal index.
deprivation. However the drop in CO2 caused a rise in
SI and a transient acceleration in cell division, indicating that the reduction of CO2 shortened the G2
phase as in minimal medium.
There is no parallel relationship between the growth
rate change and the extent of acceleration into mitosis
after the CO 2 shift down. In Fig. 4 the acceleration into
mitosis is nearly as large as in Fig. 2, while there is only
about a 10% fall in growth rate in EMM+ASP in
contrast to the 100% decrease in growth rate in EMM.
This suggested that the level of mitotic size control is
not growth rate related but it is a function of the CO2
concentration. We tried to test this in the following
way. The minimal medium was supplemented with
yeast extract. This complete medium contains the
precursors of biosynthetic reactions; the CC>2-fixing
reactions cannot therefore be rate limiting even though
they are slowed down. Fig. 5 shows the growth of cells
Fig. 6. The growth of iveel.6 cells in the presence and in
the absence of CO2. Curve A, cell number; 1 arbitrary
unit = 2'5X 10°. Curve B, cell number; 1 arbitrary unit =
6-66X 105 cells ml" 1 . Curve C, cell number; 1 arbitrary
unit = 10 cells ml" . Curve D, mean cell size.
Curve E, specific respiration rate; 1 arbitrary unit =
in EMM+YE; CO 2 deprivation has no effect on the
slope of optical density and cell number increase. The
only thing that happened was that the cells were
advanced into mitosis and there was a rise in SI and a
step in cell number. This result shows that the CO^
concentration influences the mitotic size control independently from its action on the growth rate.
It was interesting to examine the effect of CO2 on
wee cells, which have lost their mitotic size control
because of a mutation in a single gene (Nurse, 1975).
Fig. 6 shows the results with weel.6 cells in three
independent experiments. The mean cell size and the
specific respiration curves belong with the bottom cell
number curve. The cell number increase after the CO2
reduction is less than in wild type. The mean of five
experiments give a 0-39 transition point which agrees
well with the position of S phase in these cells
(Dickinson, 1983). It means that the absence of CO 2
Effect of CO2 on cell cycle events in S. pombe
437
causes a Gi block in wee cells similar to that in
wild-type cells. However the shift-down in CO2 did not
cause any increase in division rate and SI (data not
shown), thus wee cells were not advanced into mitosis.
The genetic lesion in the mitotic size control makes the
mitosis insensitive to CO2.
The bottom curve on Fig. 6 shows the specific
respiration rate of weel.6 cells before CO2 deprivation
and after the CO2 shift up. The initial low value of this
rate after CO2 deprivation indicates that respiration has
been slowed down by the reduced CO2 level. This can
be explained by the limited rate of anapleurotic reactions which fill up the TCA cycle responsible for
generating NADH. The rise in CO2 results in a fast
increase in respiration which can be due to the acceleration of the TCA cycle caused by CO2 fixation.
A comparison of Fig. 1 and Fig. 6 shows that the
specific respiration rate of wild type is almost half that
of ivee cells. It is interesting that the specific CO2
production rate of wild type is exactly double that of
wee 1.6 (Novdk & Mitchison, 1986). It means that the
respiration quotient of wee (RQ = 3) is much smaller
than that of wild type (RQ = 10). Therefore the TCA
cycle is more active compared to glycolysis in wee cells
than in wild type.
Discussion
Our results indicate that CO2 has a dual effect on the
fission yeast cell cycle in minimal medium. Reduction
of CO2 in the medium rapidly accelerates the cells into
mitosis by shortening of the G% phase of the cell cycle.
Depriving the cells of CO2 also causes them to accumulate in Gi phase as we have shown with the transition
points of wild-type and ivee cells and with the flowcytometry data. The Gi block seems to be a nutrientdeprivation response because it correlates with the
cessation of growth. COvfixing reactions, which slow
down at reduced CO2 concentration, could be behind
this effect. CO2 fixing steps occur at different points in
the metabolism of a heterotrophic cell: TCA cycle
filling up (anapleurotic) reactions, in lipid biosynthesis
and degradation, in purine and pyrimidine biosynthesis (see e.g. Biggers & Bellve, 1974). Since aspartic
acid almost completely abolishes the G\ block caused
by CO2 deprivation, anapleurotic reactions may be the
most sensitive among these reactions. This suggests
that the replenishment of the TCA cycle is necessary to
complete the Gi and to initiate the S phase.
The other observed effect of CO2 decrease is the
shortening of G2 phase. The acceleration of cells into
mitosis by shift-down of CO2 has been observed in
every medium in wild type cells, but not in wee cells. It
has been also detected when CO2 was decreased to a
value that did not lengthen the cycle time.
438
B. Novak et al.
A similar shortening of G 2 and acceleration of
mitosis has been found in nitrogen shift-down experiments (Fantes & Nurse, 1977). These were consistent
with the mitotic delay found in shift-up experiments
done by changing the nitrogen, carbon or phosphate
source. Fantes & Nurse (1977) interpreted these results
as showing that the cell size control over mitosis could
be modulated by the nutritional status or growth rate.
They did not find any mitotic acceleration or delay in
weel cells in shift experiments (Fantes & Nurse, 1978),
so they concluded that the signal generated by growth
rate is transmitted by the weeJ+ gene product to
control the entry into mitosis.
Our results confirm these findings and their interpretation with the one exception where growth rate
is not changed by CO2 shift-down in Fig. 5. Therefore
we propose that CO2 could be an element of the growth
rate monitoring system and it could mirror the nutritional status. It is well known that the specific rate of
metabolic reactions is a function of the specific growth
rate, showing a minimal (maintenance) value in the
non-growing state. It means that in faster growing and
dividing cells the CO2-production rate is larger than in
slow growing cells. The larger CO2 production leads to
a higher CO2 concentration in the cell and the intracellular CO2 concentration is proportional to the
specific growth rate. Thus the cells could 'measure'
their growth rate through the intracellular CO2 concentration. Since CO2 inhibits mitosis, the faster-growing
cells might divide their nuclei only at a larger cell size.
Although the molecular mechanism of this mitotic
inhibition caused by CO2 is completely unknown, CO2
is a well-known inhibitor of decarboxylases (Jones &
Greenfield, 1982). The TCA cycle from pyruvate to
succinyl-CoA contains three decarboxylating enzymes,
which could be the targets of CO2 action. The derepressed TCA cycle activity found in ivee 1.6 cells fits
into the picture well, because the mitosis of wee cells is
insensitive to CO2 and their TCA cycle is more active
compared to wild type.
We thank Professor Murdoch Mitchison for his continuous
interest during the work, useful suggestions and for reading
the manuscript. We thank Dr Paul Nurse and Dr P. A.
Fantes for critical reading the manuscript. We also thank Dr
Zolt<in Marcsek at United Research Organization of the
Hungarian Academy of Sciences and the Semmelweis Medical University for the flow cytometry measurements and
Liszld SzilSk for technical assistance.
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Effect of CO2 on cell cycle events in S. pombe
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