Journal of Cell Science 109, 1347-1357 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS3403
1347
A nitrogen starvation-induced dormant G0 state in fission yeast: the
establishment from uncommitted G1 state and its delay for return to
proliferation
Sophia S. Y. Su1, Yusuke Tanaka1,*, Itaru Samejima1,†, Kenji Tanaka2 and Mitsuhiro Yanagida1,‡
1Department of Biophysics, Faculty of Science, Kyoto University,Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606, Japan
2Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai, Showa-Ku, Nagoya 466,
Japan
*Present address: Primate Research Institute, Kanrin, Inuyama, Aichi-ken 484, Japan
†Present address: ICMB, University of Edinburgh, Darwin Building, Mayfield Road, Edinburgh EH9 3JR, UK
‡Author for correspondence
SUMMARY
Fission yeast cells either remain in the mitotic cell cycle or
exit to meiotic sporulation from an uncommitted G1 state
dependent on the presence or absence of nitrogen source in
the medium (Nurse and Bissett, 1981). We examined how
heterothallic haploid cells, which cannot sporulate, behave
under nitrogen-starvation for longer than 25 days at 26°C.
These cells were shown to enter a stable state (designated
the dormant G0) with nearly full viability. Maintaining the
dormant cells required glucose, suggesting that the cells
remained metabolically active although cell division had
ceased. They differed dramatically from mitotic and
uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were
investigated. The kinetics for reentry into the proliferative
state were delayed considerably, and the changes in cell
shape were enhanced particularly for those recovering
from extended nitrogen starvation. A part of the delay
could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.
INTRODUCTION
differentiation and leads to the formation of spores (Esposito
and Klapholz, 1981).
The meiotic program is triggered by the coordinate activity
of a number of gene products which relays the mating type heterozygosity and nitrogen starvation signals required for the
induction of meiosis (Beach et al., 1985; Egel et al., 1990; Iino
and Yamamoto, 1985a,b; McLeod and Beach, 1988; McLeod
et al., 1987; Nurse, 1985). In nutrient-rich medium, both heterothallic and homothallic S. pombe strains divide asexually.
Homothallic cells undergo frequent mating type switching
such that the resulting culture is a mixture of cells with
different mating types and are sexually competent. These cells
are induced to enter meiosis after conjugation in a nitrogendeficient environment, but cells of heterothallic strains without
their respective sexual partners in the same culture cannot do
so (Egel, 1989; Egel et al., 1990).
The present paper describes the fate of heterothallic cells
without sexual partners in the absence of nitrogen source. These
‘bachelor’ cells cease division, but cannot mate. Cells contain
an unreplicated complement of DNA characteristic of the G1
phase of the mitotic cell cycle (Costello et al., 1986). Cells may
remain at the uncommitted G1-like state prior to START for a
long time. Not much is known of their properties as they have
been scarcely investigated. We will show that these ‘bachelor’
cells arrested in the nitrogen-deprived condition are able to
survive very long periods of incubation, and enter a novel cell
The fission yeast Schizosaccharomyces pombe is an excellent
eukaryotic model organism to study the control of cell cycle
(Mitchison, 1970; Robinow and Hyams, 1989; Nurse, 1994a).
A large number of mutant strains defective in mitotic division
cycle and meiotic sporulation have been isolated. In S. pombe,
nitrogen source limitation triggers cells into the nonproliferative states. For sexually competent homothallic h90 strains or
for heterothallic haploid strains in the presence of both h+ and
h− mating types, cells undergo meiotic conjugation and
karyogamy after going through an uncommitted G1 stage for
mitotic or meiotic cell cycle (Nurse and Bissett, 1981). This
stage is the key point controlling cell cycle progression, and
takes place at the G1 phase of the mitotic cell cycle prior to
START (Hartwell, 1974). Cells in this uncommitted G1 stage
are offered a choice to either proceed through or to exit the
mitotic cell cycle. The duration for this uncommitted G1 stage
is not long. In the complete nutrient medium, the G1 phase of
S. pombe is very short. In the absence of nitrogen source, cells
divide and stay temporarily in this G1 stage before committing
to meiotic differentiation. Nitrogen-deprived S. pombe cells
can return to the proliferative mitotic cell cycle if nitrogen
source is resupplied within 4 hours after removal (Nurse and
Bissett, 1981). In the budding yeast Saccharomyces cerevisiae,
nitrogen starvation also triggers a/α diploids to initiate meiotic
Key words: G0, G1 phase, Cell polarity, Cell cycle control, Heat shock
1348 S. S. Y. Su and others
state if glucose is present. This stable cell state is perhaps metabolically active but remains dormant for cell growth and
division until the nitrogen source is replenished. These cells
display unique cellular structures visualized by light and
electron microscopy, and resistance to heat shock treatment.
Furthermore, the dormant cells show a significant delay for
return to the proliferative state when the nitrogen source is
replenished. The intermediary cell structures formed after
nitrogen replenishment are reminiscent of those of cells germi-
nating from spores. The structures of the dormant cells may
mimic spores though they require glucose for their establishment and maintenance. These results lead us to define dormant
cells as a third cell fate derived from the uncommitted G1 stage.
MATERIALS AND METHODS
Strains and culture conditions
The heterothallic haploid strain 972 h− isolate of S. pombe was used
10
100
Cells number/mL (x106)
A
80
60
40
% Survival
100
Cell number/mL (x106)
B
10
20
1
0
0
5
10
15
20
Days in EMM2-N
25
30
1
0
5
10
15
20
25
30
Hours in EMM2
Fig. 1. The transfer of S. pombe cells to nitrogen deficient medium, and their recovery to proliferative growth. (A) The cell number and
viability in nitrogen deficient medium. Viability of wild type h− strain 972 remains high during long-term incubation in nitrogen-deficient
medium at 26°C. Strain 972 was grown to log phase in EMM2 medium at 26°C then transferred to EMM2 lacking nitrogen (EMM2-N; see
Materials and Methods). The resulting culture was subsequently incubated at the same temperature for up to 28 days. At the indicated times
during the incubation period, samples were taken to determine the cell number and viability. Open circles represent cell number per ml of the
nitrogen-starved culture. Filled circles represent the percentage of viable cells. Each value for the survival curve is an average of three
determinations. (B-C) Cell number increase and DNA synthesis in cells recovering from nitrogen deficient medium. (B) Recovery of nitrogenstarved cells to exponential growth depends on the length of the starvation period. Cultures that were incubated in EMM2-N for either one day
or 24 days were replenished with nitrogen-enriched EMM2 medium and incubated at 26°C. At various times after EMM2 addition, cell density
was determined. Open circles represent the cell number of one-day starved culture; filled circles represent those of 24-day starved culture.
(C) DNA contents of cells recovering from nitrogen starvation. Nitrogen-enriched EMM2 was added to cultures that were nitrogen starved for
either one day (left panel) or 24 days (right). At the indicated times, samples were taken and flow cytometry analyses performed after yeast
cells were stained with propidium iodide. Zero hour indicates the time prior to EMM2 addition. 1C and 2C represent unreplicated DNA and
replicated DNA, respectively.
A dormant state in fission yeast 1349
A
B
10.5 µm
C
6.4 µm
D
5.8 µm
4.3 µm
Fig. 2. Prolonged nitrogen starvation leads to altered cell and nuclear shapes in fission yeast. Yeast cells grown to log phase in EMM2 at 26°C
were transferred to EMM2-N and incubated at the same temperature. Cells were fixed in glutaraldehyde (Toda et al., 1981) and their DNA
stained with DAPI after various times of nitrogen starvation. Each panel depicts representative cells in log phase (A), and cells starved for
nitrogen source after 6 hours (B), one day (C), and 24 days (D). Photo micrographs are identical fields observed by Nomarski optics (top) and
DAPI stained fluorescence microscopy (bottom). The average cell sizes from at least 40 cells are indicated for each panel. Bar, 10 µm.
Microscopy
S. pombe liquid cultures were collected by low speed centrifugation and
fixed with 30% formaldehyde as described by Hagan and Hyams (1988).
Alternatively, they were fixed in 2.5% glutaraldehyde as described pre-
A
18
16
14
12
Cell size (mm)
in this study (Gutz et al., 1974; Leupold, 1970). Unless otherwise
indicated, all experiments were conducted at 26°C. Liquid yeast
cultures were grown in YEPD medium (1% yeast extract, 2%
polypeptone, 2% glucose) or in synthetic EMM2 medium (Mitchison,
1970). Nutrient-deficient media EMM2-N, EMM2-NG and EMM2NGV are identical to EMM2 except that certain nutrients are missing.
EMM2-N lacks the nitrogen source NH4Cl, EMM2-NG lacks both
NH4Cl and the carbon source, glucose, and EMM2-NGV lacks
NH4Cl, and glucose, as well as the vitamin component. All plates
contain 1.5% agar. For nitrogen starvation, cells grown in EMM2 to
a density of 1-5×106 cells/ml were washed four times with sterile
distilled water to be free of growth medium, then transferred to an
equal volume of EMM2-N medium. Samples were taken for tests after
various incubation periods. For nitrogen replenishment, four volumes
of fresh EMM2 medium containing ample nitrogen source was added
to nitrogen-starved cultures. Samples were taken at various times for
assays after readdition of fresh medium.
10
8
6
Fig. 3. Cell elongation and nuclear division of fission yeast
recovering from nitrogen starvation. After EMM2 was added to
nitrogen-starved cells for one day or 24 days, aliquots were taken at
various time and cells were fixed by aldehyde fixation as described
(Hagan, 1988). (A) Cells were observed by Nomarski and the
average cell size was determined from at least 40 cells for both
cultures. (B) Cells were stained with DAPI after fixation and
observed by fluorescence microscopy. The percentage of binucleate
cells was determined by counting cells with two divided nuclei. At
least 200 bodies were observed for each determination. Open circles
are values obtained from cells recovering from one day of nitrogen
starvation. Filled circles are values obtained from cells recovering
from 24 days of nitrogen starvation.
4
2
0
0
5
10
15
20
15
20
Hours in EMM2
B
40
35
30
% Binucleate cells
Cell viability and heat shock sensitivity tests
Cell density of all cultures was estimated by the Microcellcounter
(Sysmex). Cell viability was determined by making appropriate
dilutions of samples with distilled water, then plating on YEPD agar
plates. After three days incubation at 26°C, viable colonies were
counted. Viability was expressed as a fraction of survived colonies in
the total number of cell bodies plated.
For heat shock sensitivity tests, samples were diluted ten-fold into
media prewarmed to 48°C and incubated at the same temperature for
30 minutes. After heat treatment, samples were cooled on ice for 5
minutes, plated on YEPD agar plates following appropriate dilution,
and incubated for three days at 26°C. Viability was determined as
described above.
25
20
15
10
5
0
0
5
10
Hours in EMM2
1350 S. S. Y. Su and others
A
Fig. 4. Recovery of cells after 24 days of
nitrogen starvation. (A) A nitrogen-starved
EMM2-N culture incubated for 24 days at 26°C
was supplemented with fresh EMM2 medium
and continued incubation at 26°C. At the
indicated times during incubation, cell samples
were taken, fixed with aldehyde, and stained
with DAPI. Nomarski and DAPI photo
micrographs were taken for the same field of
representative cells at each time point.
(B) Recovery of cells after one day of nitrogen
starvation. A nitrogen-starved EMM2-N culture
incubated for one day at 26°C was
supplemented with fresh EMM2 medium and
continued incubation at 26°C. Cell samples
were taken at the indicated times during
incubation, fixed with aldehyde, and stained
with DAPI. Nomarski and DAPI photo
micrographs were taken for the same field of
representative cells at each time point.
cells were collected, washed twice in sterile distilled water, and resuspended in 1.0 ml sterile water. Ice-cold ethanol was added slowly with
mixing to 70% ethanol, and then incubated at 4°C for 12 hours. Cells
were subsequently washed in 1.0 ml 50 mM sodium citrate (pH 7.0)
and digested with RNase (0.5 mg/ml) (Sigma, St Louis, MO) for 3
hours at 37°C. Cells were stained with propidium iodide (12.5 mg/ml)
and analyzed by FACScan (Beckton Dickinson, San Jose, CA).
RESULTS
viously (Toda et al., 1981). In either case, nuclei were stained with the
DNA-specific fluorescent probe 4,6-diamidino-2-phenylindole (DAPI).
For thin-section electron microscopy, the procedures described for the
freeze-substitution method were followed (Tanaka and Kanbe, 1986).
FACScan analysis
A modified procedure of Costello et al. (1986) was employed: 1-5×107
High cell viability after prolonged nitrogen
starvation
We first examined cell division growth of wild-type h− 972
haploids after long-term nitrogen starvation. Log phase cells
cultured in the synthetic EMM2 medium at 26°C were transferred to the same medium except that the nitrogen source
NH4Cl is absent. The cell number was estimated at various
times after nitrogen starvation by a Sysmex microcellcounter
A dormant state in fission yeast 1351
B
day-starved cells, we examined the DNA contents of living
cells at various times after transfer to nitrogen-enriched
medium. DNA of individual cells was stained with propidium
iodide and analyzed by flow cytometry analysis (FACScan,
Beckton-Dickinson) as shown in Fig. 1C. In one day-starved
cells, a G2 peak containing a 2C DNA content became more
prominent after 2-4 hours incubation in EMM2+N, indicating
that cells had begun to replicate their DNA (left). In the 24 daystarved cells, the 2C DNA peak increased after 6-8 hours incubation in EMM2+N (right). A small 1C peak remained visible
until 10-12 hours indicating that all cells had completed DNA
synthesis. Not only was the lag period to the onset of S-phase
longer for long-term starved cells, but the duration of all cells
to complete DNA replication seems longer as well.
(Fig. 1A). Following nitrogen starvation at 26°C, cells divided
twice, and then the cell number (open circles) maintained at a
constant level throughout 35 days of starvation at 26°C.
The viability of nitrogen-starved cells was determined to
assess whether the cell bodies had died after being deprived of
nitrogen source for such a long time. Plating efficiency was
scored at various days after transferring growing cells to
nitrogen-deficient medium. Surprisingly, extended nitrogen
starvation did not significantly affect the viability of wild-type
h− strains after 35 days. Cell viability was almost fully maintained at about 85-90% (filled circles; Fig. 1). These cells
appeared to stay at a resting state by nitrogen starvation, and
could fully resume vegetative growth in nutritionally complete
medium.
Lag in cell regeneration depends on the starvation
period
To determine whether the starvation period affects the return
to proliferative cell growth, the kinetics of cell number increase
were studied after adding fresh EMM2 medium which
contained NH4Cl as the nitrogen source to cells that had been
starved in the EMM2-N medium deficient of NH4Cl for one
day or 24 days at 26°C. We found that cell number started to
increase logarithmically after a defined lag period in each case
(Fig. 1B). The lag period (16 hours) was twice as long for cells
starved for 24 days (filled circles) than for cells starved for one
day (lag period, 8 hours; open circles). The longer the starvation period, the slower it seems to take for cells to begin vegetative growth.
Onset of S-phase is delayed
To assess when DNA synthesis first began in one day- or 24
Changes in cell size and shape
The cell size of h− strain 972 was affected by prolonged nitrogen
starvation. Vegetatively growing cells appear as hemispherically
capped rods with a diameter of approximately 3.5 µm and a
length ranging from 7-15 µm (average 10.5 µm; Fig. 2A)
(Johnson et al., 1989). Nitrogen depletion caused cells to shorten
in size and become round. After six and 24 hours incubation in
EMM2-N at 26°C, the cell length shortened from 10.5 µm to 6.4
µm and 5.8 µm, respectively (Fig. 2B,C). Cells appeared shorter
and more round after 24 days incubation (4.3 µm, Fig. 2D).
Conversely, nitrogen-starved cells regenerated the cylindrical cell shape when nitrogen source was replenished to the
medium. About two hours after transfer to EMM2, cells that
had been starved for one day began to grow lengthwise (open
circles; Fig. 3A), while cells that were starved for 24 days did
not start to grow until after a two to four hour delay. One daystarved cells reached an average size of 9 µm at approximately
5.5 hours, whereas 24 day-starved cells required at least 10
hours before reaching the same size (Fig. 3A). Therefore, the
time at which cells resumed their vegetative cell size depended
upon the duration of nitrogen starvation.
To determine when mitosis occurs for cells recovering from
nitrogen starvation, the number of cells with two nuclei was
monitored by examining DAPI-stained cells. The frequency of
binucleate cells in a one day-starved culture reached half its
maximum level at 7 hours after the addition of EMM2, which
was 4.5 hours earlier than that of 24 day-starved cells (Fig. 3B).
Nuclear division in recovering cells occurred at the maximum
cell size of 11 µm, consistent with a cell size requirement for
nuclear division (Fantes and Nurse, 1977, 1978; Fantes, 1977;
Nurse and Thuriaux, 1977).
1352 S. S. Y. Su and others
A
B
C
Fig. 5. Cell shape changes in cells recovering from nitrogen starvation are accompanied by changes in nuclear morphology. After 28 days
incubation in EMM2-N, nitrogen-enriched EMM2 was added to a nitrogen-starved culture to stimulate cells to resume proliferative growth at
26°C. Nomarski (top) and DAPI stained (bottom) photo micrographs are shown for cells deprived of nitrogen source for 28 days (A), cells
recovering from nitrogen starvation after 18 hours incubation in EMM2 (B), and log phase growing cells that had fully recovered from nitrogen
starvation after 26 hours in EMM2 (C). Bar, 10 µm.
As depicted in the regeneration of 24 day-starved cells (Fig.
4A), the transformation of round cells to rod-like cells goes
through intermediate cell shapes. Cell growth started from one
end, resulting in a pear-like structure which first appeared at 6
hours (Nishi et al., 1978; Padilla et al., 1975). Growth
extension continued unidirectionally which made the cell
appear asymmetrical, and in some cases looks like a long-neck
vase (10-13 hours). By 15 hours, cell shape was essentially
indistinguishable from vegetative rod-like cells. Thus, the
presence of nitrogen source may first stimulate the increase of
cell length through unidirectional growth. The intermediate
pear-like cells, however, were not clearly observed during the
regeneration of one day-starved cells (Fig. 4B).
Shrinkage of chromatin structure
The interphase nuclear chromatin domain of an exponentially
growing cell appeared as a hemisphere (Fig. 2A; Toda et al.,
1981). In contrast, incubation in EMM2-N for 28 days dramatically altered the nuclear chromatin region to appear flat
and adhere close to the cell periphery (Fig. 5A, also see 2D).
These flat sheets of chromatin domain seemed to appear only
when cells were nitrogen starved for a long period of time.
Cells that were starved for 6 hours or one day did not display
flat nuclear chromatin (Fig. 2B,C). Cells that are nitrogen
starved for an extended amount of time may undergo some
kind of nuclear morphological change different from briefly
nitrogen-starved cells.
Fission yeast cells were subjected to thin-sectioned electron
microscopy in order to observe fine structures unique to
nitrogen-starved cells. Electron microscopy of cells deprived
of nitrogen source for 30 days (Fig. 6A) revealed that these
cells were filled with two kinds of organelles: electron-dense
round organelles (V) reminiscent of vacuoles, and white lipid
globules (L) (Tanaka and Hirata, 1982). The cytoplasm was
greatly reduced in volume. The nucleus (N) appeared squashed
(Fig. 6A). It appeared to associate closely with the cell
membrane. The small nucleolar region (Nu) was seen. The
nuclear volume was also reduced to approximately half the
volume of growing cells (Tanaka and Kanbe, 1986; Fig. 6B).
The outer cell surface was entirely covered with a fibrous
structure as well (Fig. 6A,F). The morphological characteristics of long-term nitrogen-starved cells resembled those of
spores (Tanaka and Hirata, 1982).
Vegetative chromatin is restored upon nitrogen
replenishment
The hemispherical nuclear shape could be restored after
nitrogen source was resupplied to the medium. During
recovery from nitrogen starvation, the chromatin sheet in longterm nitrogen starved cells was transformed into a comet-like
structure (Figs 5B and 4A, 8 hours). The frequency of these
comet-like structures increased soon after pear-shaped cells
began to form and continued to be observed until normal hemispherical nuclei became more prominent after 10 hours. In Fig.
6B, the electron micrograph for a cell after 15 hours in nitrogen
rich medium is shown. The nucleus is still somewhat asymmetric but the cytoplasm appears nearly normal for the vegetative state.
Hemispherical nuclei were observed prior to the appearance
of binucleates for cells recovering from long-term nitrogen
starvation, suggesting that the restoration of nuclear shape may
be necessary before nuclear division occurs (Fig. 3B). For one
day-starved cells, most nuclei remained hemispherical at all
stages during recovery to vegetative cell division (Fig. 4B).
The flat-sheet and comet-like nuclear chromatin could be
unique for the recovery of long-term starved cells. The regeneration processes from the nitrogen-deprived state may depend
on the reformation of the hemispherical chromatin region.
Nutrient requirement for entry into a nitrogenstarved dormant state
The above results strongly suggest that haploid heterothallic S.
A dormant state in fission yeast 1353
A
B
V
Nu
pombe can enter a particular cell state (designated dormant G0
state hereafter) after an extended period of nitrogen starvation.
These cells were characterized by distinct cell and nuclear morphologies, and by a retarded response to nitrogen-enriched
medium. We addressed whether the dormant state can be established in other culture conditions to determine the minimum
nutrient requirement. We assayed the viability of cells at
various times after vegetative h− 972 cells were transferred to
four different culture media: (1) EMM2-N, (2) medium lacking
both nitrogen and glucose (EMM2-NG), (3) medium lacking
nitrogen, glucose and vitamins (EMM2-NGV), and (4)
distilled water. As shown in Fig. 7A, cells were viable up to
28 days at 26°C in EMM2-N medium only. Although the cell
number remained constant throughout the incubation period,
cells could not survive in EMM2-NG, EMM2-NGV or distilled
water.
Since nitrogen starvation causes cells to accumulate at G1 of
the mitotic cell cycle, we tested whether cells could establish
dormancy in the nutrient-deficient media after being synchronized at a common resting point. Log phase h− 972 cells were
first transferred to EMM2-N and incubated for one day at 26°C.
Then, the G1 arrested cultures were transferred to EMM-N,
EMM-NG, EMM-NGV and distilled water. Viability was
determined at various times following incubation at 26°C. We
found that EMM2-NG, EMM2-NGV and distilled water still
Fig. 6. Electron micrographs of cells after
long-term nitrogen starvation (A) and after
recovering from nitrogen starvation (B). Yeast
cells were thin-sectioned after freezesubstitution and subjected to electron
microscopy as described in Materials and
Methods. EMM2-N for 30 days (A). 30 daysstarved cells incubated for 15 hours in
nitrogen-enriched EMM2 (B). F, surface
fibrous structure; V, vacuole-like organelle; L,
lipid globule; N, nucleus; Nu, putative
nucleolus. Bars, 1 µm.
cannot sustain cell viability as high as that of EMM2-N after
an extended period of incubation time (Fig. 7B). All cells were
virtually dead after 28 days. Therefore, nitrogen deprivation
from the culture medium promotes haploid cells to enter into
dormancy, but the carbon source, glucose, is required to
maintain the dormant state. Thus, there is a parallel relationship between this dormant yeast cell and the G0 arrested cell.
Spores are highly resistant to adverse environmental
treatment such as heat shock. We examined whether dormant
cells also become heat shock resistant as well. Yeast cells were
subjected to heat treatment for 30 minutes at 48°C at various
days after nitrogen starvation. We found that cells become
more heat resistant as the duration of nitrogen starvation
extends (Fig. 7C; filled circles, heat treated; open circles, heat
untreated). Vegetative cells were highly heat sensitive as
expected (Costello et al., 1986; Egel, 1989). Cells deprived of
nitrogen source for two days were almost as sensitive to heat
shock treatment as vegetative cells; only 60% of the cells
survived heat treatment. However, cells that were starved for
7 days or longer became extremely resistant to heat shock
(about 100% survived). Thus, nitrogen starvation for at least 7
days is necessary for cells to acquire heat resistance. Note that
the dormant cells are similar to spores in heat resistance but
differed from spores in the requirement of glucose for its
viability (Egel, 1989).
1354 S. S. Y. Su and others
B
120
120
100
100
80
80
60
EMM2-N
EMM2-NG
EMM2-NGV
H2O
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20
% Survival
% Survival
A
0
0
5
10
15
20
Days (26°C)
25
30
0
5
10
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Days (26°C)
25
30
120
100
80
% Survival
EMM2-N
EMM2-NG
EMM2-NGV
H2O
40
20
0
C
60
60
40
20
0
0
5
10
15
20
Days in EMM-N (26°C)
25
30
Fig. 7. Requirement of glucose for the establishment of dormancy and heat resistance.
(A-B) Extended viability of nitrogen-starved haploid strain h− 972 requires glucose and
vitamins. (A) Log phase culture in EMM2 was transferred directly into EMM2-N (open
circles), EMM2-NG (filled circles), EMM2-NGV (open squares) or distilled water
(filled squares). (B) Log phase yeast culture in EMM2 was first transferred to EMM2N. After one day incubation at 26°C, the nitrogen-starved culture was transferred to
EMM2-N (open circles), EMM2-NG (filled circles), EMM2-NGV (open squares) or
distilled water (filled squares) as well. Arrows in B and C indicate time of transfer to
nutrient deficient media. Cultures were continued to be incubated at 26°C. At each time
point, the viability of cells incubated in the respective media was determined. Values
are averages of three determinations. (C) Haploid fission yeast cells in nitrogendeficient medium become resistant to heat shock treatment. Log phase culture in
EMM2 was transferred to EMM2-N and incubated at 26°C. At various time points, two
identical aliquots were taken. One sample was treated at 48°C for 30 minutes (filled
circles), whereas a second sample was spared of heat treatment (open circles). Plating
efficiency was determined for each sample after appropriate dilution. Values are
averages of three determinations.
DISCUSSION
Fission yeast cells either remain in the mitotic cell cycle or exit
to meiotic differentiation from an uncommitted G1 point
dependent on the presence or absence of a nitrogen source
(Nurse and Bissett, 1981; Egel, 1989). In this report, we
propose that cells enter the dormant G0 phase from this uncommitted G1 state after long-term nitrogen starvation in the
absence of sexual partners (Fig. 8A). This G0 dormant state
defines a third cell state derived from the uncommitted G1
point. The other two states represent mitotic and meiotic cells.
Maintaining the dormant cells requires glucose, but not
nitrogen source. The dormant G0 cells can survive heat
treatment, but the uncommitted G1 cells cannot (Costello et al.,
1986; Egel, 1989). Dormant cells display a distinct cell
structure which is not present in the uncommitted G1 cells, and
are resistant to metabolic as well as environmental trauma.
Little is known about the kinds of gene function that are
required for the establishment of dormancy. Rum1 and Nuc2
proteins which are involved in the inactivation of Cdc2 kinase
appeared to be required for the establishment of the G1 phase
in the absence of nitrogen source (Moreno and Nurse, 1994;
Kumada et al., 1995).
Fission yeast cannot survive long-term cultivation in media
containing a limited amount of nitrogen source: a large fraction
of the cell population is dead after two weeks growth to saturation in medium containing 10 mM NH4Cl (Costello et al.,
1986). Saturated growth in 10 mM NH4Cl yields a significant
fraction of cells with a G2-like DNA content (Costello et al.,
1986), which may not be able to establish dormancy and
survive extended incubation.
The establishment of a dormant state in the absence of NH4Cl
is accompanied by changes in cell morphology: the loss of cell
polarity, the reduction of cell size, the flattening of nuclear
chromatin, and the formation of vacuole-like organelles and
lipid globules which occupy the cytoplasm. The small cell size
associated with dormant cells may be a consequence of macromolecular breakdown. The total protein level of 24 day
nitrogen-starved cells is fourfold lower than that of cells starved
for one day (unpublished result). Prolonged nitrogen starvation
may deplete the cellular protein pool to a minimum.
Heat shock resistance, nuclear chromatin shrinkage and
organelle formation are not evident immediately upon nitrogen
depletion, but are acquired through days of extended starvation. A carbon source may be required for maintaining
nitrogen-starved cells in a metabolically active state to mediate
the changes associated with the entry into dormancy. We infer
that these properties may be important for preserving the cells’
A dormant state in fission yeast 1355
Conjugation and Meiosis
A
h-
Nitrogen
starvation
Proliferative cell
division
Spore
h+
Uncommitted G1 cell
DormantGo cell
replenishment
Vegetative cell
Nitrogen
enrichment
B
Nitrogen
enrichment
IDS
CE
ND
CD
Proliferative
cell division
N-starvation mediated
dormant state
Recovery from 1 day of N-starvation
IDS
CE ND
CD
HOURS in EMM2
0
5
IDS
10
15
CE ND
20
CD
Recovery from 24 days of N-starvation
viability when encountering environmental trauma. The
distinct flattened chromatin observed in dormant cells may
represent a higher-order structure that is transcriptionally
closed and shielded from damage. The chromosome organization of log phase and transcriptionally silent regions has been
shown to be different (Piñon, 1978; Orlando and Paro, 1995;
Rivier and Pillus, 1994; Roth, 1995; Wolffe, 1994). It remains
to be determined how chromatin structure is actually flattened
in the dormant nucleus.
The flat chromatin state is clearly not terminal. Once a
nitrogen source becomes available, it can be easily converted
back to decondensed hemispheres as cells reenter proliferative
growth. Remarkably, the flat chromatin changes into a cometlike structure during regeneration prior to S phase entry. The
change may mark a transition from a transcriptionally inactive
to an active state, and from a nonreplicative state to a replicative state.
Analogous to nitrogen-starved yeast cells, serum-starved
mammalian cells also acquire a lag before they commit to proliferative growth (Zetterberg and Larson, 1985; Augenlicht and
Baserga, 1974). Serum or growth factor exposure first leads to
a burst of gene expression from immediate early genes, and
then is followed by the induction of delayed early genes
25
Fig. 8. Three different cells states of S.
pombe. (A) Developmental pathways of S.
pombe yeast mediated by nitrogen
starvation. S. pombe cells normally
reproduce asexually in nutrient complete
media. Nitrogen starvation stimulates cells
to enter either of two developmental
pathways. In the presence of sexual
partners, cells of the opposite mating type
are induced to conjugate followed by
meiotic divisions which lead to spore
formation. Alternatively, in the absence of
sexual partners, ‘bachelor’ cells are
promoted to enter into a dormant state with
a unique chromatin structure. When either
spores or dormant cells are replenished with
nitrogen source, both are able to resume
proliferative cell division possibly through
an intermediate which display unidirectional
growth extension and comet-shaped nuclei.
(B) A diagrammatic illustration of the
kinetics for the resumption of nitrogenstarved cells to proliferative growth. The top
panel describes the return of dormant cells
to proliferative cell division by the
replenishment of nitrogen source to starved
cells. The bottom panel illustrates the
kinetics required for one day- and 24 daystarved cells to undergo the first cell
division after incubation in nitrogenenriched EMM2. The timings for the
initiation of DNA synthesis (IDS), and the
times cells reach the half maximum values
for cell elongation (CE), nuclear division
(ND), and cell division (CD) are indicated.
(Hershman, 1991). Some of these genes encode putative transcription factors, metabolic gene products and cytoskeletalmatrix proteins that may be important for triggering cell
growth and mediating morphological changes associated with
proliferating cells (Bravo, 1991; Bürger et al., 1994; Wick et
al., 1994). It is of interest to determine whether similar gene
products made by induced transcription mediate the transition
from G0-like dormancy to proliferation.
As illustrated in Fig. 8B, yeast cells stimulated to resume
mitotic cell cycle undergo cell elongation (CE), DNA synthesis
(IDS), nuclear division (ND) and cell division (CD) at a slow
rate when cells are recovering from the dormant G0 state. A
number of reasons for this delay may be considered. Small
cells cannot undergo DNA synthesis and nuclear division until
a critical cell size (or cell mass) has been attained (Fantes,
1977; Nurse and Thuriaux, 1977; Russell and Nurse, 1986,
1987). Another possibility is that cells may require a longer
time to resynthesize and reorganize proliferative cell structures, since the degree of nuclear and cytoplasmic structural
changes is extensive in long-term starved cells. The restoration
of cell shape may rely on coordinate activities of several
protein kinases as well as phosphatases, which have been
shown to affect cell shape by regulating cytoskeletal organiz-
1356 S. S. Y. Su and others
ation (reviewed by Nurse, 1994b; Matsusaka et al., 1995;
Kinoshita et al., 1996).
The kinetics of dormant cells resuming vegetative growth is
similar to those of germinating spores. The nitrogen starvationmediated resting state may in some ways resemble the resting
state in spores. The lag period for germ tube appearance (5-6
hours) coincides with the initiation of DNA replication. The
duration of germination is between 5 and 10 hours, and first
cell division occurs between 10 and 14 hours. The timings for
these events in germination (Nishi et al., 1978; Padilla et al.,
1975; Shimoda and Nishi, 1982) are consistent with the timing
for those of recovering dormant cells.
Exponentially growing cells undergo mitosis at the same
time when the cell size remains constant at about 13-14 µm
(Mitchison and Nurse, 1985; Thuriaux et al., 1978). Cells
recovering from dormancy, however, undergo the first mitosis
at approximately 11 µm while they continue to grow in size up
to 13-14 µm. Hence events that couple cell elongation and
nuclear division may be altered in recovering cells.
We are grateful to Ms Ikuyo Mizuguchi for assistance in electron
microscopy. This work was supported by a grant for Specially
Promoted Research from the Ministry of Education, Science and
Culture.
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(Received 22 December 1995 - Accepted 28 February 1996)