Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases

Article (PDF Available)inPhilosophical Transactions of The Royal Society B Biological Sciences 366(1584):3508-20 · December 2011with19 Reads
DOI: 10.1098/rstb.2011.0124 · Source: PubMed
Abstract
In dividing fission yeast Schizosaccharomyces pombe cells, the balance between Wee1 kinase and Cdc25 phosphatase which control the cyclin-dependent kinase (CDK) at the G2-M transition determines the rod-shaped cell length. Under nitrogen source starvation or glucose limitation, however, cell size determination is considerably modulated, and cell size shortening occurs for wild-type cells. For several mutants of kinases or phosphatases, including CDK, target of rapamycin complex (TORC) 1 and 2, stress-responsive mitogen-activated protein kinase (MAPK) Sty1/Spc1, MAPK kinase Wis1, calcium- and calmodulin-dependent protein kinase kinase-like Ssp1, and type 2A and 2A-related phosphatases inhibitor Sds23, this cell shortening does not normally occur. In tor1 and ssp1 mutants, cell elongation is observed. Sds23 that binds to and inhibits 2A and 2A-related phosphatases is synergistic with Ssp1 in the cell size determination and survival under low glucose and nitrogen source. Tor2 (TORC1) is required for growth, whereas Tor1 (TORC2) is needed for determining division size according to different nutrient conditions. Surprisingly, in growth-diminished tor2 mutant or rapamycin-treated cells, the requirement of separase/Cut1-securin/Cut2 essential for chromosome segregation is greatly alleviated. By contrast, defects of tor1 with secruin/cut2 or overproduction of Cut1 are additive. While Tor1 and Tor2 are opposite in their apparent functions, both may actually coordinate cell division with growth in response to the changes in nutrients.
Review
Nutrient limitations alter cell division
control and chromosome segregation
through growth-related kinases
and phosphatases
Mitsuhiro Yanagida
*
, Nobuyasu Ikai
, Mizuki Shimanuki
and Kenichi Sajiki
The G0 Cell Unit, Okinawa Institute of Science and Technology (OIST) Promotion Corporation,
Tancha 1919-1, Onna, Okinawa 904-0412, Japan
In dividing fission yeast Schizosaccharomyces pombe cells, the balance between Wee1 kinase and Cd c25
phosphatase which control the cyclin-dependent kinase (CDK) at the G2M transition deter mines
the rod-shaped cell length. Under nitrogen source starvation or glucose limitation, however, cell size
determination is considerably modulated, and cell size shortening occurs for wild-type cells. For sev-
eral mutants of kinases or phosphatases, including CDK, target of rapamycin complex (TORC) 1 and
2, stress-responsive mitogen-activated protein kinase (MAPK) Sty1/Spc1, MAPK kinase Wis1, cal-
cium- and calmodulin-dependent protein kinase kinase-like Ssp1, and type 2A and 2A-related
phosphatases inhibitor Sds23, this cell shortening does not normally occur. In tor1 and ssp1 mutants,
cell elongation is observed. Sds23 that binds to and inhibits 2A and 2A-related phosphatases is syner-
gistic with Ssp1 in the cell size determination and survival under low glucose and nitrogen source.
Tor2 (TORC1) is required for growth, whereas Tor1 (TORC2) is needed for determining division
size according to different nutrient conditions. Surprisingly, in growth-diminished tor2 mutant or
rapamycin-treated cells, the requirement of separase/Cut1-securin/Cut2 essential for chromosome
segregation is greatly alleviated. By contrast, defects of tor1 with secruin/cut2 or overproduction of
Cut1 are additive. While Tor1 and Tor2 are opposite in their apparent functions, both may actually
coordinate cell division with growth in response to the changes in nutrients.
Keywords: Tor1; Tor2; Ssp1; protein phosphatase; separase; starvation
1. INTRODUCTION
In the book ‘The biology of the cell cycle’, Mitchison [1]
wrote that one of the basic questions to ask about the
cell cycle is what is the pattern of overall cell growth
between one division and the next. This question still
remains. It is surprising how little we know about the
basic mechanism of cell growth in the relationship to
the cell cycle. Mitchison pointed out the criteria that
can be used for measuring cell growth; they are cellular
volume, constituents including water, total dry mass,
macromolecular dry mass and low molecular weight
compounds pool, protein and other macromolecules
such as RNA, DNA and carbohydrates. Precise
measurements of these parameters have actually not
advanced well technically since the time of writing the
book, so that growth data are often missing or not
satisfactory in many cell cycle studies. The fission
yeast Schizosaccharomyces pombe, a eukaryotic micro-
organism, is convenient for estimating the cell volume
by simply measuring cell length as its cell shape in the
vegetative phase is rod-like, so Mitchison pioneered
the use of S. pombe as a eukaryotic model for under-
standing growth versus cell cycle. The growing phase
(e.g. cell length increase) of S. pombe in the standard
(rich) culture medium occurs after DNA replication,
whereas the cell length is constant during the phases
of mitosis and cell division [2,3].
Thuriaux et al.[4] and Nurse & Thuriaux [5] isola-
ted S. pombe mutants that were thought to be altered in
the control coordinating cell division with cell growth.
More than 50 mutant strains—most severely altered in
this control—were isolated, which showed the same
growth rate as wild-type, but divided at a much shorter
cell size. The great majority of the mutants were
genetically mapped within the single wee1 locus (wee
means little), and the remaining one mutant turned
out to be an allele of cdc2, originally called wee2.At
that time, Wee1 and Cdc2 were predicted to be
involved in a control initiating mitosis when the cell
attains a critical cell length. The wee1
þ
gene was pos-
tulated to code for a negative element or inhibitor,
and cdc2
þ
to code for a positive element or activator
in the mitotic control. We now know that these
elements are indeed cell cycle-regulating protein kinases;
* Author for correspondence (myanagid@gmail.com).
Present address: Kobe University School of Medicine, Kusunoki-cho,
Kobe 650-0017, Japan.
One contribution of 16 to a Theme Issue ‘The cell cycle’.
Phil. Trans. R. Soc. B (2011) 366, 3508–3520
doi:10.1098/rstb.2011.0124
3508 This journal is q 2011 The Royal Society
Wee1 directly phosphorylates Cdc2 and inhibits the
kinase activity. The original mutation wee2-1 (cdc2-w1)
escapes the negative regulation by Wee1 resulting in pre-
mature division with regard to cell size. The success in
identifying Wee1 as the negative regulator of mitotic
entry came from the genetic screen for mutants display-
ing the strongest wee phenotype. In addition, the growth
rate was shown to be normal in these mutants, separating
the growth issue from the cell cycle control. In retro-
spect, there were a number of mutants that showed the
semi-wee phenotypes, which were wisely not investigated
at that time. After 30 years since the discovery of wee1
mutants, however, the time may be ripe to shed light
on broad mutations that produce the less severe, ‘wee-
like’ phenotypes, many of which may include the defects
in growth and/or cell cycle control.
Cdc25, another important regulator for mitotic
entry, was discovered by Fantes [6] through the analy-
sis of interactions between wee and various cdc (cell
division cycle) mutants. The block of mitotic entry or
the prolonged G2 interphase caused by a defective
cdc25 allele is suppressed when combined with the wee
mutants. Suppression of the temperature-sensitive (ts)
cdc25 phenotype by wee1 is almost complete. Other
cdc2-w mutations (e.g. cdc2-3w) are sensitive to Wee1
function, but largely abolish Cdc25 requirement.
Cdc25 turned out to be a protein phosphatase [7, 8]
that competes with Wee1 and is an activator of Cdc2
by dephosphorylating the tyrosine residue (Y15) of
Cdc2. Not only cdc25, cdc13 (mitotic cyclin mutant)
and most cdc2 ts alleles are blocked at the boundary
of G2 M transition. Note that the loss of Cdc25
and Cdc2 Cdc13 blocks mitotic entry but not cell
growth, leading to the formation of highly elongated
cells arrested in the G2M boundary but continuing
growth. The loss of cyclin-dependent kinase (CDK)
activation disrupts the cell cycle control and also affects
the cell size determination as clearly exemplified by
wee1 mutation. It is obvious, though often forgotten,
that the cell size is strongly affected by cell cycle control,
growth control or both. In wee1 mutant cells, growth is
not inhibited, but prematurely committed mitosis and
following cytokinesi s take precedence over growth to
produce small cells.
2. EXTENSIVE SHORTENING OF CELL SIZE
OCCURS BY DIVISION UNDER NITROGEN
DEFICIENCY
Wild-type S. pombe cells respond to nutri tional change by
changing the cell size. When S. pombe is transferred from
the complete synthetic Edinburgh Minimal Medium
(designated EMM2) to EMM2 N lacking the nitrogen
source (NH
4
Cl), cells can divide approximately twofold,
an approxima tely fourfold increase in number in the
absence of the gro wth phase, producing short and round
cells, which are arr ested at the G1 phase (figure 1a,b;
[912]). Note that the EMM2 medium has no amino
acids, so that NH
4
Clisthesolenitrogensource.These
divisions in EMM2 N are thought to occur and resulting
quiescent cells are maintained through recycling the
intra cellu lar nitrogen sources. If cell populations are com-
petent for meiosis in the EMM2 N medium, then cells
exit from the G1, conjugate with other mating type cells,
and irrev ersibly commit meiotic divisions. If cell popu-
lations are heterothalic (mono-sexual), howev er, then
solitary cells enter the G0 phase at around 12 h and
remain viable in the quiescent phase for quite long times
(grea ter than one month). The capability to mate with
opposite mating type cells is lost upon entry into the G0
phase. The size of small G0 cells remains constant. The
nitrogen-starvation-induced G0 cells thus lack both
gro wth and division, but are metabolically activ e [12,13].
During nitrogen source deficiency (designated
N-starvation hereafter)-induced divisions, the reduc-
tion of cell size occurs from the average 12 mm long
rod of vegetative cells in EMM2 to 5 mm diameter
round-shaped G0 phase cells in EMM2 N (figure 1b).
The short bars in figure 1b represent the timing of mito-
sis prior to the first and second cell division. Cell length
shortening from mother (blue) to daughter (blue, red)
and grand-daughter (red, green) is clearly shown.
Note that the growth phase (cell length increase) is miss-
ing in these divisions. The calculated ellipsoidal cell
volume and the protein content measured for these
small G0 cells are 1/3 and 1/6, respectively, of those of
vegetative cells [14]. These small G0 cell features
are not called the wee phenotype as they are seen in
N-starvation, and, besides, cells are arrested while the
wee1 mutant cells grow and divide. G0 cells are round-
shaped, suggesting that the cytoskeletal architecture is
altered from that of wee1 in the presence of nitrogen
source. Having stated these differences, there may
exist some parallel: accelerated mitoses occur twice in
the EMM2 N medium prior to the arrest. Hence,
during nutrient deficiency, wild-type cells try to make
a final division before quiescence, which occurs prema-
turely at a time when the cell size is still small. The wee
phenotype therefore may represent the commitment of
mitosis under starvation conditions. In other word,
N-starvation can produce wee-like cells.
Microarray and high-resolution analysis of tran-
scripts indicate that one half of approximately 5000
whole genome genes had significantly changed levels
by simply removing NH
4
Cl from the medium [14,15].
Mitosis under N-starvation was analysed by movies
(figure 1c). Two example of living cells (A and B) were
observed by colour-tagged histone H2A-RFP (red,
chromatin), alpha-tubulin-GFP (green, microtubule;
[16]) and Sid4-GFP protein (green) localized at the
spindle pole body (SPB; [17]). They divided at a time
interval of approximately 3 h and were arrested (e.g.
B11, B12, B21 and B22). The mode of cell division
was normal except for the absence of growth, and the
decline of cell length by division. The anaphase spindle
was short owing to the shortened cell length, or
occasionally elongated as the curved form.
How is Wee1 kinase involved dur ing the nitrogen-
deficiency-induced ‘premature’ division? Paradoxi-
cally, Wee1 kinase is required for the meiotic entry
under N-starvation, and phosphorylates the Y15 resi-
due of Cdc2 (CDK1) kinase [18]. The activity of
Wee1 is thus not lost. Hence, while not identified,
other kinases and phosphatases may be involved in
cell size shortening under N-starvation. The state of
Cdc2 kinase in the G0 phase is presumably inactive
with abundant Rum1, an inhibitor of Cdc2 [14,19],
and by Cdc2 Y15 phosphor ylation [20].
Review. Cell size control and growth M. Yanagida et al. 3509
Phil. Trans. R. Soc. B (2011)
3. Sty1 AND Ssp1 IN ADDITION TO Cdc2
ARE REQUIRED FOR SHORTENING UNDER
N-STARVATION
To identify mutants that are defective in cell size short-
ening under N-deficiency, 600 ts strains were searched
for any that remained rod-shaped while the cells divided
[13]. Only six classes of mutants remained rod-shaped
while they divided before the arrest at 268C, the per-
missive temperature. Genetic analyses indicated that
these have mutations in protein kinase genes that are
implicated in cell cycle control and stress-responsive
signalling [13]: mitogen-activated protein kinase
(MAPK) sty1/spc1-989, MAPK kinase (MAPKK)
wis1-558,-982, CDK cdc2-974, mitotic cyclin cdc13-
563, calcium- and calmodulin-dependent protein
kinase kinase (CaMKK)-like ssp1-412. In databases,
p38-like MAPK Sty1 and MAPKK Wis1 are classified
as involved in cell cycle, cell growth, gene expression,
translation and cellular response to stress, whereas Ssp1
is involved in cell cycle, cell growth, cell morphology,
actin cytoskeleton and response to str ess . Under
N-starvation, stress-responsive Wis1 and Sty1 and cal-
cium-, calmodulin-dependent Ss p1 kinases ar e th us
coordinated wi th th e cell cycle regulator Cdc2 Cdc13
to appropriately change the cell size and shape.
Examples of cells in the nitrogen-deficient medium
are shown in figure 2a in comparison with the wild-
type control. Interestingly, mutants sty1 and wis1
retained high viability immediately after the two
rounds of divisions, but greatly lost the viability during
the transition from the transient G1 to the quiescent
G0 phase [13]. Resulting long rod-shaped sty1 and
wis1 mutant cells displayed large nuclei (figure 2b).
Other mutants (cdc2, cdc13 and ssp1) also revealed
rod-shaped cells, but retained high viability in the G0
phase at the permissive temperature, suggesting that
being rod-shaped per se during the divisions under
N-starvation does not cause the loss of viability.
Nevertheless, these different classes of protein kinases
are vital for shortening divisions under N-starvation. It is
thus plausible to speculate that Ssp1, MAPK (Sty1) and
MAPKK (Wis1) may directly or indirectly regulate
the activation of Cdc2 to cause accelerated mitosis
under N-starvation. In the published movies of sty1-
989 and wis1-982 mutant cells, the growth phase clearly
exists during the division cycles in EMM2 N medium,
while cells keep their rod shape [13]. Sty1 and Wis1 are
thus essential for the cessation of growth in N-starvation.
It is truly surprising how sty1-andwis1-deficient cells
manage to commit divisions while keeping an apparent
growth phase without an outsource of nitrogen. One
possible reason for such aberrant growth might be due
to the use of an abundant carbon source (glucose)
for cell size increase, while a nitrogen source may be
G1
G0
meiosis
18
16
14
12
10
cell length (μm)
8
6
4
2
0 1234
time (h)
5678
nitrogen source
deficiency
Α
Α
Α1
Α1
Α11
Α11
Α11
Α11
Α11
Α11
Α12
Α12
Α12
Α21
Α21
Α21
Α21
Α21
Α22
Α22
Β22
Β22
Α22
Α22
Α22
Α2
Α1
Α2
Β1
Β1
Β1
Β1
Β1
Β1
Β11
Β11
Β12
Β12
Β21
Β21
Β2
Β2
Β2
Β2
Α2
Β2
Α2
Β2
Α2
Β
3.0 4.0 4.5 5.0
00(a)(c)
(b)
0.5 1.0 2.0
6.0 6.5 7.0 8.0
Β
Β
Β
Figure 1. Nitrogen source deficiency-induced cell size shortening of wild-type. (a) Schizosaccharomyces pombe wild-type cells
under the absence of nitrogen source (NH
4
Cl) divide twice and arrest at a temporal G1 phase followed by meiosis or
the entry into quiescent G0 phase dependent on the presence or absence of mating. The orange bar represents the spindle.
(b) Cell length is shortened dramatically during two divisions after transfer (time 0) from the complete EMM2 to the
NH
4
Cl-deficient EMM2 medium. (c) Light micrographs of time course changes of the two cells in the complete medium
(time 00 h) shifted to the nitrogen-deficient medium for 8.0 h. The first division produced daughter cells A1, A2 and B1,
B2 cells. The second division produced A11, A12, A21, A22 and B11, B12, B21, B22 cells. Red, histone H2A; green, tubulin
and the Sid4 SPB protein (see text).
3510 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
available by recycling. Indeed, the rate of glucose con-
sumption is high in sty1 mutant cells (L. Uehara &
A. Mori 2011, unpublished data). Based on the reason
why MAPK and MAPKK mutants post-division lose
their viability after entry into the G0 phase, we suspect
that the remodelling of nuclear chromatin required
for the survival during long-term G0 quiescence may
not occur, because the nuclei in these mutants were
abnormally expanded [13].
There have been a number of reports showing
the close relationships between S. pombe CDK (Cdc2
Cdc13) and stress-responsive pathway (SRP) through
various genes, such as protein kinase A (PKA), response
regulator Mcs4, Polo-like kinase Plo1 and target of rapa-
mycin (TOR) kinase [2429]. Plo1 may be important in
cell size shortening, as it regulates cytokinesis [3032].
Petersen & Nurse [27] reported that TOR kinase,
which is modulated by nutritional conditions and inhib-
ited by rapamycin, controls the entry into mitosis
through stress-responsive Sty1. However, overall under-
standing of the relationships among CDK, SRP and
TOR is still meagre. It is crucially important to set up
a clear-cut experimental system to relate growth with
the cell cycle. In this regard, the growth-absent,
size-shortening division under N-starvation is an excel-
lent model to understand the suppression of growth by
nutritional stress. This system may resemble the early
embryonic egg cleavage.
4. UNDER LOW GLUCOSE, THE semi-wee
IS INDUCED IN WILD-TYPE BUT NOT IN TWO
KINASE MUTANTS
Glucose is a source of energy for cells as well as the
source of cell structure, and the cellular mode of its util-
ization may be centrally important for understanding
cell growth, division and quiescence. We investigated
the cell divisionquiescence behaviour of S. pombe
under diverse glucose concentrations from excess, regu-
lar diet and starvation to fasting (1110 mM; [33]).
The division mode (observed under a microscopic per-
fusion system that constantly supplied the medium) was
surprisingly normal except for the shortening of cell
length (2030%) when glucose concentrations were
highly diluted (5.6 mM, 1/20 concentration of the stan-
dard culture medium that contains 111 mM (2%)
glucose). This semi-wee length-shortening property is
observed in a range of low glucose levels equivalent to
wild-type
(a)
(b)
(c) (i) (ii)
(d)
semi-wee
wild-type
wild-type
wild-type
26°C
2%
glucose
0.1%
glucose
ssp1
mutant
26°C
0.08%
glucose
wild-type
OP Ppe1, OP Ppa2
Dppa2 (PP2A)
Dppe1 (PP2A-like)
ssp1, tor1
sty1-989
cdc2-974
low glucose
concentration
cdc13-563
ssp1-412
wis1-982
wis1-558
nitrogen source
deficiency
wee-like
wild-type
high viability
cdc2, cdc13, ssp1
high viability
sty1, wis1
low viability
(large nucleus
with 2C DNA)
Figure 2. Genes required for cell size shortening under limited nitrogen or glucose. (a) In the absence of nitrogen source,
mutant cells sty1-989, wis1-982, -558, cdc2-974, cdc13-563 and ssp1-412 remained rod-shaped at 268C, the permissive temp-
erature. (b) Schematic of mutants that fail to shorten cell size upon the transfer to the culture deficient of the nitrogen
source. (c) Length of dividing wild-type cells is shortened in EMM2 medium containing low (0.1%) glucose instead of
standard 2% glucose concentration. Mutant ssp1-412 is elongated in 0.1% glucose at 268C rather than the shortening in
wild-type. A similar result is obtained for tor1 mutant cells at the semi-permissive temperature. (a,b) Based on Sajiki et al.
[13]; (c) based on Hanyu et al.[21]. (d) The phosphatase deletion mutant Dppe1 or Dppa2 results in the production of
small, round or short semi-wee cells, respectively. By contrast, overproduction of Ppe1 or Ppa2 causes the semi-cdc25
elongation phenotype [22,23].
Review. Cell size control and growth M. Yanagida et al. 3511
Phil. Trans. R. Soc. B (2011)
human blood sugar concentrations (figure 2c(i)).
Normal human blood glucose content is around
4 mM (0.08%) before breakfast. Schizosaccharomyces
pombe may be a good model, with regard to understand-
ing the cellular uptake and utilization mechanism of
normal blood glucose, which is defective in certain
patients with type II diabetes.
When glucose concentration is further reduced to a
level of starvation, the nature of division becomes
stochastic in addition to cell shortening, accompanied
by a curious epigenetic inheritance of division timing.
A sharp transition from division to quiescence takes
place in a narrow glucose concentration range (from
2.2 to 1.7 mM). Under severe glucose starvation
(1.1 mM), cells are mostly quiescent and only a
small population of cells divide. Under fasting
condition (0 mM), division is immediately arrested,
and fasting cells have a short chronological lifespan
(16 h) if the shift was abrupt. If, however, the shift
to fasting is slow, then the resulting lifespan greatly
increases. Various biomarker compounds specific
for different glucose concentrations have been ident-
ified. Glucose concentrations thus control the cell
size, the doubling time, the uniformity of cell division
pattern and even epigenetic behaviour among different
cell lineages.
It is surprising to find that the doubling time in 111
and 4.4 mM glucose is the same, but the semi-wee phe-
notype, 2030% reduction in cell size, par tly explains
the non-prolonged doubling time in low glucose.
Taken together, S. pombe has a very wide range of opti-
mal glucose concentrations for the rate of division with
regard to the doubling time. Schizosaccharomyces pombe
under low glucose may thus sacrifice the growth phase in
order to keep the same rate of increase in cell number.
Here, the linked regulation between growth and division
clearly exists. Note that glucose limitation or starvation,
or even fasting does not affect cell shape, whereas nitro-
gen starvation causes the deviation of cell shape (to
round) from rod.
A subsequent question is what kind of gene func-
tion converts the information about limited glucose
to cell size determination. Hanyu et al.[21] reported
that, under low glucose concentrations, Ssp1 kinase
described above plays an important role in the size
control; mutant ssp1 cells remain long rod in approxi-
mately 0.1 per cent glucose (2% in the regular
medium) at 268C, the permissive temperature, dis-
playing the semi-cdc25 phenotype (figure 2c(ii)). At
the semi-permissive temperature under low glucose,
the phenotype becomes severer as mutant cells fail to
divide. Furthermore, the measurements of remaining
glucose concentrations in the medium showed that
the rate of glucose consumption is considerably
slower in ssp1 mutant cells than that of wild-type
under limited glucose, suggesting that the utilization
of glucose is impaired in ssp1 mutant cells. Note that
ssp1 cells fail to reduce cell size under both nitrogen
and glucose limitation, so that it may have a broad
role for the utilization of nutrients, such as in incorpor-
ation or transport of nutrients. Indeed, Ssp1 is the cell
cortex protein. Ssp1 kinase may function in parallel
with Gsk3 kinase and oppose PP2A and PP2A-related
phosphatases [21].
Another protein kinase identified is Tor1, the cataly-
tic subunit of TORC2 kinase, the mutant of which fails
to reduce the cell length under limited glucose (figure
2c;[20]). A new tor1 substitution mutant tor1-L2045D
was constructed using the information of the ts tor2
mutation site that resides in the highly conserved phos-
phatidyl inositol kinase domain of the catalytic subunit
[34]. Only the substitution mutant tor1-L2045D dis-
played the ts phenotypes among the five different
substitutions made at the same site. This tor1-L2045D
mutant (tor1-D hereafter) grows normally at 268C but
fails to grow at 368C or under low glucose concentration
at the semi-restrictive temperature, displaying semi-
cdc25 phenotype at 368C or in low glucose at a semi-
permissive temperature (the deletion mutation Dtor1
is
more severe than the tor1-D mutation). Taken together,
two nutrient-sensitive protein kinases Ssp1 and Tor1 are
responsible for the cell size reduction in response to
limited glucose. Screening a large number of ts and del-
etion mutants grown under the low glucose identified
these mutants (details described elsewhere).
5. CAMKK-LIKE Ssp1 IS RELATED TO SIT4-LIKE
Ppe1 PHOSPHATASE, CORTEX ACTIN AND
AMPK-LIKE Ssp2 KINASE
The ssp1
þ
gene was origi nally identified as one of the
extragenic ts suppressors for the cold-sensitive (cs) del-
etion phenotypes of Ppe1 [35]. Ppe1 is a member of the
evolutionarily conserv ed type 2A-related phosphatase
family , similar to budding yeast SIT4 and mammalian
PP6 [3639]. Ssp1 kinase is involv ed in salt stress
responses as it is rapidly recruited to the plasma membrane
during high salt-induced osmotic pressur e [32,40]. While
the mutant phenotype resembles tha t of sty1 stress-
responsive MAPK mutants, Ssp1 and Sty1 do not seem
to act through the same pathway. Ssp1 controls the state
of cortical actin [32,35,40]: ssp1 mutant cells gr o w in a
monopolar fashion and arr est at the G2 M boundary,
but the relationship between Cdc2 and Ssp1 in the cell
cycle is unclear. Cortical actin distribution in growing
ssp1 mutant cells is also monopolar. Ssp1 is hence required
to promote the bipolar, rather than monopolar, cell
elongation. Overproduction of Ssp1 kinase caused the dis-
persion of actin, resulting in round cell shape [35]. By
contrast , the Dppe1 deletion mutant causes the dispersal
of actin, resulting in small round cells. Judging from the
mutant phenotypes, Ssp1 kinase and Ppe1 phosphatase
may be opposing. They might act on the same substrate
important for responding to nutrient limitation. One
candidate is Ssp2 (one of the two AMPKs, see below).
Ssp2 kinase was also identified as an extragenic
suppressor for Dppe1 [35]. Ssp2 is an S. pombe homol-
ogue of mammalian AMP-dependent protein kinase
(AMPK) and budding yeast Snf1, containing three dis-
tinct subunits [21]. AMPK is thought to be a central
player for carbohydrate catabolic processing. Schizosac-
charomyces pombe actually has two AMPK-like catalytic
subunits, Ppk9 and Ssp2, and 1 b and 1 g subunit hom-
ologues (Amk2/Spcc1919.03c and Cbs2, respectively).
The regulatory subunit Cbs2 is essential to maintain
the viability of N-starved G0 cells. The cell cycle pheno-
types of AMPK catalytic subunit mutants remain to
be investigated.
3512 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
6. ROLES OF PP2A Ppa2 AND PP2A-RELATED
Ppe1 PHOSPHATASES FOR MITOTIC ENTRY
AND CELL SIZE CONTROL
It has been known that PP2A and PP2A-related
catalytic subunits, Ppa2 and Ppe1, respectively, affect
the size of dividing S. pombe cells [22,23]. The cs pheno-
type of the Dppe1 mutant is rescued by overproduction
of the catalytic subunits of PP2A. Both Dppe1 and
Dppa2 mutants are small, but differ in shape, round
and rod, respectively (figure 2d). Schizosaccharomyces
pombe has the second PP2A catalytic subunit Ppa1.
The double mutant Dppa1 Dppa2 is lethal, producing
the small rod cells indistinguishable from wee1 [41]. If
okadaic acid is added to the culture of single mutant
Dppa2, then basically the same result was obtained. By
contrast, overproduction of Ppa2 results in the semi-
cdc25-like elongation, producing long rod cells. Taken
together, PP2A and PP2A-related phosphatases may
play similar roles in the cell size control. Judging from
cell shape of the mutants, Dppe1 might be more related
to growth defect, whereas Dppa2 is defective in mitotic
entry. Kinoshita et al.[22] showed that Ppa2 interacts
genetically with the cell cycle regulators Cdc25 tyrosine
phosphatase and Wee1 kinase in S. pombe: the Dppa2
mutant is lethal when combined with wee1-50, but par-
tially suppresses the phenotype of cdc25-22, suggesting
that Ppa2 and Wee1 may function in parallel.
In higher eukaryotes, PP2A is sharply do wnr egulated
during mitosis by greatwall kinase through its target
proteins, alpha-endosulphine and Arpp19, which are
inhibitors of PP2A [42,43]. Greatwall kinase phosphory-
lates and activates the inhibitors of the PP2A-B55delta
holoenzyme. In S. pombe, homologues of gr ea twall and
Arpp19/endosulphine are Ppk18 and Mug134, respect-
iv ely, judging from the database sear ch, wher eas in
Sacchar om yces cerevisiae, RIM15 kinase and IGO1/2
repr esent the counterparts, respectively [44]. RIM15 is
known to phosphorylate IGO1, which is required for
initiation of G0 phase. It is of considerable interest
whether these budding yeast and fission yeast counter-
parts of greatw all and alpha-endosulphine (and/or
Arpp19) also negativ ely regulate PP2A homologues
during mitosis. In S. pombe, the B55delta counter-
part subunit is probably P ab1. The Dpab1 deletion
phenotype suggests tha t Pab1, the regulat ory subunit of
PP2A, may control the polar actin distribution [45], poss-
ibly link ed to cell shape control. Our recent results indicate
that Dppe1, D
ekc1 and Dpab2 lose their viability in nitro-
gen-starved G0 cells (K. Sajiki 2011, unpublished data),
suggesting tha t these phosphatases may also be inv olv ed
in cell size and shape change under nitr ogen starva tion.
7. Sds23 IS RELATED TO DIVERSE FUNCTIONS
BY BINDING TO AND INHIBITING PP2A AND
PP2A-RELATED PHOSPHATASES
Hanyu et al.[21] rep orted that Sds23 is a key to linking
phosphatases with the utilization of low glucose and
related kinase Ssp1. Sds23 was identified as one of the
three high-copy suppressors for cs dis2-11 that is defective
in PP1 phosphatase [46]. The remaining two other sup-
pressors are Sds21, the second PP1 catalytic subunit
[47,48] , and Sds2 2, the positive regulatory subunit of
PP1 to promote metaphaseanaphase progression
[49 51]. The mammalian homologue of Sds22 is impli-
cated in cancer [52]. Sds23 is known to be related to
diverse functions through its ability as a high-copy sup-
pressor for mutants of PP1, APC/cyclosome subunits,
Ssp1 and others (figure 3a;[21,46]; Y. Hanyu & M.
Yanagida 2011, unpublished data). Sds23 is also
involved in inducing sexual development as Moc1 is
identical to Sds23 [53]. Conversely, high-copy plasmids
carrying the PP1 dis2
þ
,APC/Ccut9
þ
and ssp1
þ
gene
rescue the deletion of Sds23, so that the suppression is
reciprocal (figure 3a).
A critical finding to understand these diverse
functions of Sds23 is that Sds23 binds to PP2A and
PP2A-related phosphatases, and inhibits in vitro the
PP2A-related phosphatase activity of Ppe1 [21]. Ana-
lyses of mass spectroscopy and two-hybrid interactions
demonstrate that Sds23 is bound directly to the regu-
latory subunits Ekc1 (SAP-like) and Paa1 (the subunit
A-like, figure 3b;[21]). As the B and B
0
subunits (Pab1
and Par1, respectively) of PP2A are scarce or missing
in the immunoprecipitates, Sds23 might associate
with an intermediate assembly form of PP2A holo-
enzymes. Taken together, high-copy suppression of
many mutations by Sds23 appears to be due to the
collective negative modulation of two major phospha-
tases, PP2A and PP2A-related phosphatases, through
direct inhibition of the regulatory subunits Paa1 and
Ekc1. The inhibitory role of Sds23 is reminiscent of
the PP2A inhibitor alpha-endosulphine (IGO1/2,
Mus134) that is the target of greatwall kinase. However,
Sds23 acts in interphase or throughout the cell cycle.
Homologues of Sds23 are found in all fungi and in cel-
lular slime mould, but not in higher eukaryotes so far
[21]. It is similar to, but distinct from, the g subunit
of AMPK: both Sds23 and AMPK g subunit contain
the two cystathionine-b-synthase domains.
Sds23 is required to use low glucose: the deletion
mutant Dsds23 fails to proliferate in 0.1 per cent glucose
and slowly consumes glucose [21]. In sds23-deficient
cell extracts, the phosphatase activity greatly increases,
and is diminished by the addition of okadaic acid, an
inhibitor of PP2A and related phosphatases. The high
phosphatase activity of PP2A and PP2A-related may
hence be inhibitory to use low glucose. Ssp1 and the
phosphatases may be opposing, and Sds23 and Ssp1
synergistically cooperate to use low glucose. The down-
regulation of PP2A and PP2A-related phosphatases
appears to be required for using the low concentration
of glucose in the culture medium.
8. REVERSE CELL SIZE PHENOTYPES OF tor1
AND tor2 MUTANTS
The target of rapamycin complex (TORC) 1 and 2
exist in eukaryotes (figure 4a;[54]). A variety of cell
functions involved in cell growth in response to nutri-
tional cues are controlled by TORCs: TORCs are
thought to be the central regulators of growth upon
nutritional alterations. Rapamycin is an antiprolifera-
tive drug that prolongs the life of model animals, and
might be useful in the treatment of certain cancers.
Rapamycin bound to FKBP12 (a peptidyl prolyl
cistrans isomerase) inhibits TORC1 [55]. The mam-
malian TOR is the sole catalytic subunit, whereas
Review. Cell size control and growth M. Yanagida et al. 3513
Phil. Trans. R. Soc. B (2011)
S. cerevisiae and S. pombe contain two highly similar
catalytic subunits, Tor1 and Tor2 ( figure 4a).
Saccharomyces cerevisiae TORC1 and TORC2 med-
iate the control of many cellular events [5661]. The
C-terminal domain of catalytic subunits contains a
lipid kinase motif, which places them in the phospha-
tidyl inositol-kinase-related kinase (PIKK) family
[62]. Tor1 and Tor2 form two functionally distinct
TOR complexes [58]. TORC1, which is responsible
for many of the known TOR functions [63], contains
either Tor1 or Tor2 (figure 4a) . TORC2, which is
not sensitive to rapamycin, helps regulating actin cyto-
skeleton polarization [60], and its complex function is
less understood.
Similar to S. cerevisiae, S. pombe has two TOR
kinase genes, tor1
þ
and tor2
þ
[34,64 68]. However,
the nomenclature of TOR kinase in S. pombe is unfor-
tunate; S. pombe Tor2 is similar to S. cerevisiae Tor1,
whereas S. pombe Tor1 is similar to S. cerevisiae
Tor2. Accordingly, TORC1 and TORC2 in S. pombe
contain distinct catalytic subunits, Tor2 and Tor1,
respectively (figure 4a,b). Mass spectrometric analysis
with immunoprecipitation experiments indicated that
S. pombe TORC1 contains only Tor2 [20,34].
To couple extracellular nutrient signals with cell
growth, S. pombe TORC1 and TORC2 are reported
to be controlled by the small GTPases, Rheb1 [69]
and Ryh1 [70], respectively. Wild-type S. pombe is
insensitive to rapamycin [71]. However, S. pombe
becomes sensitive to rapamycin under conditions of
starvation [72]orintor2 mutants [34].
Each of S. pombe TORC1 and TORC2 contains four
evolutionarily conserved regulatory subunits (figure 4b;
[34]). Mip1 and Ste20 are homologues of mammalian
Raptor and Rictor, respectively, while Pop3/Wat1 is a
homologue of Lst8 that associates with both TORC1
and TORC2. The mutation site of a ts and rapamy-
cin-sensitive tor2-S is the substitution from L2048 to S
(figure 4b(ii); [34]). A new ts mutant tor1-D was con-
structed by introducing the D (aspartate) residue at
the conserved 2045L as described above. This tor1-D
mutant is useful for critical comparison between tor1
and tor2 phenotypes as the mutation sites are basically
identical (the inset of sequences; [20]). The cellular
phenotypes of tor1-D and tor2-S at 368C in the complete
medium are virtually opposite (figure 4b(iii); [20]). The
short, ellipsoidal-shaped phenotype was produced by
tor2-S, whereas the elongated cells were observed for
the tor1-D mutant, suggesting that their mode of invol-
vement in the cell size control, and possibly also in
their relation to the cell cycle, may be opposing.
9. NUTRIENT-DEPENDENT CELL SIZE CONTROL
BY Tor1 AND Tor2 KINASES
The opposite phenotypes of tor1-D and tor2-S in cell size
may be explained by the different roles of Tor1 and Tor2
in responding to nutritional signals (figure 4c). As the
currently held view on TORC1 [73], Tor2 facilitates
the intracellular use of nitrogen source and supports
growth (the increase of cell volume). In tor2-S mutants
even in the ample presence of environmental nitrogen
PP2A (Ppa1, Ppa2)
sexual
development
Sds23/Moc1
(a)
(b)
utilization of
low glucose
CaMKK
Ssp1
R
R
Ekc1
Ekc1
Ppe1
Ppa1
Pab1
Par1
Ppa2
Paa1
Paa1
A
A
Ppa1
Ppa2
Ppe1
C
C
C
B,B¢
C
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
sds23
sds23
sds23
PP1
APC/C
PP2A-related
inhibited
PP2A-related
active
PP2A
inhibited
PP2A
active
mitosis
GW
ENS
activation
freeing Sds23
PP2A-related (Ppe1)
Figure 3. Diverse roles of Sds23. (a) High-copy plasmid carrying the sds23
þ
gene suppresses the mutations of ssp1, protein
phosphatase PP1 and APC/cyclosome [21,46,51]. Sds23/Moc1 is required for the utilization of low glucose and sexual devel-
opment. (b) Sds23 stably associates with PP2A-related Ppe1Ekc1 and PP2A phosphatases (see text). C, R: catalytic and
regulatory subunits of PP2A-related phosphatase. C, A, B: catalytic, regulatory A and B subunits of PP2A. The phosphatase
free from Sds23 seems to be active [21]. GW and ENS represent greatwall kinase and alpha-endosulphine, respectively
(see text).
3514 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
source, protein and nucleic acid synthesis become
defective, resulting in the inhibition of growth, but
one or two rounds of the size-shortening divisions
occur, reminiscent of the wild-type cell behaviour in
N-starvation. If tor2-S cells that arrested in the G0
phase at 268C are shifted to 368C in the presence of
nitrogen source, then cells do not exit from the G0
phase [34]. These results indicate that the small cell
size of tor2-S at 368C is due to the residual rounds of
cell division in the absence of growth. As this phenotype
at 368C suggests, Tor2 is surely required for growth, but
it is uncertain how it relates to cell cycle control.
While Tor2 is essential, Tor1 is dispensable. The
phenotype of tor1-D is bec omes clearly defective under
low glucose (less than 0.1%): tor1-D mutant is defec tiv e
in shortening cell size under low glucose, like ssp1
mutants (figure 4c). The failure to reduce the cell
length under glucose limitation leads to semi-cdc25-like
cell elongation for tor1-D [20]. Tor1 and Ss p1 may
thus se nse low glucose and reduce the cell len gth through
regulating mitosis and cytokinesis. In this regard, Tor1 is
like a cell cycle-controlling gene. Indeed, the genetic
interaction of the deletion Dtor1 with cdc25 mutation
was reported [74,75].
The timing of cell cycle events in tor1-D after the
release from the G0 phase was monitored. DNA repli-
cation occurred with the same timing as the wild-type,
but the entry into mitosis and subsequent cytokinesis
is delayed, resulting in the increase of cell size [20].
The delay is due to the delay in Cdc2 mitotic acti-
vation, as Y15 phosphorylation of Cdc2 remains in
elongated cells. Interestingly, actin was abundant at
the single cell tip in the interphase of tor1-D mutants.
Bipolar cell elongation seems to be absent in tor1-D
mutant cells. The intensity of actin at the equator
was also high, whereas myosin makes a normal-looking
contractile ring. Interphase monopolar growth of
tor1-D mutants that accompanied abundant actin
localization at one single tip might cause the delay in
Cdc2 activation. Proper localization of actin may be
required for signalling the transition of nutrient state
or utilization, which may be the upstream event for
regulating the G2M transition.
10. REQUIREMENT OF SEPARASE/
Cut1-SECURIN/Cut2 IS ALLEVIATED IN
TOR2-DIMINISHED CELLS
Surprisingly, the complex of securin/Cut2separase/,
essential for chromosome segregation, strongly interacts
with both TORC1 and 2. Alleles of cut1 were syntheti-
cally rescued by tor2-S mutation (one example of the
spot test is shown in figure 4d;[20]). The addition of
rapamycin to cut1 mutants also strongly suppressed
Tor2 2029
Tor1 2025
Raptor
mTOR
(a)(c)
(d)
(b)
mTOR
Tor1,2
Tor2
Tor2
Tor1
mTORC1 mTORC2
mammal
TORC1(i)
(ii)
(iii)
Toc1
Tco89
Ste20/
Rictor
Mip1/
Raptor
Tor2
Tor1
Sin1
S
D
PI3K
WT
tor2-2048S
tor1-2045D
PI3K
Pop3/Lst
8
Pop3/Lst
8
TORC2
Bit61
S. cerevisiae
S. pombe
scTORC1 scTORC2 spTORC1 spTORC2
Rictor
KOG1
TSC11
Mip1
Ste20
G0
VEG late G2
VEG early G2
Tor2
+
(TORC1)
2% glc
separase
cut1, cut2
cut1, cut2
cut1, cut2
OP Cut1
wild-type
tor2-S
cut1-693 tor2-S
cut1-693
wild-type
cut2-364
cut2-364 tor1-D
tor1-D
33°C 3 days
34.5°C 3 days
TOR
tor2
tor1
ste20, pop3
rapamycin
effect
synth rescue
synth rescue
synth defect
synth defect
0.08% glc
0.08% glc at 36°C
Tor1
+
(TORC2)
+N medium at 36°C
tor2-S mutant
tor1-D mutant
Figure 4. Functional relationship of TORCs with securin and separase. (a) Two TORCs exist in mammals, Saccharomyces cer-
evisiae and Schizosaccharomyces pombe. Mammalian TOR is the sole catalytic subunit in mammals, while budding and fission
yeasts have two distinct catalytic subunits. (b)(i) The subunit constituents of TORC1 and 2 in S. pombe. The crosses indicate
the mutation sites of tor1-D and tor2-S in the PI3K domain of Tor1 and Tor2. (b)(ii) The mutated residues are indicated in red
colour in the conserved amino acid sequences in the PI3K domain. (b)(iii) DAPI-stained micrographs of wild-type, tor2-S and
tor1-D cultured at 368C for 6 h. (c) Schematic of the role of Tor2 and Tor1 in cell size determination (see text). glc, glucose.
(d) Synthetic rescue or defect observed in the pair of mutations in securin/cut2, separase/cut1 or overproduction (OP) of Cut1
and mutations of tor1, tor2, regulatory subunits (ste20 and pop3) or the addition of rapamycin. See text. Two examples of the
spot test at the semi-permissive temperature, showing the (left) synthetic rescue and (right) defect, are shown (see text).
Review. Cell size control and growth M. Yanagida et al. 3515
Phil. Trans. R. Soc. B (2011)
the ts phenotype. As the main target of rapamycin is
Tor2 in S. pombe, these results suggest that the down-
regulation of Tor2 lessens the necessity for Cut1.
Consistently, the synthetic rescue by tor2-S mutation
and rapamycin was also found for the mutant alleles of
cut2. As the levels of Cut1 and Cut2 are not restored
at all in cells suppressed by tor2-S or rapamycin, the
necessity of Cut1 Cut2 is greatly alleviated, or the
mode of chromosome segregation under the dimin-
ished Tor2 situation may be drastically altered,
regarding the requirement of Cut1Cut2. It remains
to be determined which of the Cut1 functions that
include proteolytic and non-proteolytic activities in
S. pombe [64,65,76] are actually suppressed.
The additive defects were observed between cut2
and tor1 (the spot test result is shown in figure 4d).
Between cut1 and tor1, the additive effect also exists.
These results suggest that the Cut2 Cut1 complex
shares the essential function with Tor1. The synthetic
defect was also found between the overproduction of
Cut1 and the tor1-D mutant. The previous study
[77] showed that overproduction of Cut1 causes the
synthetic defect with ste20 and pop3 mutations that
are defective in the regulatory subunits of TORCs.
These apparently opposite results between Tor1 and
Tor2 in the interaction with Cut1 Cut2 mutations
again suggest that the roles of Tor1 and Tor2 are
opposing (discussed below).
Other unexpected finding is that the ts phenotype
of the cut2 mutant is partly suppressed by Dfkh1, the del-
etion of FKBP12-like Fkh1, in the absence of rapamycin
[20]. Fkh1 encodes a peptidylproline cistrans isomerase
enzyme, which accelerates the folding of proteins. While
Fkh1 is necessary for tor2-S and tor1-D mutants to be
sensitive to rapamycin, suppression of the ts phenotype
of cut2 by the deletion Dfkh1 occurs in the absence of
rapamycin. Fkh1 appears to be needed for the ts pheno-
type of the cut2 mutant. Since Fkh1 affects the protein
conformation, the result might fit with a notion that
Cut2 is a chaperone-inhibitor of Cut1 [54]: Fkh1 may
cause instability of mutant Cut2 protein.
11. DISCUSSION
The aim of this review is to discuss the perspective
regarding facts and hypotheses on size control during
the cell division cycle under limited nutrients and
their implication in the mode of mitosis. First, it
should be emphasized that cell size control is the meet-
ing point for cell division cycle and growth control.
When considering growth control, it is not surprising
that glucose and nitrogen source are determinant fac-
tors for the cell size. The cell length of S. pombe at the
time of division is pre-determined, depending on
different concentrations of nitrogen source and glu-
cose in the culture medium. The wee or semi-wee
cellular phenomenon occurs in wild-type cells under
limited nutrients. Second, protein phosphorylation
and dephosphorylation are closely implicated in the
cell size control. Several kinases and phosphatases
are found to control cell size under the nutritional
limitations. Discussions are mostly restricted to the
cases of S. pombe rather than budding yeast and mam-
malian systems. The reason is that critical examination
of mammalian cell size control during the somatic cell
division cycle has been scarce and that the role of
SWE1 (Wee1 homologue) and MIH1 (Cdc25 homol-
ogue) in cell size control at the time of S. cerevisiae
division is unclear.
As is the case of S. pombe Cdc25 and Wee1, which are
opposing phosphatase and kinase, but actually coordi-
nate the timing of Cdc2 activation for mitotic entry
through the change of their activities, TORC1 (Tor2)
and TORC2 (Tor1) may be opposing, but actually coor-
dinate growth, mitosis and cell size control in response
to nutritional cues. Premature mitosis is observed in
the tor2-S mutant, whereas cells are elongated in the
tor1-D mutant, reminiscent of wee1 and cdc25 mutants.
TORC1 and TORC2 are both kinases, so that they
are unlikely to target the same substrate to control the
cell size by their opposing functions. It is possible that
TORC1 and TORC2 are the upstream regulators for
Cdc2 activation, and affect directly or indirectly Wee1
and Cdc25 depending on the levels of nutrients. Note
that S. pombe has the second Wee1-like kinase, Mik1,
which may also be involved in the nutritional control
for Cdc2 activation. The hypothesis that TORC1 and
TORC2 control Cdc2 activation at the G2M bound-
ary in opposing manners remains to be tested.
Nutrient-sensitive Ssp1 kinase (and possibly also Ssp2
AMPK) and PP2A (Ppa2), PP2A-related (Ppe1) phos-
phatases are also opposing. The Ssp1Ppa2/Ppe1
signalling may also be considered as the upstream regu-
lators for Cdc2 activation. They are implicated in
calcium signalling in addition to nutrient uptake. How-
ever, their relationship to TOR kinases, dependent or
independent, remains unclear. The Ssp1Ppa2/Ppe1
signalling may target the same substrate(s) to control
the cell size under different nutrients.
We argue that Sds23 is a key molecule to understand
the role of Ppa2 and Ppe1 phosphatases for the nutri-
tional utilizations because Sds23 restrains the
phosphatases by stable binding. The loss of Sds23
leads to the hyperactivation of the phosphatases, causing
the failure to consume the low concentration of glucose
for cell proliferation [21] and resulting in loss of viability
of the G0 cells under nitrogen starvation [14]. The
effect of Dsds23 deletion on the glucose consumption
is exceptionally strong among thousands of mutant
strains examined. Measurement indicates that the con-
sumption rate of glucose in Dsds23 cells is extremely
slow (L. Uehara & A. Mori 2011, unpublished data).
The biomarker compound cytidine diphosphate-
choline, the level of which greatly increases under
glucose fasting in S. pombe [33], considerably increases
in the extracts of Dsds23 deletion mutants (T. Pluskal
2011, unpublished data), strongly suggesting that the
intracellular nutrient state of Dsds23 is close to glucose
starvation, whereas glucose is abundant in the culture
medium. The uptake of low glucose is clearly defective
in Dsds23. In higher glucose concentration (2%),
Dsds23 cells can proliferate, suggesting that the glucose
transport becomes very inefficient. We consider that
this kind of analysis might be useful for understanding
the human disease type II diabetes.
The cell length of Dsds23 is long and rod-shaped [46],
resembling the overproduction phenotype of Ppa2 and
Ppe1 phosphatases. Sds23 is highly phosphorylated.
3516 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
Judging from the phosphorylated residues determined
by mass spectrometry, candidate kinases are PKA,
protein kinase C and MAPK. It is important to deter-
mine whether phosphorylation controls the binding to
phosphatase. We consider that Sds23 acts in parallel
with PP1 and APC/cyclosome, and negatively regulates
PP2A and PP2A-related phosphatases in interphase or
throughout the cell cycle. The role of Sds23 or the
downregulation of PP2A and PP2A-related phospha-
tases becomes essential when the levels of glucose and
nitrogen source are low. The transcript level of
Sds23 sharply increases by oxidative (H
2
O
2
) stress,
and transiently increases by cadmium, heat and sorbitol
treatments [78].
The role of PP2A in cell size control and its
interactions with Cdc25 and Wee1 was documented
many years ago in S. pombe [22,23,41], but their nega-
tive role for glucose consumption has only recently been
realized through the study on Sds23. Furthermore, the
discovery of mitotic inhibition of PP2A by phosphoryl-
ation of alpha-endosulphine and Arpp19 by greatwall
kinase [42,43] strongly suggested the importance of
PP2A downregulation during mitotic progression. It
is conceivable that the downregulation of PP2A and
PP2A-related phosphatases during mitosis enhances
the utilization of glucose and the maximal production
of energy source.
The role of CaMKK-like Ssp1 kinase is similar to
that of Sds23. Ssp1 is essential to properly respond
to the starvation of nitrogen source and also glucose
[13,14,21], suggesting that Ssp1 may broadly contrib-
ute to survival under limited nutrients. However, its
molecular function, particularly in the relation to cal-
cium and calmodulin, has been little studied. It is
scarcely understood how actin localization, and glu-
cose and nitrogen source utilization are integrated
with Ssp1 kinase. The short, conserved stretch
sequence present between the kinase- and calmodu-
lin-binding domains is essential for maintaining rod-
like cell shape [21]. Mass spectrometric analysis
shows that Ssp1 is bound to 14-3-3 homologues,
Rad24 and Rad25, suggesting that Ssp1 shuttles
between the nucleus and cytoplasm. Ssp1 is highly
phosphorylated: seven phosphopeptides have been
identified [21]. Overproduction of Ssp1 disperses
cortex actin, resulting in the production of round
cells. By contrast, under nitrogen starvation, ssp1
mutants keep the rod cell shape and fail to reduce
cell size. The role of Ssp1 based on the interaction
with Sty1 and Wis1 is of considerable interest. Our
knowledge is still scarce for the relationship between
Ssp1 and Sty1 in the regulation of cell size under
nitrogen starvation.
To critically compare the phenotypes between tor1
and tor2 mutants, the ts mutant tor1-D that has the
substitution at the same PI3K site as that of the pre-
viously isolated tor2-S mutant is useful [20]. Their
phenotypic comparison indicates that Tor1 and Tor2
are opposite in many aspects including the cell size
and also in the mode of interactions with securin and
separase mutations. Rapamycin is highly inhibitory
to tor2-S, but only slightly to tor1-D. As mutant
TORC1 and TORC2 kinases are purified and their
TOR kinase activities are found to be greatly
diminished [20], different phenotypes may be due to
the substrate specificities of TORCs kinases.
We interpreted that the opposite cell size phenotypes
of tor1-D and tor2-S are due to their distinct responses to
nutritional conditions. One important role of Tor1 is to
reduce the cell size for division upon decrease of glucose
concentration. Effects of other nutrients on the cell size
control by Tor1 remain investigated. By contrast, the
role of Tor2 is to support growth by increasing the cell
size until cells reach the critical size for division.
Hence, Tor1 and Tor2 may be considered to coordinate
growth with the determination of the timing of cell
division depending on nutritional conditions, although
their apparent phenotypes and deduced functions
look to be opposing. To substantiate such a hypo-
thesis, more mechanistic studies are necessary in
future, particularly, in relation to Cdc2 activation as
discussed above.
The final issue is how to interpret the synthetic rescue
of cut1 and cut2 mutations by tor2-S or rapamycin. The
suppression by rapamycin requires Fkh1, a FKBP hom-
ologue. This unexpected rescue is strong so that the
functional loss of Cut1 Cut2 complex is clearly alle-
viated if Tor2 (TORC1) is diminished. Chromosome
segregation looks quite normal in the suppressed
mutant cells, but the low level of separase and securin
does not increase at all, as if only a small amount of
Cut1Cut2 is needed in tor2 or rapamycin-treated
cells [20]. The complex of Cut1Cut2 is absolutely
needed for proper chromosome segregation in wild-
type cells cultured in the regular medium, but the
necessity is known to be greatly lessened in the cut1
and cut2 mutant cells in medium containing high con-
centrations of salt or sorbitol [64]. However, effect of
salt differs from that of rapamycin, as the high salt
increases the level of Cut1Cut2 in a Sty1-dependent
manner. It is well known in mammalian cells that
immunological response (transplant rejection) is sup-
pressed after rapamycin treatment. Additionally, the
progression of certain cancers is prevented, and the life-
span is extended in mice. Although the mechanism is
scarcely understood, diminished Tor2 (TORC1) greatly
lessens the necessity of both Cut1 and Cut2, suggesting
that the mode of chromosome segregation might be dra-
matically altered by the nutritional change via Tor2
(TORC1).
Our results suggest that the segregation role of Cut1
Cut2 is opposite to TORC1 (Tor2), but in parallel with
TORC2 (Tor1). There is a popular concept that growth
opposes cell division. Conversely, in S. pombe, the
period of mitosis restrains growth (cell elongation).
TORC1 may restrain the Cut2Cut1 function in
order to prevent premature mitosis, while TORC2 is
in parallel with Cut2Cut1 as its role is to determine
the critical timing of division under different nutritional
conditions. The close interaction of the TORC com-
plexes with Cut2 Cut1 suggests that nutritional
control may be mediated at the metaphaseanaphase
transition. This is a surprising possibility, and calls for
further study. The nutritional control might be exerted
on the metaphaseanaphase progression through the
functions of Cut1Cut2, which might require the
balanced regulation of TORC1 function. Cut2 is phos-
phorylated ([64]; Y. Hanyu & M. Yanagida 2011,
Review. Cell size control and growth M. Yanagida et al. 3517
Phil. Trans. R. Soc. B (2011)
unpublished data). Further study is definitively needed
for understanding these interactions of Cut1Cut2 with
TORC1 and TORC2.
In summary, we identified Sty1, Wis1, Ssp1, Tor1
and Tor2 kinases, and Ppa2 and Ppe1 protein phos-
phatases to be important for regulating the cell
division cycle control under different nutritional
conditions. Their contributions become strikingly
apparent in mutant cells under nutritional limitations.
These kinases and phosphatases seem to affect Cdc2
kinase activation, though the mechanisms are little
understood. Our results revealed the surprising depen-
dency of mitotic metaphase to anaphase transition on
the nutrient-sensing TORCs. The block of chromo-
some segregation by diminished Cut1 Cut2 by
mutations is restored by diminished TORC1 by
mutation or rapamycin. The mode of chromosome
segregation may have to be controlled by TORCs in
order to respond to nutritional changes.
This review is dedicated to John Murdoch Mitchison, the
great founder of the S. pombe growth and cell cycle control
field. The authors thank all the members of the G0 cell
unit of OIST, particularly to Ayaka Mori, Lisa Uehara and
Nobuyasu Ikai, a former member of the laboratory in
Kyoto University. The generous support of OIST is
gratefully acknowledged. The present study was partly
conducted by the CREST research project of the Japan
Science and Technology (JST Corporation) grant while
M.Y. was in Kyoto University.
REFERENCES
1 Mitchison, J. 1971 The biology of the cell cycle. London,
UK: Cambridge University Press.
2 Mitchison, J. M. 1957 The growth of single
cells. I. Schizosaccharomyces pombe. Exp. Cell Res. 13,
244262. (doi:10.1016/0014-4827(57)90005-8)
3 Nurse, P., Thuriaux, P. & Nasmyth, K. 1976 Genetic
control of the cell division cycle in the fission yeast Schi-
zosaccharomyces pombe. Mol. Gen. Genet. 146, 167178.
(doi:10.1007/BF00268085)
4 Thuriaux, P., Nurse, P. & Carter, B. 1978 Mutants
altered in the control co-ordinating cell division with
cell growth in the fission yeast Schizosaccharomyces
pombe. Mol. Gen. Genet. 161, 215220. (doi:10.1007/
BF00274190)
5 Nurse, P. & Thuriaux, P. 1980 Regulatory genes control-
ling mitosis in the fission yeast Schizosaccharomyces
pombe. Genetics 96, 627 637.
6 Fantes, P. 1979 Epistatic gene interactions in the control
of division in fission yeast. Nature 279, 428430. (doi:10.
1038/279428a0)
7 Dunphy, W. G. & Kumagai, A. 1991 The cdc25 protein
contains an intrinsic phosphatase activity. Cell 67,
189196. (doi:10.1016/0092-8674(91)90582-J)
8 Gould, K. L., Moreno, S., Tonks, N. K. & Nurse, P.
1990 Complementation of the mitotic activator,
p80cdc25, by a human protein-tyrosine phosphatase.
Science 250, 15731576. (doi:10.1126/science.1703321)
9 Costello, G., Rodgers, L. & Beach, D. 1986 Fission yeast
enters the stationary phase G0 state from either mitotic
G1 or G2. Curr. Genet. 11, 119 125. (doi:10.1007/
BF00378203)
10 Nurse, P. & Bissett, Y. 1981 Gene required in G1 for
commitment to cell cycle and in G2 for control of mitosis
in fission yeast. Nature 292, 558 560. (doi:10.1038/
292558a0)
11 Su, S. S., Tanaka, Y., Samejima, I., Tanaka, K. &
Yanagida, M. 1996 A nitrogen starvation-induced dor-
mant G0 state in fission yeast: the establishment from
uncommitted G1 state and its delay for return to prolifer-
ation. J. Cell Sci. 109, 1347 1357.
12 Yanagida, M. 2009 Cellular quiescence: are controlling
genes conserved? Trends Cell Biol. 19, 705 715.
(doi:10.1016/j.tcb.2009.09.006)
13 Sajiki, K. et al. 2009 Genetic control of cellular quies-
cence in S. pombe. J. Cell Sci. 122, 1418 1429.
(doi:10.1242/jcs.046466)
14 Shimanuki, M. et al. 2007 Two-step, extensive alterations
in the transcriptome from G0 arrest to cell division in
Schizosaccharomyces pombe. Genes Cells 12, 677692.
(doi:10.1111/j.1365-2443.2007.01079.x)
15 Wilhelm, B. T., Marguerat, S., Watt, S., Schubert, F.,
Wood, V., Goodhead, I., Penkett, C. J., Rogers, J. &
Bahler, J. 2008 Dynamic repertoire of a eukaryotic tran-
scriptome surveyed at single-nucleotide resolution.
Nature 453, 1239 1243. (doi:10.1038/nature07002)
16 Tatebe, H., Goshima, G., Takeda, K., Nakagawa, T.,
Kinoshita, K. & Yanagida, M. 2001 Fission yeast living
mitosis visualized by GFP-tagged gene products. Micron
32, 6774. (doi:10.1016/S0968-4328(00)00023-8)
17 Morrell, J. L. et al. 2004 Sid4p-Cdc11p assembles the
septation initiation network and its regulators at the
S. pombe SPB. Curr. Biol. 14, 579584. (doi:10.1016/j.
cub.2004.03.036)
18 Wu, L. & Russell, P. 1997 Roles of Wee1 and Nim1
protein kinases in regulating the switch from mitotic div-
ision to sexual development in Schizosaccharomyces
pombe. Mol. Cell Biol. 17, 10 17.
19 Labib, K. & Moreno, S. 1996 r um1: a CDK inhibitor
regulating G1 progression in fission yeast. Trends Cell
Biol. 6, 62 66. (doi:10.1016/0962-8924(96)81016-6)
20 Ikai, N., Nakazawa, N., Hayashi, T. & Yanagida, M.
In press The reverse, but coordinated, roles of Tor2
(TORC1) and Tor1 (TORC2) kinases for growth, cell
cycle and separase-mediated mitosis in S. pombe. Open
Biol. (doi:10.1098/rsob.110007)
21 Hanyu, Y. et al. 2009 Schizosaccharomyces pombe cell div-
ision cycle under limited glucose requires Ssp1 kinase,
the putative CaMKK, and Sds23, a PP2A-related phos-
phatase inhibitor. Genes Cells 14, 539 554. (doi:10.1111/
j.1365-2443.2009.01290.x)
22 Kinoshita, N., Yamano, H., Niwa, H., Yoshida, T. &
Yanagida, M. 1993 Negative regulation of mitosis by
the fission yeast protein phosphatase ppa2. Genes Dev.
7, 10591071. (doi:10.1101/gad.7.6.1059)
23 Shimanuki, M., Kinoshita, N., Ohkura, H., Yoshida, T.,
Toda, T. & Yanagida, M. 1993 Isolation and characteriz-
ation of the fission yeast protein phosphatase gene ppe1þ
involved in cell shape control and mitosis.
Mol. Biol. Cell
4, 303313.
24 George, V. T., Brooks, G. & Humphrey, T. C. 2007
Regulation of cell cycle and stress responses to hydro-
static pressure in fission yeast. Mol. Biol. Cell 18,
41684179. (doi:10.1091/mbc.E06-12-1141)
25 Kishimoto, N. & Yamashita, I. 2000 Cyclic AMP regulates
cell size of Schizosaccharomyces pombe through Cdc25 mito-
tic inducer. Ye a s t 16, 523529. (doi:10.1002/(SICI)1097-
0061(200004)16:6,523::AID-YEA546.3.0.CO;2-5)
26 Petersen, J. & Hagan, I. M. 2005 Polo kinase links
the stress pathway to cell cycle control and tip growth in
fission yeast. Nature 435, 507 512. (doi:10.1038/
nature03590)
27 Petersen, J. & Nurse, P. 2007 TOR signalling regulates
mitotic commitment through the stress MAP kinase
pathway and the Polo and Cdc2 kinases. Nat. Cell Biol.
9, 12631272. (doi:10.1038/ncb1646)
3518 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
28 Shieh, J. C., Wilkinson, M. G., Buck, V., Morgan, B. A.,
Makino, K. & Millar, J. B. 1997 The Mcs4 response reg-
ulator coordinately controls the stress-activated Wak1-
Wis1-Sty1 MAP kinase pathway and fission yeast cell
cycle. Genes Dev. 11, 10081022. (doi:10.1101/gad.11.
8.1008)
29 Shiozaki, K., Shiozaki, M. & Russell, P. 1997 Mcs4 mito-
tic catastrophe suppressor regulates the fission yeast cell
cycle through the Wik1-Wis1-Spc1 kinase cascade.
Mol. Biol. Cell 8, 409 419.
30 Johnson, A. E. & Gould, K. L. 2011 Dma1 ubiquitinates
the SIN scaffold, Sid4, to impede the mitotic localization
of Plo1 kinase. EMBO J. 30, 341 354. (doi:10.1038/
emboj.2010.317)
31 Ng, S. S., Papadopoulou, K. & McInerny, C. J. 2006
Regulation of gene expression and cell division by Polo-
like kinases. Curr. Genet. 50, 7380. (doi:10.1007/
s00294-006-0077-y)
32 Robertson, A. M. & Hagan, I. M. 2008 Stress-regulated
kinase pathways in the recovery of tip growth and micro-
tubule dynamics following osmotic stress in S. pombe.
J. Cell Sci. 121, 4055 4068. (doi:10.1242/jcs.034488)
33 Pluskal, T., Hayashi, T., Saitoh, S., Fujisawa, A. &
Yanagida, M. 2011 Specific biomarkers for stochastic
division patterns and starvation-induced quiescence
under limited glucose levels in fission yeast. FEBS J. 278,
1299 1315. (doi:10.1111/j.1742-4658.2011.08050.x)
34 Hayashi, T., Hatanaka, M., Nagao, K., Nakaseko, Y.,
Kanoh, J., Kokubu, A., Ebe, M. & Yanagida, M. 2007
Rapamycin sensitivity of the Schizosaccharomyces pombe
tor2 mutant and organization of two highly phosphory-
lated TOR complexes by specific and common
subunits. Genes Cells 12, 13571370. (doi:10.1111/j.
1365-2443.2007.01141.x)
35 Matsusaka, T., Hirata, D., Yanagida, M. & Toda, T. 1995
A novel protein kinase gene ssp1þ is required for altera-
tion of growth polarity and actin localization in fission
yeast. EMBO J. 14, 3325 3338.
36 Afshar, K., Werner, M. E., Tse, Y. C., Glotzer, M. &
Gonczy, P. 2010 Regulation of cortical contractility
and spindle positioning by the protein phosphatase 6
PPH-6 in one-cell stage C. elegans embryos. Development
137, 237247. (doi:10.1242/dev.042754)
37 Bastians, H. & Ponstingl, H. 1996 The novel human
protein serine/threonine phosphatase 6 is a functional
homologue of budding yeast Sit4p and fission yeast
ppe1, which are involved in cell cycle regulation. J. Cell
Sci. 109, 2865 2874.
38 Cherkasova, V., Qiu, H. & Hinnebusch, A. G. 2010 Snf1
promotes phosphorylation of the alpha subunit of
eukaryotic translation initiation factor 2 by activating
Gcn2 and inhibiting phosphatases Glc7 and Sit4.
Mol. Cell Biol. 30, 2862 2873. (doi:10.1128/MCB.
00183-10)
39 Sutton, A., Immanuel, D. & Arndt, K. T. 1991 The SIT4
protein phosphatase functions in late G1 for progression
into S phase. Mol. Cell Biol.
11, 21332148.
40 Rupes, I., Jia, Z. & Young, P. G. 1999 Ssp1 promotes
actin depolymerization and is involved in stress response
and new end take-off control in fission yeast. Mol. Biol.
Cell 10, 14951510.
41 Kinoshita, N., Ohkura, H. & Yanagida, M. 1990
Distinct, essential roles of type 1 and 2A protein phos-
phatases in the control of the fission yeast cell division
cycle. Cell 63, 405 415. (doi:10.1016/0092-8674(90)
90173-C)
42 Gharbi-Ayachi, A., Labbe, J. C., Burgess, A., Vigneron,
S., Strub, J. M., Brioudes, E., Van-Dorsselaer, A.,
Castro, A. & Lorca, T. 2011 The substrate of Greatwall
kinase, Arpp19, controls mitosis by inhibiting protein
phosphatase 2A. Science 330, 16731677. (doi:10.
1126/science.1197048)
43 Mochida, S., Maslen, S. L., Skehel, M. & Hunt, T. 2011
Greatwall phosphorylates an inhibitor of protein phos-
phatase 2A that is essential for mitosis. Science 330,
16701673. (doi:10.1126/science.1195689)
44 Talarek, N. et al. 2010 Initiation of the TORC1-
regulated G0 program requires Igo1/2, which license
specific mRNAs to evade degradation via the 5
0
–3
0
mRNA decay pathway. Mol. Cell 38, 345355. (doi:10.
1016/j.molcel.2010.02.039)
45 Kinoshita, K., Nemoto, T., Nabeshima, K., Kondoh, H.,
Niwa, H. & Yanagida, M. 1996 The regulatory sub-
units of fission yeast protein phosphatase 2A (PP2A)
affect cell morphogenesis, cell wall synthesis and cytokin-
esis. Genes Cells 1, 29 45. (doi:10.1046/j.1365-2443.
1996.02002.x)
46 Ishii, K., Kumada, K., Toda, T. & Yanagida, M. 1996
Requirement for PP1 phosphatase and 20S cyclosome/
APC for the onset of anaphase is lessened by the
dosage increase of a novel gene sds23þ. EMBO J. 15,
66296640.
47 Dawson, J. F. & Holmes, C. F. 1999 Identification of
sds21 in fission yeast in an inhibitor-resistant high mol-
ecular mass protein phosphatase-1 complex. Biochem.
Cell Biol. 77, 551558. (doi:10.1139/o99-062)
48 Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T. &
Yanagida, M. 1989 The fission yeast dis2
þ
gene required
for chromosome disjoining encodes one of two putative
type 1 protein phosphatases. Cell 57, 9971007.
(doi:10.1016/0092-8674(89)90338-3)
49 Posch, M., Khoudoli, G. A., Swift, S., King , E. M.,
Deluca, J. G. & Swedlow, J. R. 2010 Sds22 regulates
aurora B activity and microtubule kinetochore inter-
actions at mitosis. J. Cell Biol. 191, 61 74. (doi:10.
1083/jcb.200912046)
50 Stone, E. M., Yamano, H., Kinoshita, N. & Yanagida, M.
1993 Mitotic regulation of protein phosphatases by the
fission yeast sds22 protein. Curr. Biol. 3, 1326.
(doi:10.1016/0960-9822(93)90140-J)
51 Ohkura, H. & Yanagida, M. 1991 S. pombe gene sds22
þ
essential for a midmitotic transition encodes a leucine-
rich repeat protein that positively modulates protein
phosphatase-1. Cell 64, 149157. (doi:10.1016/0092-
8674(91)90216-L)
52 Jiang, Y., Scott, K. L., Kwak, S. J., Chen, R. & Mardon, G.
2011 Sds22/PP1 links epithelial integrity and tumor sup-
pression via regulation of myosin II and JNK signaling.
Oncogene 30, 32483260. (doi:10.1038/onc.2011.46)
53 Goldar, M. M., Nishie, T., Ishikura, Y., Fukuda, T.,
Takegawa, K. & Kawamukai, M. 2005 Functional con-
servation between fission yeast moc1/sds23 and its two
orthologs, budding yeast SDS23 and SDS24, and phe-
notypic differences in their disruptants. Biosci.
Biotechnol. Biochem. 69, 14221426. (doi:10.1271/bbb.
69.1422)
54 Nagao, K. & Yanagida, M. 2006 Securin can have a
separase cleavage site by substitution mutations in the
domain required for stabilization and inhibition of separ-
ase. Genes Cells 11, 247260. (doi:10.1111/j.1365-2443.
2006.00941.x)
55 Zheng, X. F., Florentino, D., Chen, J., Crabtree, G. R. &
Schreiber, S. L. 1995 TOR kinase domains are required
for two distinct functions, only one of which is inhibited
by rapamycin. Cell 82, 121 130. (doi:10.1016/0092-
8674(95)90058-6)
56 Beck, T. & Hall, M. N. 1999 The TOR signalling path-
way controls nuclear localization of nutrient-regulated
transcription factors. Nature 402, 689692. (doi:10.
1038/45287)
Review. Cell size control and growth M. Yanagida et al. 3519
Phil. Trans. R. Soc. B (2011)
57 Cardenas, M. E., Cutler, N. S., Lorenz, M. C., Di Como,
C. J. & Heitman, J. 1999 The TOR signaling cascade regu-
lates gene expression in response to nutrients. Genes Dev.
13, 3271 3279. (doi:10.1101/gad.13.24.3271)
58 Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A.,
Crespo, J. L., Bonenfant, D., Oppliger, W., Jenoe, P. &
Hall, M. N. 2002 Two TOR complexes, only one of
which is rapamycin sensitive, have distinct roles in cell
growth control. Mol. Cell 10, 457 468. (doi:10.1016/
S1097-2765(02)00636-6)
59 Powers, T. & Walter, P. 1999 Regulation of ribosome bio-
genesis by the rapamycin-sensitive TOR-signaling
pathway in Saccharomyces cerevisiae. Mol. Biol. Cell 10,
9871000.
60 Schmidt, A., Kunz, J. & Hall, M. N. 1996 TOR2 is
required for organization of the actin cytoskeleton in
yeast. Proc. Natl Acad. Sci. USA 93, 13 78013 785.
(doi:10.1073/pnas.93.24.13780)
61 Kamada, Y., Fujioka, Y., Suzuki, N. N., Inagaki, F.,
Wullschleger, S., Loewith, R., Hall, M. N. & Ohsumi, Y.
2005 Tor2 directly phosphorylates the AGC kinase
Ypk2 to regulate actin polarization. Mol. Cell Biol. 25,
7239 7248. (doi:10.1128/MCB.25.16.7239-7248.2005)
62 Lempiainen, H. & Halazonetis, T. D. 2009 Emerging
common themes in regulation of PIKKs and PI3Ks.
EMBO J. 28, 30673073. (doi:10.1038/emboj.2009.281)
63 Martin, D. E. & Hall, M. N. 2005 The expanding TOR
signaling network. Curr. Opin. Cell Biol. 17, 158 166.
(doi:10.1016/j.ceb.2005.02.008)
64 Kawasaki, Y., Nagao, K., Nakamura, T. & Yanagida, M.
2006 Fission yeast MAP kinase is required for the
increased securin separase interaction that rescues
separase mutants under stresses. Cell Cycle 5,
18311839. (doi:10.4161/cc.5.16.3010)
65 Nakamura, T., Nagao, K., Nakaseko, Y. & Yanagida, M.
2002 Cut1/separase C-terminus affects spindle pole body
positioning in interphase of fission yeast: pointed nuclear
formation. Genes Cells 7, 11131124. (doi:10.1046/j.
1365-2443.2002.00586.x)
66 Alvarez, B. & Moreno, S. 2006 Fission yeast Tor2 pro-
motes cell growth and represses cell differentiation.
J. Cell Sci. 119, 4475 4485. (doi:10.1242/jcs.03241)
67 Matsuo, T., Otsubo, Y., Urano, J., Tamanoi, F. &
Yamamoto, M. 2007 Loss of the TOR kinase Tor2
mimics nitrogen starvation and activates the sexual devel-
opment pathway in fission yeast. Mol. Cell Biol. 27,
31543164. (doi:10.1128/MCB.01039-06)
68 Uritani, M., Hidaka, H., Hotta, Y., Ueno, M.,
Ushimaru, T. & Toda, T. 2006 Fission yeast Tor2 links
nitrogen signals to cell proliferation and acts downstream
of the Rheb GTPase. Genes Cells 11, 13671379.
(doi:10.1111/j.1365-2443.2006.01025.x)
69 Urano, J., Sato, T., Matsuo, T., Otsubo, Y., Yamamoto,
M. & Tamanoi, F. 2007 Point mutations in TOR
confer Rheb-independent growth in fission yeast and
nutrient-independent mammalian TOR signaling in
mammalian cells. Proc. Natl Acad. Sci. USA 104,
35143519. (doi:10.1073/pnas.0608510104)
70 Tatebe, H., Morigasaki, S., Murayama, S., Zeng, C. T. &
Shiozaki, K. 2010 Rab-family GTPase regulates TOR
complex 2 signaling in fission yeast. Curr. Biol. 20,
19751982. (doi:10.1016/j.cub.2010.10.026)
71 Weisman, R., Choder, M. & Koltin, Y. 1997 Rapa-
mycin specifically interferes with the developmental
response of fission yeast to star vation. J. Bacteriol. 179,
63256334.
72 Weisman, R., Finkelstein, S. & Choder, M. 2001 Rapa-
mycin blocks sexual development in fission yeast
through inhibition of the cellular function of an
FKBP12 homolog. J. Biol. Chem. 276, 24 73624 742.
(doi:10.1074/jbc.M102090200)
73 Wullschleger, S., Loewith, R. & Hall, M. N. 2006 TOR
signaling in growth and metabolism. Cell 124, 471484.
(doi:10.1016/j.cell.2006.01.016)
74 Ikeda, K., Morigasaki, S., Tatebe, H., Tamanoi, F. &
Shiozaki, K. 2008 Fission yeast TOR complex 2 activates
the AGC-family Gad8 kinase essential for stress resist-
ance and cell cycle control. Cell Cycle 7, 358364.
(doi:10.4161/cc.7.3.5245)
75 Schonbrun, M., Laor, D., Lopez-Maury, L., Bahler, J.,
Kupiec, M. & Weisman, R. 2009 TOR complex 2 con-
trols gene silencing, telomere length maintenance, and
survival under DNA-damaging conditions. Mol. Cell
Biol. 29, 4584 4594. (doi:10.1128/MCB.01879-08)
76 Tomonaga, T. et al. 2000 Characterization of fission yeast
cohesin: essential anaphase proteolysis of Rad21 phos-
phorylated in the S phase. Genes Dev. 14, 2757 2770.
(doi:10.1101/gad.832000)
77 Yuasa, T. et al. 2004 An interactive gene network for
securin-separase, condensin, cohesin, Dis1/Mtc1 and
histones constructed by mass transformation. Genes
Cells 9, 10691082. (doi:10.1111/j.1365-2443.2004.
00790.x)
78 Chen, D., Toone, W. M., Mata, J., Lyne, R., Burns, G.,
Kivinen, K., Brazma, A., Jones, N. & Bahler, J. 2003
Global transcriptional responses of fission yeast to
environmental stress. Mol. Biol. Cell 14, 214229.
(doi:10.1091/mbc.E02-08-0499)
3520 M. Yanagida et al. Review. Cell size control and growth
Phil. Trans. R. Soc. B (2011)
    • "We found that Sts5 has a role in the morphological response to nutritional stress. Wild type S. pombe cells mount characteristic morphological responses to changing environmental conditions: increased temperature (Mitchison and Nurse, 1985), decreased nutrient availability (Costello et al., 1986; Su et al., 1996; Yanagida, 2009; Yanagida et al., 2011), and hyper-osmotic stress decrease the incidence of bipolar growth activation and alter overall cell dimensions (Rupes et al., 1999; Robertson and Hagan, 2008 ). The mechanisms that modulate cell morphogenesis and polarized cell growth in response to varying growth and environmental conditions are still poorly understood . "
    [Show abstract] [Hide abstract] ABSTRACT: RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables.
    Full-text · Article · Jul 2016
    • "These environmental changes also accelerated mitotic commitment to reduce cell size at division after 120 min (Figure 1B). Changes in carbon availability can also be sensed to adjust cell size [18]. All of the minimal growth media used in this study contained identical glucose levels (2%) unless otherwise stated. "
    [Show abstract] [Hide abstract] ABSTRACT: Cell growth and cell-cycle progression are tightly coordinated to enable cells to adjust their size (timing of division) to the demands of proliferation in varying nutritional environments. In fission yeast, nitrogen stress results in sustained proliferation at a reduced size. Here, we show that cells can sense nitrogen stress to reduce target of rapamycin complex-1 (TORC1) activity. Nitrogen-stress-induced TORC1 inhibition differs from amino-acid-dependent control of TORC1 and requires the Ssp2 (AMPKα) kinase, the Tsc1/2 complex, and Rhb1 GTPase. Importantly, the β and γ regulatory subunits of AMPK are not required to control cell division in response to nitrogen stress, providing evidence for a nitrogen-sensing mechanism that is independent of changes in intracellular ATP/AMP levels. The CaMKK homolog Ssp1 is constitutively required for phosphorylation of the AMPKα(Ssp2) T loop. However, we find that a second homolog CaMKK(Ppk34) is specifically required to stimulate AMPKα(Ssp2) activation in response to nitrogen stress. Finally, ammonia also controls mTORC1 activity in human cells; mTORC1 is activated upon the addition of ammonium to glutamine-starved Hep3B cancer cells. The alternative nitrogen source ammonia can simulate TORC1 activity to support growth and division under challenging nutrient settings, a situation often seen in cancer. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015
    • "development in yeast when mating partners avail. On the other hand, it also G0-arrest in heterothallic background when mating partners are not available (Yamamoto et al. 1997; Yanagida 2009; Yanagida et al. 2011). In this study, we show that the two NS-induced processes hardly share common signaling molecules (seeFig. "
    [Show abstract] [Hide abstract] ABSTRACT: Nitrogen starvation (NS) induces sexual development when mating partners are available or enter into quiescent state (G0) in heterothallic background in fission yeast. However, little is known whether the two processes share common signaling molecules or cells defective in the two processes share common transcriptional signatures. To address these questions, we first assessed 77 kinase-deletion strains for NS-induced G0-arrest phenotypes. Our result indicated that 10 out of 77 kinase-deletion strains exhibited defect in G0-arrest, only 3 of which were defective in sexual development based on a previous study, suggesting that the two processes hardly share common signaling components. We subsequently performed transcriptional profiling analysis. Our result indicated that NS-induced transcriptional change was so robust that it prevailed the alteration by individual kinase-deletion alleles. Based on comparison between kinase-deletion strains proficient and deficient in sexual development or G0-arrest, we identified subsets of genes that were associated with sexual development-deficient or G0-arrest-deficient kinase-deletion strains. Multiple pairing analyses allowed grouping of functional related kinases. Furthermore, we showed that Pka1-mediated pathways were required for upregulation of NS-induced genes upon NS and downregulation of the same set of genes under the N-replete conditions. Taken together, our analyses indicate that sexual development and NS-induced G0-arrest are unrelated; and sexual development-deficient and G0-arrest-deficient kinase-deletion strains possess distinct transcriptional signatures. We propose that Pka1 is a key regulator of nitrogen metabolic pathways and Pka1-mediated signaling pathways play roles in regulation of NS-induced genes under both N-depleted and N-replete conditions.
    Full-text · Article · Dec 2014
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