Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.108.094797
A G2-Phase Microtubule-Damage Response in Fission Yeast
Fernando R. Balestra and Juan Jimenez1
Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide/CSIC, Carretera de Utrera, Km1, 41013 Sevilla, Spain
Manuscript received August 2, 2008
Accepted for publication October 9, 2008
ABSTRACT
Microtubules assume a variety of structures throughout the different stages of the cell cycle. Ensuring the
correct assembly of such structures is essential to guarantee cell division. During mitosis, it is well established
that the spindle assembly checkpoint monitors the correct attachment of sister chromatids to the mitotic
spindle. However, the role that microtubule cytoskeleton integrity plays for cell-cycle progression during
interphase is uncertain. Here we describe the existence of a mechanism, independent of the mitotic
checkpoint, that delays entry into mitosis in response to G2-phase microtubule damage. Disassembly of the
G2-phase microtubule array leads to the stabilization of the universal mitotic inhibitor Wee1, thus actively
delaying entry into mitosis via inhibitory Cdc2 Tyr15 phosphorylation.
I
N eukaryotic cells, reliable transmission of genetic
information requires the replication of DNA during S
phase and segregation of the sister chromatids during
mitosis. To permit the completion and correct progression
of these two critical events, a variety of mechanisms exist by
which the cell actively halts cell-cycle progression until
earlier processes have been completed. These mechanisms, known as ‘‘checkpoints,’’ monitor key processes and
can delay cell-cycle progression in the event of a problem
(Weinert and Hartwell 1988; May and Hardwick
2006; Callegari and Kelly 2007). In the fission yeast
Schizosaccharomyces pombe, the machinery regulating entry
into mitosis has been thoroughly characterized (Nurse
1994), and a variety of stress and checkpoint signaling
pathways that converge upon the proteins that govern the
G2/M transition have been identified (Suda et al. 2000;
Lopez-Aviles et al. 2005; Callegari and Kelly2007). The
key regulator of this transition is the conserved cyclinB/
Cdc2 complex. Activation of this complex, and entry into
mitosis, is inhibited by Wee1 kinase, which negatively
regulates Cdc2 kinase by phosphorylation of its Tyr15
residue, a phosphorylation step reversed by Cdc25 phosphatase (Nurse 1994).
In S. pombe, the best-characterized checkpoint operating at the G2/M transition is the G2-phase DNA damage
checkpoint. UV irradiation of fission yeast induces a G2phase delay prior to mitosis onset (al-Khodairy and
Carr 1992; al-Khodairy et al. 1994). Detection of DNA
damage or DNA synthesis intermediates results in the
activation of a signal transduction cascade mediated by
the Rad3 and Chk1 protein kinases that inhibits entry
into mitosis (al-Khodairy and Carr 1992; Walworth
1
Corresponding author: Centro Andaluz de Biologı́a del Desarrollo,
Universidad Pablo de Olavide/CSIC, Carretera de Utrera, Km 1, 41013
Sevilla, Spain. E-mail: [email protected]
Genetics 180: 2073–2080 (December 2008)
et al. 1993; Saka et al. 1997). The link between these
checkpoints and the enzymes controlling mitosis is also
well established. Chk1 can phosphorylate Cdc25 to
create a binding site for 14-3-3 proteins, which in turn
inactivates Cdc25 and modifies its subcellular localization (Furnari et al. 1997; Lopez-Girona et al. 1999).
Chk1 also mediates an activating phosphorylation of
Wee1, thus providing an additional direct link between
the DNA damage checkpoint and core cell-cycle regulation (O’Connell et al. 1997; Lee et al. 2001).
In addition to those mechanisms involved in verifying
the replication and integrity of DNA, cells must possess
mechanisms to correctly segregate genetic material into
each daughter cell. This task depends on the formation
and organization of complex microtubule-based structures. During the fission yeast cell cycle, striking changes
take place in the organization of the cytoplasmic
microtubule cytoskeleton (Hagan 1998; Sawin and Tran
2006). At G2 phase, fission yeast cells typically have three
to four bundles of microtubules running along the cell
axis. As cells enter mitosis, the cytoplasmic microtubules
are disassembled and intranuclear microtubules form
the mitotic spindle, which is responsible for separating
each set of sister chromatids toward opposite poles that
will be divided into the two daughter cells. By the end of
mitosis, microtubules are nucleated from the cell division site to form a transient post-anaphase array
(Heitz et al. 2001).
It is well established that the integrity of these
microtubule structures is monitored during the M
phase. The mitotic checkpoint senses correct spindle
assembly, triggering a delay in mitotic progression in the
case of unattached kinetochores or a lack of tension
between sister chromatids (Asakawa et al. 2006; May
and Hardwick 2006). The mitotic arrest deficiency (Mad)
and budding uninhibited by benomyl (Bub) proteins
2074
F. R. Balestra and J. Jimenez
are key components of the mitotic spindle checkpoint
conserved in organisms ranging from yeast to humans.
The conserved components of the mitotic spindle checkpoint act at various stages of mitosis to coordinate spindle
assembly and sister-chromatid separation as well as to
delay mitotic exit and cytokinesis in response to spindle
defects (Booher and Beach 1988; Mulvihill and
Hyams 2002; Musacchio and Salmon 2007). However,
the existence of surveillance mechanisms capable of
verifying microtubule integrity prior to the commitment
to mitosis is controversial (Uetake and Sluder 2007).
On the basis of the observation that wee1-deficient S.
pombe cells were extremely sensitive to microtubuledamaging agents, we initiated the analysis of a link connecting microtubule structures and cell-cycle control,
an issue of interest given the number of microtubuletargeting drugs that are currently used as chemotherapeutic
agents for cancer patients (Pellegrini and Budman
2005). Here we report the existence of a novel mechanism in fission yeasts that monitors microtubule integrity during the G2 phase. This mechanism transiently
inhibits entry into mitosis when interphase microtubule
structures are compromised.
methylsulfonyl fluoride, 10% glycerol, 1% Nonidet P-40, and
5 mg each of aprotinin, leupeptin, and pepstatin/ml], broken
up with a Fast-Prep machine (Bio101) using acid-washed glass
beads (Sigma) and centrifuged to prepare a cleared whole-cell
extract. For Wee1 detection, the cell pellets were resuspended
in 100 ml of RIPA buffer (10 mm sodium phosphate, 1% Triton
X-100, 0.1% SDS, 10 mm EDTA, 150 mm NaCl, pH 7.0) containing the following protease inhibitors: 10 mg/ml leupeptin,
10 mg/ml aprotinin, 10 mg/ml pepstatin, 10 mg/ml soybean
trypsin inhibitor, 100 mm 1-chloro-3-tosylamido-7-amino-l-2heptanone, 100 mm N-tosyl-l-phenylalanine chloromethyl ketone,
100 mm phenylmethylsulfonyl fluoride, 1 mm phenanthroline,
and 100 mm N-acetyl-leu-leu-norleucinal. Cells were boiled for
5 min and broken using 500 mg of acid-washed glass beads
(Sigma) in a Fast-Prep machine (Bio101); the crude extract
was recovered by washing with 100 ml of RIPA. All steps were
carried out at 4°. For Western analysis, samples were electrophoresed on 12 or 8% polyacrylamide gels and transferred to
nitrocellulose membrane. The blots were probed with antibodies against tyrosine-15-phosphorylated Cdc2 (Cell SignalingTechnnology) with mouse monoclonal antibodies against
the PSTAIR peptide or anti-HA monoclonal antibody. Goat
anti-rabbit or anti-mouse antibody conjugated to horseradish
peroxidase (1:1000) was used as a secondary antibody. Immunoblots were developed using the ECL kit (Amersham)
and protein levels were quantified using Image J (National
Institutes of Health, Bethesda, MD).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Cell culture and strains: Cells were cultured in supplemented minimal medium (EMM) (Moreno et al. 1991).
Synchronous cultures were generated by centrifugal elutriation (Mulvihill et al. 1999) or by a block-and-release strategy
using the cdc25-22 mutation (Booher et al. 1989). When
centrifugal elutriation was used, the synchronized culture was
monitored during the first cell cycle and early in G2 during the
second cell division round split into two. A total of 75 mg/ml of
carbendazim (MBC) dissolved in dimethyl sulfoxide (DMSO)
was added to one culture, and the same volume of DMSO, as a
negative control, was added to the other. When the block-andrelease strategy was used, the culture was split into two after
release; 75 mg/ml MBC dissolved in DMSO was added to onehalf and DMSO alone, as a negative control, was added to the
other half. For the spot test, yeast extract medium supplemented as required (YES) was used. Serial dilutions of cells
were spotted onto plates with MBC (4 mg/ml) or DMSO as a
negative control.
Fluorescence microscopy: DAPI and calcofluor staining
were used to stain nuclei and calculate the percentage of
septated cells, respectively (Moreno et al. 1991). Standard
anti-b-tubulin immunofluorescence procedures were used to
stain microtubule arrays (Woods et al. 1989). For fixation of
histone 3 serine-phosphorylated (PSH3) cells glutaldehyde
was omitted from the fixation step and fixation performed
with 3% formaldehyde for 5 min (Petersen et al. 2001). H3SP
was detected using the UPSTATE anti-H3SP antibody. Cells
were examined using a Leica fluorescence microscope equipped with a Plan Apo 3100 lens.
Western blot: For tyrosine-15-phosphorylated Cdc2 detection, cell extracts were made from exponential growing
cultures harvested by centrifugation, washed once in ice-cold
Stop buffer (150 mm NaCl, 50 mm NaF, 10 mm EDTA, 1 mm
NaN3) and frozen as cell pellets at 80°. Cell pellets were
thawed in 200 ml of lysis buffer [150 mm NaCl, 50 mm Tris (pH
8.0), 50 mm NaF, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenyl-
Microtubule damage delays entry into mitosis:
Checkpoints are essential for cell-cycle progression. A
single defective checkpoint can severely affect the ability
of the cell to respond to damage-inducing agents. For
example, in budding and fission yeast, mutations in genes
required for DNA-damage checkpoints can result in hypersensitivity to UV irradiation (Weinert and Hartwell
1988; al-Khodairy and Carr 1992; al-Khodairy et al.
1994; Callegari and Kelly 2007). Similarly, deficiencies in the spindle checkpoint can confer hypersensitivity to treatments that disrupt microtubule structures
(Asakawa et al. 2006; May and Hardwick 2006).
Interestingly, we observed that wee1-deficient S. pombe
cells were extremely sensitive to the presence of the
microtubule-depolymerizing drug MBC (Figure 1A). In
comparison to wild-type cells, wee1D mutants accumulated aberrant nuclei and presented a high percentage
of septated cells (cut phenotype) in the presence of
MBC (see Figure 1, B and C). This observation appears
to uncover a role for Wee1 in modulating the cell cycle
in response to microtubule damage. Wee1 is thought to
act primarily at the G2/M transition (Nurse 1994).
Therefore, aside from a possible role of this cell-cycle
regulator in the mitotic spindle checkpoint (currently
under study), we initiated the analysis of a possible
mechanism connecting microtubule G2 structures and
cell-cycle control.
To investigate the possibility of a G2/M microtubuledamage control in fission yeasts, we prepared S. pombe
cells synchronized in early G2 by centrifugal elutriation.
These cells were allowed to progress to early G2 of the
Microtubule-Damage Response in Fission Yeast
2075
Figure 1.—G2 microtubule disassembly triggers a mitotic onset delay. (A) MBC growth hypersensitivity of wee1-deleted (wee1D)
S. pombe cells. Successive dilutions of wild-type (wt) and wee1D cells were spotted onto solid YES medium containing MBC (4 mg/ml)
or DMSO. (B) Wild-type and wee1D cells were DAPI (nucleus) and calcofluor (cell wall) stained after 2 hr of incubation in EMM
plus MBC (75 mg/ml) or DMSO and examined by microscopy. (C) The percentage of septation was calculated in these conditions.
(D) Positive PSH3 inmunostained cell (arrow, left) among nonpositive cells. The nucleus of these cells was also visualized (DAPI
staining, right). (E) Wild-type (top) or nda3-TB101 (bottom) cells were grown in EMM liquid medium at 25° and synchronized by
centrifugal elutriation. The synchronized culture was divided early in G2 of the second round of cell division with MBC (75 mg/ml)
added to one-half and DMSO added to the other half (time 0 min). Aliquots were taken periodically and the percentage of cells
positively stained for phosphorylated histone H3 and the percentage of septated cells were determined. (F) Samples of cells of
both strains were also fixed for microtubule inmunostaining. After MBC addition (time 0 min), microtubule depolymerization was
observed in wild-type cells while the microtubule cytoskeleton remained intact in nda3-TB101 cells (right). Identical images were
visualized during the course of the experiment, ensuring that the microtubule cytoskeleton damage remained in MBC treated
wild-type cells.
second synchronous cycle (to avoid any effect caused by
the elutriation stress), split into two, and treated either
with MBC dissolved in DMSO or with DMSO alone as a
control. Samples were taken over time and the septation
index and histone H3 phosphorylation—an early event
of the mitotic prophase (Petersen et al. 2001)—was
monitored (Figure 1D). As shown in Figure 1, E and F
(top), interphase microtubules were completely disassembled in wild-type MBC-treated cells, and these cells
exhibited a delayed entry into mitosis relative to untreated cells, suggesting the existence of a mechanism
that transiently halts the cell cycle during G2 in response
to microtubule damage.
Certain microtubule-damaging agents such as thiabendazole have been shown to also affect actin structures (Sawin and Snaith 2004). In budding yeast, the
morphogenesis checkpoint senses actin or septin struc-
ture integrity and can induce a cell-cycle delay via Swe1/
Wee1 (Sia et al. 1998; Keaton and Lew 2006). Similarly,
a size checkpoint control that links actin filaments to a
Cdc2 control has been proposed for S. pombe (Rupes
et al. 2001). Thus, although we have used MBC as a
highly specific antimicrotubule drug, it is possible that
unknown nontubulin targets of MBC could be responsible for the observed G2/M delay. To discard this
possibility, we used the nda3-TB101 mutant, a strain
harboring a point mutation in the nda3 gene (coding
for b-tubuline), which confers MBC resistance (Sawin
and Snaith 2004). In this mutant background, the
microtubule array is now resistant to the effect of MBC
but any other possible MBC target would remain
sensitive. As shown in Figure 1, E and F (bottom), cells
possessing the MBC-resistant tubulin completely bypassed the MBC-induced delay. Thus, the G2-phase delay
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F. R. Balestra and J. Jimenez
following MBC treatment is wholly dependent on its
depolymerization of microtubules.
Microtubule-damage signaling operates through
Wee1 but not through Cdc25: The canonical G2-phase
cell-cycle arrest requires inhibitory phosphorylation of
Cdc2 catalyzed by Wee1 kinase and opposed by Cdc25
phosphatase (Nurse 1994). The MBC hypersensitivity
of wee1-deficient cells implicates Wee1 as a potential
target of the microtubule-damage signaling pathway,
but we cannot exclude Cdc25 inhibition from a role in
triggering the G2 delay. To address this possibility, we
assessed MBC sensitivity in the cdc2-1w, cdc2-3w, and cdc23w cdc25D mutant strains. cdc2-3w is an active allele of
cdc2 containing a mutation that relieves the requirement for Cdc25 but retains sensitivity to Wee1. Conversely, cdc2-1w is an active allele containing mutations
that result in a Cdc2 variant insensitive to the inhibitory
effect of Wee1 but still sensitive to Cdc25 dephosphorylation (Enoch and Nurse 1990). If Cdc25 is required
for the MBC-induced delay, then the cdc2-3w strain and
particularly the cdc2-3w cdc25D strain, should manifest
MBC hypersensitivity. As shown in Figure 2A, wee1D and
cdc2-1w but not cdc2-3w or cdc2-3w cdc25D mutant cells
were hypersensitive to MBC treatment, suggesting that
Wee1 but not Cdc25 is involved in this microtubuledamage signaling mechanism.
To determine the role of Wee1 in this delay, we
analyzed the effect of MBC during the G2/M transition
in wee1-deficient cells. Synchronization by centrifugal
elutriation of wee1D cells is difficult due to their reduced
size. However, by using the thermo-sensitive wee1-50
mutant strain, cells could be synchronized at 25° (permissive temperature) before shifting them to the restrictive
temperature for wee1-50, permitting cell-cycle progression and carrying out MBC treatment as described above.
The MBC-induced G2 delay was less prominent in wildtype cells grown at 35°. Thus, the assay was carried out at
30°, where the MBC-induced G2 delay occurred as at 25°
in wild-type cells (supplemental Figure S1) and wee1-50
strains are still defective for Wee1 activity. At this temperature, synchronous wee1-deficient cells bypassed the
G2 delay triggered by MBC (Figure 2B), indicating that
Wee1 is required to induce the G2 delay caused by
microtubule damage. To confirm that Cdc25 is not required for the G2/M delay, we performed similar experiments using synchronized cdc2-3w cdc25D fission yeast
cells. We found that S. pombe cells lacking the Cdc25 pathway retained their response to MBC treatment (Figure
2C). Taken together, these observations strongly suggest
that the G2-phase delay induced by microtubule damage
is mediated primarily via Wee1 inhibition of Cdc2.
The cdc25-22 thermo-sensitive allele encodes a Cdc25
protein active at 25° and completely inactive at 35°
(Nurse et al. 1976). At intermediate permissive temperatures (27°–29°), the reduced Cdc25 activity results in a
Cdc2-Tyr15 regulation that relies mainly on the Wee1
side of the control and therefore any Wee1 regulatory
effect is amplified in this genetic background. Interestingly, growth of this strain was partially sensitive to MBC
at 27° (Figure 2D), but contrary to wee1-deficient cells,
MBC treatment in the cdc25-22 strain produced elongated cells with a conventional G2-block phenotype
(Figure 2E). This cell-cycle block was reverted by removing the drug and completely abolished in cdc25-22
wee1D double-mutant cells (data not shown). Thus in a
cdc25-22 genetic background, the G2-phase delay triggered by MBC became an efficient G2 arrest, supporting
the notion of a microtubule-damage-sensing mechanism that exerts a delay at G2/M transition through
Wee1. The enhancement of the transient G2 delay
under MBC treatment makes the cdc25-22 mutation a
particularly useful tool for studying this novel microtubule-damage signaling response.
A mechanism for the Wee1-mediated inhibition of
Cdc2 by G2-damaged microtubules: To determine if the
Wee1-dependent MBC-induced cell-cycle delay is mediated via the canonical phosphorylation and inhibition
of Cdc2, we examined Tyr15 phosphorylation in G2
synchronized cells by using a cdc25-22 block-and-release
strategy (Alfa et al. 1990). After release at 25°, Tyr15
phosphorylation and septation were determined over
time in MBC- or DMSO- (control) treated cells. As shown
in Figure 3, A and B, DMSO-treated cells (control) progressed normally through the cell cycle and the G2/M
transition was accompanied by a reduction of the level
of Tyr15 phosphorylation as expected (Gould and
Nurse 1989). However, tyrosine dephosphorylation of
Cdc2 associated with mitotic activation was severely
delayed in MBC-treated cells (see Figure 3A). Thus,
the microtubule-damage-dependent G2-phase delay
acts via Wee1 to promote maintenance of Cdc2 Tyr15
phosphorylation.
Since Wee1 is the direct regulator of Cdc2 Tyr15
phosphorylation in this microtubule-damage response,
the Cdc2 inhibitory effect mediated by Wee1 in MBCtreated cells could be exerted by increasing Wee1
activity and/or Wee1 abundance. To gain insight into
the mechanism by which microtubule depolymerization
inhibits Cdc2, we examined synchronized cells to determine the levels of Wee1 protein during the G2/M
transition. Using the cdc25-22 block-and-release strategy,
the amount of tagged Wee1-HA protein was monitored
during the G2/M transition in cells treated with either
MBC or DMSO (control) (Figure 3C). Samples were
taken to monitor the progression of the cells through
mitosis via PSH3 staining and their septation index
(Figure 3D). As shown in Figure 3, C and E, levels of
Wee1 protein decreased as cells entered into mitosis in
control cells but, significantly, remained high in MBCtreated cells. This result indicates that Wee1 protein
levels are involved in triggering a G2-phase delay in the
response to damage to microtubule structures. On the
basis of this observation one might expect that fission
yeast cells with higher levels of Wee1 protein would have
Microtubule-Damage Response in Fission Yeast
2077
Figure 2.—Wee1p mediates the microtubuledamage-induced G2-phase delay. (A) MBC
(4 mg/ml) growth sensitivity of wild-type S. pombe
cells and mutants defective for the Wee1-mediated inhibition of Cdc2 (wee1D and cdc2-1w) or
Cdc25-mediated Cdc2 activation (cdc2-3w and
cdc2-3w cdc25D). Assays were carried out in YES
medium at 25°. (B) Cell-cycle progression with
MBC (75 mg/ml) or DMSO of synchronized
wee1-50 cells (thermo-sensitive allele). Cells were
grown in EMM medium at 25° and after elutriation were shifted to 30° (semirestrictive temperature) and the percentage of phosphorylated
histone H3 (left) and the percentage of septated
cells (right) was monitored as described in Figure
1E. (C) Cell-cycle progression of synchronized
Cdc25-deficient cells (cdc2-3w cdc25D cells) treated with MBC (75 mg/ml) or DMSO as described
in Figure 1E. (D) MBC (4 mg/ml) growth hypersensitivity of cdc25-22 S. pombe cells at 27° (semirestrictive temperature). Successive dilutions of
wild-type and cdc25-22 cells were spotted onto
YES solid medium containing MBC (4 mg/ml)
or DMSO. (E) Cell length of each strain and condition was visualized under the microscope.
an enhanced G2-phase delay in response to MBC treatment. As shown in Figure 3F, wild-type cells (a single
wee1 gene copy per genome) are slightly elongated fol-
lowing MBC treatment, consistent with a transient G2
delay; however, in cells expressing higher levels of Wee1
(harboring three wee1 gene copies) (Russell and Nurse
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F. R. Balestra and J. Jimenez
Figure 3.—Inhibitory Cdc2-Tyr15 phosphorylation and its regulatory mechanism under microtubule-damaging conditions. (A
and C) Cells were synchronized by a block-and-release strategy. The synchronized culture was split into two, MBC (75 mg/ml) or
DMSO were added to the cells after release, and aliquots were taken periodically. Extracts were prepared from each aliquot and
analyzed by Western blot. (A) Cdc2-Tyr15 phosphorylation (PCdc2) was detected along the G2/M transition in the synchronized
cdc25-22 strain. Phospho-cdc2 (tyr15) antibody or anti-PSTAIR antibody were used to determine Cdc2-Tyr15 phosphorylation or
total Cdc2 protein as a loading control, respectively. (B) The percentage of septated cells shown in A was determined under the
microscope to monitor cell-cycle progression. (C) Abundance of Wee1 protein along the G2/M transition in synchronized cdc2522 wee1-HA cells. Anti-HA or anti-PSTAIR antibodies were used to determine Wee1-HA or total Cdc2 protein as a control, respectively. (D) The percentage of PSH3-positive cells and the percentage of septated cells shown in C were determined under the
microscope to monitor entry into mitosis and cell-cycle progression. (E) Wee1 levels detected in C with MBC (solid bars) or DMSO
(open bars) were quantified. Cdc2 was used as a loading control. (F) Wild-type S. pombe cells and 3x wee1 cells were grown on liquid
medium supplemented with MBC (75 mg/ml) or DMSO for 3 hr at 25° and DAPI/calcofluor-stained cells were visualized under
the microscope.
1987), this delay was significantly enhanced. Thus the
G2-phase microtubule-damage response appears to be
regulated, at least in part, by controlling Wee1 protein
levels. Degradation of Wee1 is the main mechanism
triggering the decrease in Wee1 levels as cells enter mitosis
(McGowan and Russell 1995; Aligue et al. 1997;
Muñoz et al. 1999; Watanabe et al. 2005), suggesting
that stabilization of Wee1 is likely involved in the
maintenance of this protein during the G2/M transi-
tion in the presence of microtubule-depolymerizing
agents.
The spindle checkpoint represents a well-established
signaling mechanism able to detect defects in microtubule structures. Since microtubule disassembly appears
to be the cause of the MBC-induced G2 delay in S. pombe
cells, we tested whether this delay was dependent on
aspects of the mitotic spindle checkpoint apparatus. We
examined the MBC sensitivity of several deletion mu-
Microtubule-Damage Response in Fission Yeast
tants for components of the mitotic spindle checkpoint,
including the spindle checkpoint response genes mad2
and bub1 (mad2D and bub1D, respectively) (Asakawa
et al. 2006; May and Hardwick 2006), as well as in cells
deleted for dma1 (dma1D), which is essential in S. pombe
for the septation initiation network inactivation in the
presence of spindle damage (Murone and Simanis
1996). Significantly, its mammalian ortholog, Chfr, is implicated in a very early mitotic response to microtubule
damage (Matsusaka and Pines 2004). All of these
mitotic checkpoint components were hypersensitive to
MBC treatment in a similar fashion to that exhibited by
wee1-deficient mutants (see supplemental Figure S2).
However, according to the Cdc2 Tyr15-phosphorylation
assay in synchronous fission yeast cells, the G2 delay
triggered by MBC treatment remained intact in these
mutant strains, indicating that the G2-phase microtubuledamage response described here is independent of
mitotic spindle damage signaling pathways (supplemental Figure S3).
In budding yeasts, a G2-phase arrest has been reported
in response to microtubule perturbation during meiosis
(Hochwagen et al. 2005), but equivalent mechanisms
operating during the mitotic cycle has not been described so far. In mammalian cells, the ‘‘antephase
checkpoint’’ seems to regulate early steps of the mitotic
phase during the mitotic cycle in response to microtubule disassembly (Matsusaka and Pines 2004). However, whether this checkpoint is independent of the
spindle checkpoint remains unclear. It has been recently described that the integrity of the microtubule
cytoskeleton is not subjected to checkpoint surveillance
in normal human cells (Uetake and Sluder 2007). In
this study, the authors describe an interphase duration
of 4 hr longer than that of the control (24 hr vs. 20 hr on
average) in nocodazole-treated populations of primary
fibroblasts. This delay, which is consistent with the MBCinduced delay described in this study for S. pombe cells, is
attributed to a stress caused by the loss of microtubules.
In S. pombe, stress delays entry into mitosis by activation
of the p38/Sty1 MAP kinase pathway (Suda et al. 2000;
Lopez-Aviles et al. 2005). As occurred with mutants for
the spindle checkpoint, the MBC-mediated delay in G2
was also observed in sty1D cells defective for these MAPkinase-signaling stress conditions (supplemental Figure
S4), thereby excluding this pathway of the mechanism
delaying the G2/M transition by microtubule injuries.
Thus, we conclude that the results described in our study
uncover a mechanism (a G2-phase microtubule-damage
checkpoint) that links interphase microtubule integrity
with entry into mitosis in fission yeasts.
Checkpoint defects are often associated with cancer
cells (Kastan and Bartek 2004). The identification of
new damage-sensing mechanisms in different cell structures, capable of arresting the cell cycle, could lead to
new approaches to cancer treatment ( Janetka et al. 2007).
In the mechanism described in this study, stabilization
2079
of the universal Wee1 kinase is essential for the cell response to microtubule perturbation. Several microtubuletargeting drugs are used as chemotherapeutic drugs for
human cancer patients (Pellegrini and Budman 2005).
On the basis of our observations, selective Wee1 inhibition or degradation could be used to sensitize tumor
cells to antimicrotubule agents for therapeutic applications. The ability to manipulate such a novel checkpoint
may even provide the basis for completely novel anticancer treatments.
We thank Anabel Lopez and Victor Carranco for excellent technical
assistance, Victor Alvarez and Rafael R. Daga for useful discussions,
Paul Nurse and Paul Russell for yeast strains, Jon Chung and Iain
Hagan for their help in centrifugal elutriation experiments, and John
R. Pearson for critical reading of the manuscript. This work was
supported by grants from the Spanish Ministerio de Educacion y
Ciencia (BFU2006-11672). The institutional support from the Junta
de Andalucı́a is acknowledged.
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Communicating editor: M. D. Rose