1 Departments of Radiation Oncology and Cell Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Graduate School of Biostudies, Department of Gene Mechanisms, Kyoto
University, Sakyo-ku, Kyoto, 606-8502, Japan
3 Radiation Biology Center, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku,
Kyoto, 606-8501, Japan
* These authors contributed equally to this work
Present address: MRC Cell Mutation Unit, University of Sussex, Falmer,
Brighton, BN1 9RR, UK
Author for correspondence (e-mail:
tmatsumo{at}aecom.yu.edu
)
Accepted 19 January 2002
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Summary |
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Key words: Spindle checkpoint, Mad2, Slp1
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Introduction |
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The cohesin protein complex (Guacci et
al., 1997; Michaelis et al.,
1997
) maintains the link between the sister chromatids. At
anaphase, cohesin is cleaved by a specific protease separase (budding yeast
Esp1/fission yeast Cut1), which triggers sister chromatid separation
(Uhlmann et al., 2000
).
Throughout most of the cell cycle, separase forms a complex with securin
(budding yeast Pds1/fission yeast Cut2) and is kept as an inactive form
(Nasmyth et al., 2000
;
Yanagida, 2000
). Separase is
released from the complex and activated after securin is removed and degraded
by ubiquitin-dependent proteolysis (Ciosk
et al., 1998
; Kumada et al.,
1998
). Therefore, the destruction of securin leads to the
activation of separase and to the subsequent sister chromatid separation.
Securin contains a short amino-acid motif termed the `destruction box', a
signature sequence for proteins to be selectively destroyed at or after the
onset of anaphase. Proteins that contain a destruction box are ubiquitinated
by the action of the APC/Cyclosome protein complex
(King et al., 1995
;
Sudakin et al., 1995
).
Following ubiquitination, proteins with the destruction box are degraded by
the 26S proteasome. The ubiquitination of securin also requires another
protein, budding yeast Cdc20 (Schwab et
al., 1997
; Visintin et al.,
1997
)/fission yeast Slp1
(Matsumoto, 1997
)/human p55CDC
(Weinstein et al., 1994
),
which serves as a substrate-specific activator of APC. When Cdc20 is
overexpressed, securin can be degraded during interphase, whereas other
destruction box proteins will remain stable. In cells lacking Cdc20, securin
remains stable, and sister chromatids will not separate
(Visintin et al., 1997
).
Mad2 mediates the cross-talk between the spindle checkpoint and the
mechanism that regulates securin proteolysis as it physically interacts with
Slp1/Cdc20/p55CDC. In yeast, expression of a mutant allele of
slp1+/CDC20, which is defective in binding to Mad2
abolishes the spindle checkpoint in a dominant manner
(Hwang et al., 1998;
Kim et al., 1998
). In humans,
Mad2 and p55CDC associate with each other in early mitosis
(Fang et al., 1998
). The
association is transient, and the Mad2-p55CDC complex dissociates in late
mitosis (Wassmann and Benezra,
1998
). Biochemical experiments have demonstrated that Mad2
inhibits APC-dependent ubiquitination
(Fang et al., 1998
;
Li et al., 1997
). These
results indicate that Mad2 targets Slp1/Cdc20/p55CDC and prevents them from
promoting the securin proteolysis at such time when the spindle attachment to
kinetochores is incomplete.
In higher eukaryotes Mad2 changes its localization in a
cell-cycle-dependent manner (Chen et al.,
1996; Howell et al.,
2000
; Li and Benezra,
1996
). It is preferentially found on the nuclear periphery,
although it is also distributed throughout the cell
(Kallio et al., 1998
). At or
near the onset of mitosis, Mad2 translocates into the nucleus. From prophase
to prometaphase if the spindle attachment is incomplete, it localizes to
kinetochores. Similarly, if spindle formation is inhibited by a poison such as
nocodazole, Mad2 can be found associated with unattached kinetochores. The
localization of Mad2 on unattached kinetochores would suggest that unattached
kinetochores play an important role in activating the spindle checkpoint so as
to delay the onset of sister chromatid separation.
Bub1, another component of the spindle checkpoint, has also been shown to
translocate in the cell cycle. From prophase to prometaphase, mouse Bub1 is
localized to kinetochores (Taylor and
McKeon, 1997). Once kinetochores are aligned on the metaphase
plate, it diffuses throughout the nucleus. Fission yeast Bub1 behaves
similarly. It, however, remains at kinetochores in later mitosis
(Bernard et al., 1998
) as well
as meiosis (Bernard et al.,
2001
). It has been proposed that fission yeast Bub1, besides its
role in the spindle checkpoint, plays an additional role in maintenance of
sister kinetochore cohesion in meiosis I.
Mad2 forms protein complexes with other components of the spindle
checkpoint. The Mad1-Mad2 complex, which exists throughout the cell cycle
(Chen et al., 1999), is
necessary for localization of Mad2 on unattached kinetochores
(Chen et al., 1998
). Mad2 also
forms a complex with Mad3 in budding yeast
(Hardwick et al., 2000
).
Because the Mad2-Mad3 complex also contains Cdc20, this complex may function
at the endpoint of the checkpoint signaling cascade.
In this study we have used a simple tractable organism, fission yeast, to investigate localization of Mad2 in conjunction with its target, Slp1. When the spindle attachment is not complete, these two proteins form a complex and are colocalized on unattached kinetochores. During later stages of mitosis, the complex dissociates and the majority of Mad2, but not Slp1, is found on the spindle. Additionally we have also shown that Mad1 is required for the localization of Mad2 to the nuclear periphery during interphase.
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Materials and Methods |
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Cloning of mad1+ gene, its disruption and
tagging
With the full-length mad2+ gene as bait, a part of the
mad1+ gene was identified through the yeast two-hybrid
screen. Nucleotide sequence analysis indicated that a cosmid clone, c1055,
contains the full-length mad1+. A
SalI-SphI fragment of the cosmid, which contained the
mad1+ gene, was subcloned into pUC119 to construct
119gMad1. The ura4+ was inserted between NcoI and
NruI sites of the Sall-SphI fragment on 119gMad1.
This insertion would allow expression of the first 34 amino acids, at most, of
the Mad1 protein. The linear DNA fragment, which contained the
ura4+ gene in place of a part of the
mad1+ gene, was used for transformation. Stable
Ura+ diploids were examined by southern blot as well as tetrad
analysis. The examination indicated that deletion of the
mad1+ gene does not affect cell growth under normal
conditions.
The gene encoding the HA epitope, flanked by NotI sites at both ends, was inserted into a NotI site generated right before the termination codon of the mad1+ gene on 119gMad1. The linear DNA fragment resulting from digest with SalI and SphI was used for replacement of the disrupted allele of mad1 with the HA-tagged allele by double homologous recombination.
Protein analysis
Cells were washed and suspended in modified HB buffer as described by
Yamada et al. (Yamada et al.,
2000). Yeast extract was prepared by beating cells in the presence
of glass beads for 30 seconds, five times. The cell extracts were centrifuged
at 10,000 g for 5 minutes, with aliquots of the supernatant
either used for western blot or immunoprecipitation. For SDS-PAGE, protein
extracts were loaded at 150 to 200 µg/lane, whereas 1.5 to 2.0 mg of
protein extract was used for immunoprecipitation.
Cytological techniques
PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) was
added to the fixed cells at 30% (v/v) of the final methanol concentration.
Cells were then washed with PBS three times and stained with 1 µg/ml DAPI.
For tubulin staining, the methanol fixation method was employed. Cells were
fixed with methanol as described above and were treated with 0.1 mg/ml
zymolase 100T in PEMS buffer (PEM, 1.2 M sorbitol) at 37°C for 10 minutes.
The cells were then washed with PEMS containing 0.1% TritonX-100 for 2 minutes
and washed with PEM three times. For blocking, PEMBAL buffer (PEM, 0.1% bovine
serum albumin) was used. After 1 hour of blocking, cells were stained with the
primary antibody (tat1) and incubated over night. Cells were washed with
PEMBAL for 30 minutes three times, followed by incubation with a CY3
conjugated secondary antibody for 4 hours. Finally cells were washed with
PEMBAL three times and stained with 1 µg/ml of DAPI. All steps were
performed at room temperature. For Slp1 staining, cells were first fixed by
formaldehyde at the final concentration of 3.7% for 30 minutes. After
performing two wash steps with PEM, spheroplasts were obtained by incubation
at 37°C for 7 minutes using 0.5 mg/ml zymolase and 0.3 mg/ml novozyme. The
following steps are identical to those for tubulin staining, except that the
Slp1 antibody was used as a primary antibody. The Mad2-GFP signal was observed
under the microscope using the same cells as was used for tubulin or Slp1
immunostaining.
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Results |
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|
Mad2 in a ß-tubulin mutant, nda3-KM311
In higher eukaryotes, Mad2 will localize to unattached kinetochores if the
spindle-kinetochore attachment is incomplete
(Chen et al., 1996;
Waters et al., 1998
). Using
the synchronous culture of the cdc25 mutant, mitosis proceeded
normally, but we were unable to anticipate at what time point
spindle-kinetochore attachment failed. In order to capture Mad2-GFP
localization to unattached kinetochore, we employed a cold-sensitive
nda3-KM311 mutant, which is unable to form a spindle because of a
mutation in the ß-tubulin gene
(Hiraoka et al., 1984
). At the
restrictive temperature (20°C), the mutant will arrest during mitosis with
condensed chromosomes but with no spindle
(Hiraoka et al., 1984
).
Importantly, the arrest induced by the nda3 mutation requires a
functional spindle checkpoint (He et al.,
1997
). As shown in Fig.
2, Mad2-GFP was concentrated as one or two speckles in the mutant
after 6 hours of incubation at the restrictive temperature. When the mutants
were incubated longer at the restrictive temperature, some of them exhibited
three speckles, which might represent the three chromosomes of fission yeast
(not shown). In the same cells, Mis6 a kinetochore-specific protein
(Saitoh et al., 1997
) was
colocalized with Mad2-GFP, indicating that the fission yeast Mad2 is localized
to unattached kinetochores.
|
The cell cycle arrest caused by the nda3 mutation is reversible.
If the arrested cells are shifted back to the permissive temperature
(33°C), cell cycle progression is re-initiated and the cells will proceed
synchronously (Hiraoka et al.,
1984). We examined localization of Mad2-GFP in the cells that were
released from the nda3-induced arrest. The progression of mitosis was
monitored both by visualizing chromosomes using DAPI staining and by indirect
immunofluorescent staining of the spindle. We also determined the relative
abundance of Slp1, which drops in late mitosis, as an additional means of
monitoring cell cycle progression (Yamada
et al., 2000
). After the nda3 mutant was arrested at
20°C for 6 hours, approximately 80% of the cells exhibited the bright
speckles of Mad2-GFP (Figs
3A,B). Three minutes after the release, in response to a shift to
the permissive temperature, most of the bright speckles became less intense.
At this time point, short spindles became visible in those cells
(Fig. 3B), although separation
of sister chromatids was not apparent. The reduction in the intensity of the
Mad2-GFP signal during the 3 minutes immediately following release from
metaphase arrest was highly reproducible. We analyzed the digital images in a
quantitative manner and categorized each speckle into either of two groups. In
one group, speckles remained as intense as they had at time 0, whereas the
second group was scored for a reduced intensity. The analysis indicated that
in 30% of the cells, Mad2-GFP speckle intensity did not change
(Fig. 3A). Although we could
not demonstrate the biological significance of the change in the speckle
intensity, it might be possible that the bright speckles represented Mad2-GFP
localized to unattached kinetochores. The sudden reduction in the intensity of
the Mad2-GFP is similar to that of Bub1
(Bernard et al., 2001
). Six
minutes after the release, anaphase was initiated and Mad2-GFP was localized
to the spindle. During the following 9 minutes, the GFP-Mad2 remained with the
spindle. When the spindle disappeared 18 minutes after the release, Mad2-GFP
was not observed localized to any distinctive structure
(Fig. 3B). Analysis of the
levels of Mad2 and Slp1 indicated that although Mad2 was constant throughout
mitosis, Slp1 abundance dropped after anaphase was initiated
(Fig. 3C).
|
Mad2 in other mitotic mutants
We also examined the localization of Mad2-GFP in other mutants that arrest
at specific stages of mitosis. In the temperature-sensitive cut7
mutant, spindle formation is blocked shortly after duplication of the SPB. The
mitotic spindle radiating from the opposite poles cannot interdigitate and
thus remains as a V shape (Hagan and
Yanagida, 1992). The cut7-specific defect generates a
condition whereby the bipolar attachment of the spindle to kinetochores is not
achieved. Previously, we demonstrated that this defect causes an arrest in a
mad2+-dependent manner
(Kim et al., 1998
). At the
restrictive temperature the majority of Mad2-GFP was found on condensed
chromosomes as one or more speckles (Fig.
4). Although we did not examine the colocalization with Mis6,
these speckles of Mad2-GFP would represent the position of unattached
kinetochores. The dis1+ gene encodes an M-phase-specific
kinetochore protein with a presumed role in establishing a connection between
kinetochores and microtubules (Nakaseko et
al., 2001
). Its defect would result in an anomaly in the
spindle-kinetochore interaction. Indeed, sister chromatids do not separate in
the dis1 cold-sensitive mutant, although the spindle will elongate
(Ohkura et al., 1988
). In the
dis1 mutant, Mad2-GFP was localized as speckles in the chromatin
domain (Fig. 4). Mad2-GFP was
localized similarly in the three mutants nda3, cut7 and
dis1, in which the spindle-kinetohore attachment are defective. Other
mutants, such as the cut4 and nuc2, display an arrest of the
cell cycle in response to a lack of APC activity
(Yamada et al., 1997
;
Yamashita et al., 1996
). In
these mutants the interaction between the spindle and kinetochores is
presumably normal, and we found that the majority of the Mad2-GFP was
localized to the spindle (Fig.
4).
|
Interaction between Slp1 and Mad2
Slp1 is a target of the spindle checkpoint
(Kim et al., 1998). When
spindle formation is incomplete, Mad2 is thought to bind to Slp1 and prevent
proteolysis by Slp1; proteolysis is necessary for the onset of anaphase. In
order to determine at which mitotic stages Mad2 is interacting with Slp1, we
performed immunoprecipitation using cell extracts prepared from various cell
cycle mutants. In late G2 extracts prepared from the cdc25 mutant,
Slp1 was not detectable. Extracts, immunoprecipitated with the antibody to
Slp1, did not reveal the presence of either Slp1 or Mad2, although the
extracts did contain Mad2 (Fig.
5A). These results demonstrated that the antibody to Slp1 did not
directly interact with Mad2 and thus could serve as a negative control. In
contrast, precipitates from the nda3 mutant contained both Slp1 and
Mad2, indicating that the two proteins formed a complex when spindle formation
was not completed (Fig. 5A). In
extracts prepared from the nuc2 mutant, which causes an arrest owing
to defective APC subunit, the levels of Mad2 and Slp1 were similar to those
found in the nda3 mutant. However the immunoprecipitates using the
antibody to Slp1 contained Slp1 but very little Mad2. Similar results were
obtained using extracts prepared from another APC mutant (cut9) and
the mts3 mutant defective in a subunit of the 26 S proteasome
(Fig. 5A). These results would
suggest that the Mad2-Slp1 complex, which is stable when the spindle formation
is incomplete, is disassembled prior to the onset of anaphase.
|
We next examined localization of Slp1 using indirect immunofluorescent staining. First, in the G2-phase-arrested cdc25 mutant, detection of the antibody to Slp1 revealed speckle-like signals (Fig. 5B). Considering that in the cdc25 mutant, Slp1 was not detectable by immunoblotting (Fig. 5A), the staining may represent a non-specific recognition by the antibody. Second, in the nda3 mutant, Slp1 was observed as speckles in the chromatin domain. These speckles were colocalized with Mad2-GFP, indicating that in the nda3 mutant, Slp1 is also localized to unattached kinetochores (Fig. 5B). In other mitotic mutants such as nuc2 and mts3, we did not observe Slp1 localized to any distinctive cellular location, although the Slp1 protein was detectable by western blot (Figs 5A,B).
Interaction between Mad1 and Mad2
In species such as budding yeast, frog and human
(Chen et al., 1999;
Chen et al., 1998
;
Jin et al., 1998
), Mad2 also
interacts with Mad1, which is another component of the spindle checkpoint. We
isolated a part of the fission yeast mad1+ gene by the
yeast two-hybrid screen using Mad2 as bait. The fulllength
mad1+ gene was identified in a cosmid clone 1055
(Mizukami et al., 1993
). In
order to confirm the physical interaction between Mad1 and Mad2, we tagged the
C-terminus of Mad1 with an HA epitope. Immunoprecipitates with the antibody to
Mad2 contained Mad2 as well as Mad1-HA
(Fig. 6A). If a similar
experiment was performed with extracts prepared from cells that did not
express Mad2, the precipitates did not contain Mad1-HA. Thus, the
immunoprecipitation demonstrated a specific interaction between Mad1 and
Mad2.
|
To determine the role of Mad1 in localization of Mad2, we examined
localization of Mad2-GFP in cells lacking Mad1 (mad1). As
shown in Fig. 6B, although
Mad2-GFP was localized to the nuclear periphery and chromatin domain in the
wild-type cells during interphase, it was not found in the corresponding
locations in
mad1. To confirm that the abundance of Mad2-GFP was
similar in the two cell lines, we examined its levels by western blot. We
found that both the wild-type strain and
mad1 expressed Mad2-GFP at
similar levels (Fig. 6C). These
results indicate that Mad1 is required for localization of Mad2.
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Discussion |
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Upon release from the nda3 block, the kinetochore-associated
Mad2-GFP signal rapidly decreased. Three minutes after the release, the
spindle can be recognized as a very short fiber. At this early time point, the
intensity of Mad2-GFP has already decreased. We would speculate that the
attachment of the spindle to the kinetochore can be completed within three
minutes of the release and that this decrease in intensity of Mad2-GFP
reflects the movement of Mad2 away from kinetochores upon completion of the
spindle attachment. Unlike in higher eukaryotes, all three kinetochores of
fission yeast are clustered near the SPB, a structure equivalent to the
centrosome (Funabiki et al.,
1993). This configuration may be beneficial, as it allows the
spindle to be attached in such a short period of time.
In order to demonstrate Mad2 localization to kinetochores, we have used
Mis6 as a kinetochore-specific marker
(Saitoh et al., 1997). CHIP
(chromatin immunoprecipitation) is another method by which localization can
more precisely be shown. If successful, one could determine in which region of
the kinetochore a protein is localized to. However, this method proved
unsuccessful in examining Mad2 localization. As recently shown in PtK1 cells,
Mad2 is a transient component of the kinetochore with a half-life of
24
to 28 seconds (Howell et al.,
2000
). A CHIP assay may require a more stable interaction between
the protein and its target site.
The role of unattached kinetochores and catalytic model
In this study we have also examined the localization of Slp1, a target of
Mad2, and found that Slp1 and Mad2 colocalize to unattached kinetochores when
formation of the spindle is inhibited by the nda3 mutation. The
colocalization coincides with the formation of the Mad2-Slp1 complex. Although
the complex is readily detectable in extracts prepared from cells arrested by
the nda3 mutation, it is not detectable in extracts prepared from
cells arrested by other mutations such as the nuc2, cut9 or
mts3. These mutations affect ubiquitin-dependent proteolysis, causing
arrest of a cell cycle progression at a later stage of mitosis. These results
strongly support the catalytic model
(Kallio et al., 1998), in
which unattached kinetochores are believed to catalyze the formation of the
Mad2-Slp1 complex.
Mad2 is localized to the spindle after the transition from metaphase to anaphase. The role of Mad2 at the spindle is still to be revealed. Because the Mad2-Slp1 complex is not detectable after the transition, we would speculate that Mad2 on the spindle plays a role unrelated to the regulation of Slp1.
Silencing the spindle checkpoint
Although the levels of Mad2 and Slp1 are similar among cell extracts
prepared from the nda3, nuc2, cut9 and mts3 mutants, the two
proteins are only found assembled into the complex when extracts are prepared
from the nda3 mutant. One would speculate that the complex, which is
assembled when attachment of the spindle is incomplete, is disassembled upon
the completion of the attachment. The disassembly would be necessary for Slp1
to promote ubiquitin-dependent proteolysis. We therefore postulate that an
active mechanism is responsible for silencing the spindle checkpoint and
disassembly of the Mad2-Slp1 complex. Closer examination of Mad2, as well as
Slp1, would reveal the molecular mechanism behind the silencing.
The role of Mad1
In cells lacking Mad1, Mad2 fails to localize to the nuclear periphery or
the chromatin domain. it has recently been demonstrated that Mad1 recruits
Mad2 to unattached kinetochores in frogs
(Chen et al., 1998). Our result
indicates that Mad1 plays an additional role that of anchoring Mad2 to
the nuclear periphery and probably regulating its entry into the nucleus.
Every time a cell enters mitosis, kinetochores are unattached for a certain
period of time. Thus, the spindle checkpoint is activated as part of the
normal cell cycle. The Mad1-Mad2 complex, which is stable throughout the cell
cycle (Chen et al., 1999
),
would have to be regulated in such a way, so it would be able to enter into
the nucleus at or around the onset of mitosis.
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Acknowledgments |
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