Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh, Scotland, EH9 3JR, UK
* Author for correspondence (e-mail: Kevin.Hardwick{at}ed.ac.uk)
Accepted 4 November 2002
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Summary |
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Key words: Bub3, Spindle checkpoint, Kinetochore
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Introduction |
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Budding yeast genetics originally identified the Mad and Bub proteins, and
later the Mps 1 kinase, as components of this checkpoint
(Hoyt et al., 1991;
Li and Murray, 1991
;
Weiss and Winey, 1996
). These
have since been shown to be conserved across species from yeast to man. Their
precise functions in the checkpoint pathway are now the subject of intense
study in many model organisms. More recently a number of kinetochore proteins,
including the CENP-E microtubule motor protein
(Chan et al., 1998
;
Yao et al., 2000
), and ROD and
Zw 10 (Basto et al., 2000
;
Chan et al., 2000
), have also
been shown to play roles in the checkpoint.
The Bub3 protein binds constitutively to two other checkpoint proteins, the
Bub1 kinase (Brady and Hardwick,
2000; Martinez-Exposito et
al., 1999
; Roberts et al.,
1994
; Taylor et al.,
1998
) and Mad3 (Fraschini et
al., 2001
; Hardwick et al.,
2000
; Millband and Hardwick,
2002
). Mad3 is the yeast orthologue of vertebrate BubR1, which
also binds to Bub3 (Chan et al.,
1999
; Chen, 2002
;
Yao et al., 2000
). The precise
function of Bub3 remains unclear but it has been suggested to target Bub1 and
BubR1/Mad3 to kinetochores (Millband and
Hardwick, 2002
; Taylor et al.,
2001
). In Saccharomyces cerevisiae, there are two quite
distinct Bub3-containing complexes. Upon checkpoint activation, Bub3 and Bub1
bind to Mad1 (Brady and Hardwick,
2000
), and Bub3 is also found in a large complex associated with
Mad3, Mad2 and the spindle checkpoint effector Cdc20
(Fraschini et al., 2001
;
Hardwick et al., 2000
). In
HeLa cells a similar complex containing BubR1, Bub3, Mad2 and Cdc20 has been
detected (Fang, 2002
;
Sudakin et al., 2001
;
Tang et al., 2001
). Thus it is
possible that Bub3 has a number of distinct functions in the spindle
checkpoint.
To prevent anaphase onset the Mad and Bub proteins inhibit the function of
the checkpoint effector Cdc20/fizzy (Fang
et al., 1998; Hwang et al.,
1998
; Kallio et al.,
1998
; Kim et al.,
1998
; Wassmann and Benezra,
1998
). Cdc20 targets specific substrates to an E3 ubiquitin ligase
known as the cyclosome/anaphase promoting complex (APC)
(Visintin et al., 1997
), which
marks them for destruction by the proteasome (for reviews, see
Page and Hieter, 1999
;
Zachariae and Nasmyth, 1999
).
A key APC substrate that needs to be destroyed for anaphase onset is securin
Pds1/Cut2 (Funabiki et al.,
1996
; Yamamoto et al.,
1996
; Zou et al.,
1999
), which complexes with and inhibits the separase Esp1
(Ciosk et al., 1998
). Once
Esp1 is released it cleaves a cohesin, Scc1, in budding yeast
(Uhlmann et al., 1999
) and
sister-chromatid separation and anaphase ensue.
The spindle checkpoint pathway has been reconstituted in Xenopus
egg extracts and shown to be MAP kinase dependent
(Minshull et al., 1994). It
has since been shown to require the XMad1
(Chen et al., 1998
), XMad2
(Chen et al., 1996
), XBub1
(Sharp-Baker and Chen, 2001
),
XBubR1 (Chen, 2002
), XMps1
(Abrieu et al., 2001
), Aurora B
(Kallio et al., 2002
) and
CENPE proteins (Abrieu et al.,
2000
). Here we have produced a number of antibodies specific for
the XBub3 protein. With these antibodies we show that XBub3 function is
required both for spindle checkpoint activation and for maintenance of a
checkpoint arrest in Xenopus egg extracts. We find that XBub3 is
complexed with both XBub1 and XBubR1 kinases, and we localise XBub3 throughout
the cell cycle in Xenopus tissue culture (XTC) cells. During
interphase XBub3 has a diffuse nuclear localisation, and it is recruited to
kinetochores during early prophase. Once chromosomes align at the metaphase
plate, XBub3 staining is lost. In egg extracts, our antibodies do not
interfere with kinetochore localisation of XBub3 or of the other checkpoint
proteins tested. We discuss their possible mechanisms of interference with the
spindle checkpoint and different roles that XBub3 might play.
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Materials and Methods |
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Expression of GST-XBub3 and production of XBub3 antibodies
The full-length XBub3 DNA was subcloned into pGEX-6P-2 (Pharmacia) for the
expression of GST-tagged XBub3 in the E. coli strain BL21.DE3
(Stratagene). GST-XBub3 was induced overnight at 16°C and purified from
E. coli lysates on glutathione-agarose resin (Sigma). The protein was
eluted using 20 mM glutathione and dialysed into TBS (25 mM Tris, pH 8.0, 150
mM NaCl). Antibodies were raised in rabbits and sheep against full-length
GST-XBub3 protein (Diagnostics Scotland) and affinity purified against
GST-XBub3 protein coupled to Affigel 10 resin (BioRad). Antibodies were also
raised in rabbits and sheep against a peptide corresponding to the C-terminal
amino acids 316-330 (C-AIYIRQVTDAETKPK) of XBub3. The peptide was coupled with
glutaraldehyde to keyhole limpet hemocyanin for immunisation in rabbits and
sheep and to Affigel-10 resin (BioRad) for affinity purification of XBub3
peptide antibodies (Harlow and Lane,
1988).
Immunoprecipitation of XBub1 and XBubR1 and protein
phosphatase treatment
Polyclonal XBub1 and XBubR1 antibodies were a gift from Rey-Huei Chen
(Department of Molecular Biology and Genetics, Cornell University, Ithaca,
NY). To immunoprecipitate XBub1 or XBubR1 20 µl of Affi-prep protein A
support beads (BioRad, Hercules, CA) were incubated with 3 µg of XBub1
antibody for 1 hour at room temperature in TBS. The beads were washed into
Extract Buffer (XB; 10 mM HEPES, pH 7.8, 50 mM sucrose, 100 mM potassium
chloride, 10 mM magnesium chloride, 1 mM calcium chloride and 5 mM EGTA)
containing 10 µg/ml each of leupeptin, pepstatin and chymostatin (LPC) as
protease inhibitors, and 50 mM sodium fluoride, 1 mM sodium vanadate and 80 mM
sodium-ß-glycerophosphate as phosphatase inhibitors. Excess buffer was
removed and the washed beads were then incubated with 80 µl of CSF-arrested
Xenopus egg extract for 1 hour at 4°C. The beads were then washed
three times with XB, containing protease and phosphatase inhibitors, twice in
XB and twice with protein phosphatase buffer containing 10 mM
MnCl2. The XBub1 beads were then split into two aliquots, which
were incubated in the presence and absence of 20 units of
protein
phosphatase (New England Biolabs Inc., Beverly, MA) for 30 minutes at
30°C. The bound proteins were eluted in SDS-PAGE sample buffer, resolved
by SDS PAGE and detected by immunoblotting using rabbit XBub1 antibody or
sheep XBub3 peptide antibody.
Immunoprecipitation of Xenopus Bub3 protein from denatured
egg extracts
40 µl of CSF-arrested Xenopus egg extract was denatured in 1 ml
of TCA buffer (20 mM Tris pH 8.0, 10% TCA, 50 mM ammonium acetate, 2 mM EDTA).
The denatured proteins were pelleted at 14,000 g for 10 minutes at
4°C in a microfuge. The pellets were then resuspended in 100 µl of
resuspension buffer (100 mM Tris, pH 11, 3% SDS, 3 mM DTT) by heating to
65°C for 5 minutes and 95°C for 10 minutes. 1 ml of
immunoprecipitation buffer suspension (10 mM Tris pH 8.0, 150 mM NaCl, 1%
Triton X-100) was then added to the extract protein suspension. 3 µg of
affinity-purified rabbit XBub3 peptide antibodies were coupled to 20 µl of
Affi-prep protein A support beads (Bio-Rad, Hercules, CA) in TBS (25 mM Tris,
150 mM sodium chloride, pH 8.0), and the washed beads were incubated with the
above protein suspension for 90 minutes at 4°C. The beads were washed
three times in immunoprecipitation buffer and the bound proteins eluted in SDS
PAGE sample buffer. The proteins were resolved by SDS PAGE and detected using
sheep XBub3 peptide antibodies.
Xenopus egg extracts and kinase assays
Fresh CSF-arrested Xenopus egg extracts were prepared from
unfertilised Xenopus eggs as previously described
(Murray, 1991). For analysis
of spindle checkpoint activation the CSF egg extracts were pre-incubated with
XBub3 antibodies to a final concentration of 50 µg/ml for 1 hour at
4°C. The extracts were released from metaphase arrest by addition of 0.8
mM CaCl2 in the presence or absence of 10 µg/ml nocodazole and
10,000 sperm nuclei/µl extract. For analysis of spindle checkpoint
maintenance the checkpoint was activated by addition of 10 µg/ml nocodazole
and 10,000 sperm nuclei/µl extract for 20 minutes at room temperature,
antibodies added and the extracts incubated for a further 30 minutes at room
temperature. 0 time points were taken and then CaCl2 added to 0.8
mM. The reactions were incubated at room temperature for 60 minutes and 1
µl samples were removed from the extracts during this time for histone H1
kinase activity analysis. The products of the histone H1 kinase reaction were
resolved by 15% SDS-PAGE and the dried gels exposed to hyperßmax X-ray
film (Amersham). Nuclear morphology in egg extracts was analysed by fixing
aliquots of extract in an equal volume of fix/stain solution (10 mM HEPES, 200
mM sucrose, 7.4% formaldehyde containing 10 µg/ml Hoechst 33258) and the
DNA visualised by fluorescence microscopy. Images were captured using a
Photometrics Sensys charge-coupled device (CCD) camera linked to a using a
Zeiss Axioskop fluorescence microscope using Smartcapture imaging software
(Vysis).
Gel filtration analysis
Aliquots of CSF egg extract were incubated in the presence of 10,000
sperm/µl egg extract, diluted fivefold in XB containing 10 µg/ml each of
leupeptin, pepstatin and chymostatin and 50 mM sodium fluoride, 1 mM sodium
vanadate, 80 mM sodium-ß-glycerophosphate, 1mM DTT and then filtered
through a 0.22 µm filter unit (Millipore). The diluted samples were
resolved on Superose-6 gel filtration column (Pharmacia) equilibrated in XB
and 1 mM DTT at a flow rate of 0.4 ml/minute, and 500 µl fractions were
collected. The samples were precipitated in 15% TCA, washed in acetone and
resuspended in SDS PAGE sample loading buffer.
Immunofluorescent staining of XTC cells
Asynchronous and nocodazole-treated (10 µg/ml for 4 hours)
Xenopus tissue culture cells were grown on coverslips for 24 hours,
rinsed in MMR buffer (100 mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM
CaCl2, 10 mM EDTA, 5 mM HEPES, pH 7.8) and incubated in 25% Hanks
balanced salt solution (Gibco-BRL, Paisley, Scotland) for 10 minutes at room
temperature. The cells were fixed in MMR containing 3% paraformaldehyde,
rinsed in MMR and then blocked in TBS containing 5% milk. Primary antibodies
were added to a final concentration of 1 µg/ml in TBS/5% milk and detected
using fluorescent Alexa-labelled secondary antibodies (Molecular Probes).
Nuclear morphology was analysed by placing the coverslips on slides containing
mounting medium containing DAPI (Vector Labs).
Immunofluorescent staining of chromosomes assembled in
Xenopus egg extracts
Replicated metaphase sperm chromatin was produced in Xenopus egg
extracts using the method previously described
(Chen et al., 1998). Briefly,
20 µl of CSF egg extract was released into interphase by addition of 0.8 mM
CaCl2 in the presence of 1500 sperm/µl extract. The extracts
were incubated at room temperature for 90 minutes to replicate the DNA and
then induced to enter mitosis by addition of 10 µl of CSF egg extract and
incubated at room temperature for a further 90 minutes. Affinity-purified
XBub3 antibodies were added to 0.1 µg/ml during this incubation. The
extracts were then incubated in the presence or absence of 10 µg/ml
nocodazole for 10 minutes at room temperature and followed by dilution with 10
volumes of XB containing 0.5% Triton X-100. The diluted extracts were then
layered over 5ml of 30% glycerol made in XB plus 0.5% Triton-X100 with a
coverslip placed at the bottom of the tube. The chromosomes were collected by
centrifugation at 8000 rpm in a Beckman JS13.1 rotor and the coverslips
processed for immunofluorescence as described for staining of XTC cells.
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Results |
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Fig. 1 shows an alignment between the Bub3 proteins of Xenopus, human, Drosophila and budding yeast. The bulk of the Bub3 protein is highly conserved across species. This is likely to be because of its 3D structure, which consists almost entirely of seven WD repeats that fold together to form a ß-propeller-like structure (David Wilson, UC Davis, personal communication).
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As the Xenopus Bub3 protein remained uncharacterised, we raised antibodies against full-length bacterially expressed GST-XBub3 and a peptide corresponding to the C-terminal residues 316-330 of XBub3 in both rabbits and sheep. The affinity-purified antibodies recognise bacterially expressed GST-XBub3 by western blotting (data not shown), confirming that the antibodies were capable of recognising XBub3 protein and detected a single band in Xenopus tissue culture cell (XTC) lysates running slightly larger than the predicted molecular weight of XBub3 (37kDa) (Fig. 2A). The antibodies recognised a doublet of bands in both CSF and interphase egg extracts (Fig. 2A). Although our antibodies can immunoprecipitate XBub3 protein expressed in the rabbit reticulocyte lysate system and a small amount of XBub3 from egg extracts (data not shown), none of the full-length or peptide antibodies can efficiently immunodeplete XBub3 from egg extracts unless the extract is first denatured. After TCA precipitation and renaturation, both bands of the doublet could be immunoprecipitated by the rabbit XBub3 peptide antibody (Fig. 2B).
|
Xenopus Bub1 protein has recently been isolated and shown to
undergo a mobility decrease on SDS-PAGE consistent with the active form of
XBub1 being a phosphoprotein in Xenopus egg extracts
(Schwab et al., 2001;
Sharp-Baker and Chen, 2001
).
We immunoprecipitated XBub1 and XBubR1 using affinity-purified antibodies and
we probed the immunoprecipitates with our XBub3 antibodies. Both bands of the
XBub3 protein doublet were present in XBub1 and XBubR1 immunoprecipitates
(Fig. 2C,E), demonstrating that
both forms of XBub3 associate with each of these kinases. We found no evidence
for a complex containing both XBub1 and XBubR1 (data not shown) (see also
Chen, 2002
).
To determine whether the two forms of XBub3 found in egg extracts were due
to modification by phosphorylation, we treated the XBub1 immunoprecipitates
with protein phosphatase. Such treatment led to an increase in the
gel mobility of XBub1, consistent with dephosphorylation of XBub1
(Fig. 2C,D) but the doublet of
XBub3 remained. These results demonstrate that both forms of the XBub3 protein
are capable of binding XBub 1 and XBubR1 in egg extracts and that the XBub3
doublet is unlikely to be the result of phosphorylation. Thus, while only one
major form of XBub3 is present in XTC cells, there appear to be two different
forms in Xenopus egg extracts.
We next analysed how the two forms of XBub3 fractionate upon gel filtration chromatography of CSF egg extracts containing sperm nuclei. We observed that both bands of the doublet of XBub3 behave identically upon gel filtration analysis, forming two major peaks consisting of free Bub3 (Fig. 3, fractions 35-38) and Bub3 complexes (fractions 25-31). Although a substantial proportion of the XBub3 protein co-elutes with Bub1 and BubR1 in complexes of around 669kDa, a major proportion of XBub3 is found in complexes of 440-232 and does not co-elute with either of these proteins (fractions 29-31). A small amount of the Mad1 protein overlaps with the elution profile of the XBub3, but we observed no differences in the distribution or the amounts of any of these checkpoint proteins upon checkpoint activation or in the absence of sperm chromatin.
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Xenopus Bub3 is required to establish and maintain spindle
checkpoint arrest in Xenopus egg extracts
Xenopus eggs are arrested in metaphase of their second meiotic
division by cytostatic factor (CSF) when they are laid. Fertilisation produces
a transient peak of calcium, which induces exit from this meiotic arrest and
the initiation of mitotic divisions. The spindle checkpoint can be
reconstituted in Xenopus egg extracts by releasing the extracts from
metaphase arrest with CaCl2 in the presence of 10,000 sperm/µl
extract and 10 µg/ml nocodazole
(Minshull et al., 1994).
Activation of the spindle checkpoint in these egg extracts produces a mitotic
arrest that cannot be overridden by the addition of calcium and is
characterised by the presence of high histone H1 kinase activity and highly
condensed chromosomes. We wished to investigate the role of XBub3 in the
spindle checkpoint. To do this we tested whether our XBub3 antibodies had any
effect on activation of the spindle checkpoint in egg extracts. CSF-arrested
extracts were preincubated with affinity-purified full-length rabbit
anti-XBub3 antibodies or mock affinity-purified rabbit pre-immune serum and
then released from metaphase by calcium addition in the presence of 10
µg/ml nocodazole and 10,000 sperm/µl of extract
(Fig. 4B). It is important to
note that pre-incubation of CSF-arrested egg extracts with our rabbit
full-length XBub3 antibody had no effect on the CSF arrest of the extract:
histone H1 kinase levels were still high at the zero time point, before
calcium addition (Fig. 4B).
Thus XBub3 is not required to maintain a CSF arrest. This fits well with the
findings that neither XBub1 (Sharp-Baker
and Chen, 2001
; Tunquist et
al., 2002
) nor XBubR1 (Chen,
2002
) are required to maintain a CSF arrest.
|
In the presence of a pre-immune antibody the spindle checkpoint was activated, as shown by the presence of condensed chromatin and maintenance of high levels of histone H1 kinase activity up to 60 minutes after calcium addition (Fig. 4A,B). However, upon addition of calcium, H1 kinase levels failed to be maintained in the extracts containing XBub3 antibodies. We conclude that these antibodies are `function-blocking' and that XBub3 is required to establish a spindle checkpoint arrest. Interphase nuclei were formed in the presence of nocodazole and XBub3 antibody within 20 minutes, and these grew to normal proportions by 60 minutes (Fig. 4Aa,c), indicating that inhibition of XBub3 function does not cause major disruption of nuclear structure.
We next tested whether our full-length XBub3 antibodies were capable of overriding the maintenance of a spindle checkpoint arrest if the checkpoint is activated prior to antibody addition. The spindle checkpoint was activated by the addition of nocodazole and 10,000 sperm/µl extract for 20 minutes at room temperature. Full-length rabbit XBub3 antibodies were then added to the extract for 30 minutes at room temperature. Following this incubation calcium was added to release the egg extracts from CSF arrest. Under these conditions, addition of our XBub3 antibody disrupted the maintenance of the previously established spindle checkpoint, observed as a decrease in histone H1 kinase activity (Fig. 4C) and the formation of interphase nuclei (data not shown) after calcium addition. When taken together these results show that Xenopus Bub3 function is required for the activation and for the maintenance of a spindle checkpoint arrest in egg extracts.
XBub3 binds to kinetochores prior to XMad2 during early prophase
Bub3p has been localised to kinetochores in a number of species when the
spindle checkpoint is active (Basu et al.,
1998; Martinez-Exposito et
al., 1999
; Taylor et al.,
1998
). XMad2 localises to kinetochores in XTC cells treated with
the microtubule-depolymerising drug, nocodazole, and during late
prophase/prometaphase of the normal cell cycle
(Chen et al., 1996
).
Although Mad1 is required to recruit Mad2 to kinetochores
(Chen et al., 1996) and Bub3
required for Bub1 kinetochore recruitment
(Taylor et al., 1998
),
analysis of the timing of Mad1-Mad2 and Bub1-Bub3 kinetochore recruitment
during the cell cycle and upon spindle checkpoint activation has not been
carefully examined. To do this we first analysed XBub3 localisation in XTC
cells in which the checkpoint had been activated using the
microtubule-depolymerising drug, nocodazole. XBub3 was detected using our
affinity-purified sheep anti-XBub3 peptide antibodies. The chromosomes of
cells arrested in metaphase by nocodazole showed highly condensed chromosomes
with strong punctate staining for XBub3, and this staining overlapped exactly
with the kinetochore staining of XMad2 protein, indicating that XBub3 protein
localises to kinetochores when the spindle checkpoint is activated in XTC
cells (Fig. 5). We next
examined the localisation of XBub3 protein in an asynchronous population of
XTC cells. XBub3 protein localised exclusively to the nucleus during
interphase, becoming bound to kinetochores in early prophase and persisting
there until all the chromosomes had aligned at metaphase
(Fig. 6a-e). At metaphase
(Fig. 6e) a single chromosome
stained brightly for XBub3 and XMad2, presumably because this chromosome had
not yet, or had only just, aligned on the metaphase plate. XBub3 and XMad2
were not detectable at the kinetochores of chromosomes at the metaphase to
anaphase transition or during anaphase, with cells simply showing general
cytoplasamic staining at this time. Thus the bulk of both checkpoint proteins
appear to leave the kinetochore at metaphase in XTC cells. By telophase, XBub3
was once again concentrated in the nucleus
(Fig. 6g).
|
|
The XBub3 and XMad2 staining were equally intense at kinetochores in nocodazole-treated cells (Fig. 5). However, within the asynchronous culture of XTC cells, we observed intense XBub3 staining during early prophase at a time when a high proportion of the XMad2 protein was still diffusely nuclear and associated with the nuclear membrane. We only observed clear XBub3 and XMad2 colocalisation during late prophase and prometaphase (Fig. 6c,d). Thus we have consistently observed XBub3 at kinetochores before XMad2 kinetochore staining (Fig. 6, panel b) and before XMad1 kinetochore staining (data not shown).
XBub3 antibodies do not override the spindle checkpoint by preventing
kinetochore recruitment of Bub3 or Mad2 proteins
Both XMad1 and XMad2 have been shown to localise to replicated chromosomes
when the spindle checkpoint is activated in Xenopus egg extracts
(Chen et al., 1998). Addition
of XMad1 antibodies to these extracts prevents kinetochore binding of both
XMad1 and XMad2 proteins and prevented the establishment of the spindle
checkpoint. Addition of XMad2 antibodies did not affect the kinetochore
localisation of either protein, yet interfered with checkpoint function
(Chen et al., 1998
).
As XBub3 appears to be recruited to kinetochores before XMad2, we wished to determine whether our XBub3 antibodies would inhibit the establishment of the spindle checkpoint in egg extracts by preventing XBub3 and/or XMad1/Mad2 binding to kinetochores. To do this we examined the kinetochore localisation of XBub3 on chromosomes assembled in egg extracts in the presence of our XBub3 antibodies. Metaphase chromosomes and spindles were first allowed to assemble in egg extracts, and then nocodazole was added to induce microtubule depolymerisation. When chromosomes were isolated (see Materials and Methods) during the metaphase arrest we found no XBub3 detectable at kinetochores. Chromosomes isolated after nocodazole treatment showed bright, punctate XBub3 staining, which was confirmed as kinetochore staining by colocalisation with XMad2 (Fig. 7, top 2 rows). Pre-incubation of the egg extracts with our full-length or peptide XBub3 antibodies did not disrupt the kinetochore localisation of XBub3 or XMad2 as the kinetochore staining obtained is identical to that seen in the absence of pre-incubation with XBub3 antibodies (Fig. 7, bottom two rows). We also observed no disruption of XBub1 kinetochore staining on chromosomes isolated from egg extracts containing XBub3 antibodies (data not shown). These results show that although our XBub3 antibodies disrupt the establishment and maintenance of a spindle checkpoint arrest (Fig. 4) they do not do this by preventing XBub3, XMad2 or XBub1 binding to kinetochores in the egg extracts.
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Discussion |
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In Xenopus egg extracts, spindle checkpoint activation and
maintenance both require the functions of XMad1 and XMad2
(Chen et al., 1998;
Chen et al., 1996
), the
kinesin-related microtubule motor protein CENP-E
(Abrieu et al., 2000
), the
protein kinases XBub1 (Sharp-Baker and
Chen, 2001
), XBubR1 (Chen,
2002
), XMps1 (Abrieu et al.,
2001
), Aurora B (Kallio et
al., 2002
) and a Xenopus MAP kinase
(Cross and Smythe, 1998
;
Minshull et al., 1994
;
Takenaka et al., 1997
). Here
we have shown that Xenopus Bub3 is also required for spindle
checkpoint activation and for maintenance of a spindle checkpoint signal in
egg extracts (Fig. 4). This is
the first demonstration that Bub3 is required to maintain a spindle checkpoint
arrest in any system.
Our work (Fig. 4) also
demonstrates that XBub3 function is not required for the maintenance of a
meiotic metaphase arrest by CSF. This agrees with experiments showing that
XBub1 and XBubR1 depletion have no effect on the maintenance of a CSF arrest
(Chen, 2002;
Sharp-Baker and Chen, 2001
;
Tunquist et al., 2002
). Note,
it has recently been shown that XBub1 is required to establish a CSF arrest
(Tunquist et al., 2002
), but a
similar role for XBub3 has yet to be analysed.
XBub3 complexes
Bub3 is a small WD-repeat protein, and the budding yeast homologue has been
crystallised and shown to consist of seven WD-repeat domains that fold
together to form a ß-propeller structure (David Wilson, UC Davis,
personal communication). Many WD-repeat-containing proteins are known to form
multi-protein complexes (for a review, see
Smith et al., 1999). Bub3p
forms complexes with Bub 1p and Mad3p in both S. cerevisiae and
Schizosaccharomyces pombe
(Hardwick et al., 2000
;
Millband and Hardwick, 2002
;
Roberts et al., 1994
) and the
kinases Bub1 and BubR1 in vertebrates, the second of which is homologous to
yeast Mad3 (Chan et al., 1999
;
Chan et al., 1998
;
Martinez-Exposito et al.,
1999
; Taylor et al.,
1998
). In budding yeast, two distinct Bub3-containing
multi-protein complexes are formed during metaphase and upon spindle
checkpoint activation. The first of these complexes is a Bub3-Bub1-Mad1
complex containing phosphorylated Mad1, the formation of which has been
suggested to cause the release of Mad2 from Mad1
(Brady and Hardwick, 2000
;
Sironi et al., 2001
). The
second complex contains Bub3-Mad3-Mad2-Cdc20
(Fraschini et al., 2001
;
Hardwick et al., 2000
). In
vertebrates a similar complex has been detected between Bub3-BubR1, Cdc20 and
Mad2 proteins. However, it is currently unclear as to whether Bub3-BubR1 and
Mad2 are part of one APC inhibitory complex or function as distinct complexes
both of which bind Cdc20 and inhibit APC activation
(Fang, 2002
;
Sudakin et al., 2001
;
Tang et al., 2001
).
Our gel filtration and co-immunoprecipitation analyses show that the
Mad1-Mad2, Bub1-Bub3 and BubR1-Bub3 complexes are present in
metaphase-arrested egg extracts (Fig.
3). We do not observe any major change in the elution profiles of
these proteins upon checkpoint activation (data not shown). Thus, if spindle
checkpoint activation in egg extracts requires the formation of a
Mad1-Bub1-Bub3 complex or a Bub3-BubR1-Mad2 complex then the amount of protein
found in such complexes corresponds to a small proportion of the total protein
present in the egg extract. Alternatively, such complexes may be kinetochore
bound and therefore not detectable by our gel filtration analysis. Consistent
with the former explanation, less than 1% of Mad2 forms a complex with Cdc20
when the checkpoint is active in Xenopus egg extracts
(Chung and Chen, 2002), and
gel filtration analysis in budding yeast shows no change in the elution
profiles of Bub1-Bub3, Mad1-Mad2 and Mad3 between cycling yeast cell cultures
and spindle-checkpoint-arrested cultures
(Fraschini et al., 2001
).
Our gel filtration analysis also revealed that although a substantial
proportion of the XBub3 protein co-elutes with Bub1 and BubR1 in complexes of
around 670 kDa, a major proportion of XBub3 is found in complexes of 440-232
where it does not co-elute with the XBub kinases. The precise composition of
these complexes remains to be determined, but we find no co-enrichment of
Xenopus Cdc20 protein, which is present in most of the fractions
between 670 and 67 kDa (L.C. and K.G.H., unpublished)
(Lorca et al., 1998).
XBub3 localisation
Drosophila, mouse and human Bub3 homologues are known to bind to
kinetochores during prophase and prometaphase of the cell cycle and are
associated with lagging chromosomes that have not yet formed correct bipolar
attachment to the mitotic spindle (Basu et
al., 1999; Martinez-Exposito
et al., 1999
; Taylor et al.,
1998
). Here we show that XBub3 localises to kinetochores in XTC
cells during prophase and prometaphase and is undetectable on kinetochores
that have aligned on the spindle at metaphase
(Fig. 5). In HeLa cells, Bub1
and BubR1 have been shown to decrease 3.7- and 3.9-fold respectively upon
kinetochore attachment, whereas Mad2 decreases 152-fold
(Zhou et al., 2002
). We
estimate that the decrease in XBub3 binding upon kinetochore attachment is at
least fivefold from analysis of kinetochore pixel intensities during our
immunofluorescence assay (data not shown). This is consistent with the
decrease in Bub3 binding reported at mouse kinetochores at metaphase upon
kinetochore attachment of three- to fivefold
(Martinez-Exposito et al.,
1999
). Although the authors in that study still detected small
amounts of Bub3 at metaphase we were unable to detect any Bub3 or Mad2 protein
associated with kinetochores in the high general cytoplasmic staining of XBub3
present in metaphase cells (Fig.
6).
In tissue culture cells, Mad1 and Mad2 proteins are localised to
kinetochores during prophase and prometaphase and to unattached kinetochores
during metaphase (Chen et al.,
1998; Chen et al.,
1996
). Mad1 is required to recruit Mad2 kinetochore localisation
in this system (Chen et al.,
1998
). Interestingly, we have consistently observed XBub3 binding
to kinetochores very early in prophase, before we observe XMad2
(Fig. 6) or XMad1 (L.C. and
K.G.H., unpublished). Human Bub1 is known to localise to kinetochores before
BubR1 (Jablonski et al.,
1998
). Such observations suggest that the Bub1 and Bub3 proteins
could be required for the recruitment of the Mad and BubR1 proteins to
kinetochores. We attempted to test this in egg extracts using our XBub3
antibodies, but they did not interfere with the kinetochore binding of any
checkpoint component tested, including XBub3 itself
(Fig. 7). Unfortunately, it has
not been possible to efficiently immunodeplete XBub3 from egg extracts with
any of our antibodies. Our antibodies do recognise native XBub3 as shown by
our immunofluorescence data (Fig.
7), but they are only able to immumunoprecipitate a small
proportion of XBub3 from native egg extracts (L.C. and K.G.H., unpublished).
However, since these antibodies override the spindle checkpoint when added to
egg extracts, this limited binding is clearly capable of disrupting XBub3
spindle checkpoint function. Sharp-Baker and Chen have recently reported that
immunodepletion of XBub1 from egg extracts prevents the recruitment of XBub3,
XMad1, XMad2, CENP-E and XBubR1 to kinetochores
(Chen, 2002
;
Sharp-Baker and Chen, 2001
).
This is consistent with our analysis that Bub3 kinetochore binding occurs
before Mad1-Mad2 recruitment during cell division and suggests that Bub3-Bub1
binding to kinetochores may occur at an early stage in spindle checkpoint
activation, facilitating Mad1-Mad2 kinetochore recruitment
(Sharp-Baker and Chen, 2001
).
In mammalian cells Bub1 associates with kinetochores before BubR1 and the
kinesin-like protein, CENP-E (Jablonski et
al., 1998
). CENP-E binds to BubR1
(Chan et al., 1999
;
Yao et al., 2000
), and
immunodepletion of CENP-E prevents XMad1 and XMad2 kinetochore recruitment and
checkpoint activation (Abrieu et al.,
2000
). Furthermore, immunodepletion of XMps1 also prevents the
kinetochore association of Mad1 and Mad2 at kinetochores, perhaps by
preventing CENP-E binding to kinetochores
(Abrieu et al., 2001
). Thus
Mad1 and Mad2 appear to require a complex platform of checkpoint proteins at
Xenopus kinetochores before they can be recruited.
Although XBub3 and XMad2 are both nuclear during interphase we never found
XBub3 concentrated at the nuclear envelope, as observed for XMad2
(Fig. 5)
(Chen et al., 1996) and XMad1
(Chen et al., 1998
). It has
recently been shown that human Mad1 and Mad2 are associated with nuclear pore
complexes in interphase (Campbell et al.,
2001
). In this regard it is interesting to note that most
organisms contain Bub3-related proteins (named Rae1 or Gle2), which have been
shown to be involved in nuclear-cytoplasmic transport
(Bharathi et al., 1997
;
Murphy et al., 1996
). In
addition, it has recently become clear that the region of Bub1 and Mad3 that
interacts with Bub3 (Hardwick et al.,
2000
; Taylor et al.,
1998
) is closely related to the GLEBS (for GLE2p-binding sequence)
motif of the nuclear pore complex protein hNUP98
(Wang et al., 2001
). However,
why the Mad proteins are associated with nuclear pores throughout interphase,
when Bub3 and Bub1 are not, remains unclear. Perhaps it is important to keep
the Mad and Bub proteins apart, in relatively restricted cellular locations,
until the onset of mitosis.
Possible modes of action of XBub3 antibody
Our analysis of XBub3 function has relied upon the addition of
function-interfering antibodies to egg extracts. Many efforts were also made
to immunodeplete XBub3, but it proved impossible to do so efficiently. Others
have had similar problems (Chen,
2002), and it is not clear why immunodepletion has proven so
difficult to achieve, although the XBub3 protein is certainly quite abundant.
We are confident that our antibody interference experiments, which are often
used in analyses of Xenopus spindle checkpoint function
(Chen et al., 1998
;
Abrieu et al., 2001
;
Kallio et al., 2002
), have
clearly demonstrated a role for XBub3 in spindle checkpoint activation and
maintenance.
Our XBub3 antibodies prevent spindle checkpoint activation, without
preventing the kinetochore association of XBub3, XMad2
(Fig. 7), XMad1 or XBub1
kinetochore association (L.C. and K.G.H., unpublished). In this regard they
behave like -Mad2 antibodies (Chen
et al., 1998
). It is possible that our XBub3 antibodies prevent
kinetochore association of XBubR1, CENP-E or other spindle checkpoint
components, but this seems unlikely as immunodepletion of CENP-E prevents Mad1
and Mad2 kinetochore recruitment (Abrieu et
al., 2000
) and immunodepletion of XBubR1 reduces the kinetochore
association of several checkpoint components
(Chen, 2002
). Our antibodies
have no obvious effect on Mad/Bub protein recruitment to kinetochores
(Fig. 7) and thus it seems most
likely to us that they interfere with a `downstream' function, after
kinetochore recruitment of the checkpoint proteins has taken place. For
example, they could act by preventing Bub3-BubR1 from forming inhibitory
complexes with the APC activator, Cdc20/fizzy
(Fang, 2002
;
Tang et al., 2001
). We
attempted to test this by immunoprecipitation of Cdc20 or XBubR1 from egg
extracts in the presence and absence of our XBub3 antibody. As yet no
convincing effects have been observed: we can detect no difference in the
XBub3, XBubR1 or XMad2 levels associated with Cdc20 in the presence and
absence of our function-blocking XBub3 antibodies (data not shown).
The effect of our antibodies may be more subtle. For example they could
disrupt transmission of the checkpoint signal from unattached kinetochores
throughout the cell, which is necessary to ensure that anaphase onset is
inhibited globally. The molecular nature of this global signal and its mode of
transmission remain a mystery. Real-time studies of hsMad2
(Howell et al., 2000) have
demonstrated that Mad2 is only a transient component of kinetochores, with a
t1/2 of 24-28 seconds. In addition, Mad2-binding sites were
observed to move from kinetochores to spindle poles via microtubules. It will
be of great interest to see whether Bub3 and its associated kinases, Bub1 and
BubR1, also turnover rapidly at kinetochores and whether some components of
the spindle checkpoint pathway are more stably associated with kinetochores,
providing a platform for the rapid turnover of Mad2 protein.
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