1 Chromosome Structure Group, Wellcome Trust Centre for Cell Biology, Institute
of Cell and Molecular Biology, University of Edinburgh, King's Buildings,
Mayfield Road, Edinburgh EH9 3JR, Scotland, UK
2 University of Porto, IPATIMUP Institute of Molecular Pathology and Immunology
of the University of Porto, Porto, Portugal
3 Faculty of Medicine, University of Porto, Porto, Portugal
* Author for correspondence (e-mail: bill.earnshaw{at}ed.ac.uk)
Accepted 11 April 2003
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Summary |
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Key words: Mitosis, Chromosomal passenger proteins, Survivin, Aurora-B, INCENP, RNA interference, BubR1
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Introduction |
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Recently Survivin has been shown to be a chromosomal passenger protein
(Skoufias et al., 2000;
Uren et al., 2000
;
Jiang et al., 2001
;
Wheatley et al., 2001a
). These
highly conserved proteins are present at centromeres during prometaphase, then
transfer to the central spindle and presumptive cleavage furrow at the cell
cortex during early anaphase (see Earnshaw
and Bernat, 1990
; Earnshaw and
Cooke, 1991
; Adams et al.,
2001a
). Chromosomal passenger proteins cloned to date include
INCENP/Sli-15, Aurora-B/Ipl-1/AIR-2 and Survivin/BIR1 (for reviews, see
Adams et al., 2001a
;
Terada, 2001
), but at least
one other protein with similar behaviour also exists, TD60 (telophase-disc 60)
(Andreassen et al., 1991
).
INCENP is a multidomain protein that interacts with heterochromatin
protein-1 (Ainsztein et al.,
1998) and microtubules
(Wheatley et al., 2001b
), and
it is thought to be a targeting or scaffolding subunit for Aurora-B kinase
(Adams et al., 2000
;
Bolton et al., 2002
). In
contrast, relatively little is known about the mitotic role of Survivin other
than that it can stimulate Aurora-B kinase activity in vitro
(Bolton et al., 2002
;
Bishop and Schumacher, 2002
).
It is likely that the chromosomal passengers act in concert to execute their
role(s) during mitosis, and they are stockpiled as a complex in
Xenopus eggs and in yeast (Adams
et al., 2000
; Bolton et al.,
2002
; Cheeseman et al.,
2002
). Interactions between the three proteins have also been
detected by two-hybrid and in vitro binding experiments
(Kaitna et al., 2000
;
Wheatley et al., 2001a
).
INCENP and Aurora-B are required for phosphorylation of histone H3 on
serine 10 (Hsu et al., 2000;
Adams et al., 2001b
;
Murnion et al., 2001
) and, in
Drosophila and S. pombe [though not Xenopus
(MacCallum et al., 2002
)], for
targeting of condensin to chromosomes
(Giet and Glover, 2001
;
Morishita et al., 2001
). The
passengers are essential for chromosomes to achieve a stable metaphase
biorientation (Adams et al.,
2001b
; Kaitna et al.,
2002
), possibly by regulating the interaction of kinetochores with
microtubules (Yoon and Carbon,
1999
; Biggins et al.,
1999
; Biggins and Murray,
2001
; Kang et al.,
2001
; Tanaka et al.,
2002
; Kaitna et al.,
2002
). Ipl-1p (the budding yeast Aurora kinase) is essential for
the tension-sensitive arm of the spindle assembly checkpoint
(Biggins and Murray, 2001
),
and studies using dominant-negative mutants and antibody injection have
implicated mammalian Survivin and Aurora-B in the spindle assembly checkpoint
(Murata-Hori et al., 2002
;
Kallio et al., 2001
;
Kallio et al., 2002
). Aurora-B
has been implicated in anaphase spindle midzone organization in many species
(Speliotes et al., 2000
;
Giet and Glover, 2001
;
Murata-Hori et al., 2002
;
Murata-Hori and Wang, 2002
;
Giodini et al., 2002
) and
spindle elongation in yeast (Rajagopalan
and Balasubramanian, 2002
). Survivin may also contribute to the
regulation of microtubule dynamics (Li et
al., 1998
; Giodini et al.,
2002
) and has been reported to be involved in spindle assembly
(Giodini et al., 2002
). In
C. elegans, Aurora-B/AIR-2 is also required for separation of
homologous chromosomes in meiosis I
(Rogers et al., 2002
;
Kaitna et al., 2002
). Lastly,
the passengers are required for the execution of cytokinesis
(Mackay et al., 1998
;
Schumacher et al., 1998
;
Terada et al., 1998
;
Cutts et al., 1999
;
Fraser et al., 1999
;
Li et al., 1999
;
Speliotes et al., 2000
;
Uren et al., 2000
;
Oegema et al., 2001
;
Leverson et al., 2002
).
In the present study we have used siRNA
(Elbashir et al., 2001) to
silence Survivin expression in human cells. Our results indicate that Survivin
is required for metaphase chromosome alignment and maintenance of the spindle
assembly checkpoint arrest in the presence of taxol and cytokinesis.
Furthermore, we show that a monoclonal antibody
(Fortugno et al., 2002
) used
in many studies of Survivin localization apparently recognizes another
spindle-associated epitope(s) in addition to Survivin.
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Materials and Methods |
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Cell culture, RNAi application and drug treatments
HeLa cells in exponential growth were seeded onto poly-lysine-coated glass
coverslips in 24-well plates at 3x104 cells per well and
grown overnight in RPMI/10% FBS (Gibco-BRL) and maintained in 5%
CO2 at 37°C. HeLa cells stably expressing Survivin-GFP were
described previously (Wheatley et al.,
2001a). RNAi was performed according to Elbashir et al.,
(Elbashir et al., 2001
) (see
also
www.mpibpc.gwdg.de/abteilungen/100/105/).
Briefly, a single pulse of 60 pmoles of siRNA was administered to the cells at
50% confluency by transfection with OligoFectamine
(www.invitrogen.com)
in complete medium without antibiotics. Cells were maintained in this medium
for the duration of the experiment and assayed for Survivin silencing by
indirect immunofluorescence and immunoblotting. For synchronisation in
S-phase, cells were treated with 2 mM thymidine for 20 hours at 37°C then
released into fresh medium. To depolymerize microtubules, cells were treated
with either 0.1 µg/ml colcemid (Sigma) for 2-24 hours or 0.1 µg/ml
nocodazole (Sigma) for 12-18 hours. To stabilise microtubules, cells were
incubated with 33 nM taxol (Sigma) for 12-18 hours.
Immunoblotting
Total extracts of 5x105 cells treated with control or
Survivin-specific oligonucleotides for 60 hours or 84 hours were boiled for 5
minutes in Laemmli buffer containing ß-mercaptoethanol, run in a 15%
SDS-PAGE gel and blotted onto a nitrocellulose membrane (Amersham;
www.apbiotech.com).
The membrane was blocked with 5% skimmed milk in PBS/0.1% Tween 20 and
incubated with an anti-Survivin antibody (diluted 1/500; NB500-201
www.novus-biologicals.com)
in 3% milk/PBS/0.1% Tween 20 for 3 hours at room temperature. After washing in
1.5% milk/PBS/0.1% Tween 20, the membrane was incubated for 1 hour with
anti-rabbit horseradish-peroxidase-linked secondary antibody (diluted
1/10,000,
www.apbiotech.com)
in 3% milk/PBS/0.1% Tween 20. After washing, the membrane was labelled using
the enhanced chemiluminescence protocol
(www.apbiotech.com).
Indirect immunofluorescence microscopy and quantification
At various time points after transfection, cells were fixed with 4%
formaldehyde and permeabilized with 0.15% Triton X-100 as described previously
(Wheatley and Wang, 1996),
unless otherwise stated. Cells were probed with antibodies against Survivin
(1/500, rabbit; NB500-201
www.novus-biologicals.com),
Aurora-B (1/250, mouse; AIM1
www.translab.com),
INCENP (1/500; polyclonal antibody rabbit D), 8E2 (1/500, mouse, gift of D.
Altieri, Yale University, USA), BubR1 (1/500, sheep; gift of S. S. Taylor,
University of Manchester, UK), and
-tubulin (1/2000, mouse; B512
www.sigma-aldrich.com).
Anti-centromeric antibodies (ACA) were used to stain the centromeres (1/1000,
human), and chromosomes were stained with DAPI (Sigma). Mad2 staining was
performed according to a previous protocol
(Waters et al., 1998
), using a
polyclonal Mad2 antibody (1/50, rabbit; gift from E. D. Salmon, University of
North Carolina, USA). Secondary antibodies were used at 1/200
(www.jacksonimmuno.com).
Quadruple labelling was performed using a suitable combination of primary
antibodies, with secondary antibodies conjugated to Texas Red, fluorescein and
Cy5, and samples counterstained with DAPI. Chromosome spreads were made by
hypotonically swelling colcemid-treated cells with 75 mM KCl for 20 minutes at
room temperature prior to fixation.
Image stacks were taken using an Olympus IX-70 microscope controlled by Delta Vision SoftWorx (Applied Precision, Issequa, WA, USA) and a 60x objective (NA 1.4). Image stacks were deconvolved, quick-projected and captured as tiff images. For quantification, the 10 central sections of an image stack were deconvolved and projected using an averaging algorithm. The total integrated intensity of a 20x20 pixel box was measured at the appropriate wavelengths using the Data Inspector tool. For each prometaphase cell analysed, three measurements were taken on the chromosomes within the cell and three of the background outside the cell. Values were corrected by subtracting the background of the appropriate wavelength. Quantifications were made at the 48 hour or 60 hour time points so that Survivin-depleted cells could be compared directly with Survivin-positive cells on the same coverslip.
Time-lapse imaging
Cells were seeded onto poly-lysine-coated grid coverslips in 24-well plates
at a density of 7.5x103 cells per well. Cells were blocked in
S-phase with 2 mM thymidine for 20 hours, 40 hours after transfection.
Cultures were washed several times to remove mitotic and apoptotic cells
before adding medium with or without a drug (33 nM taxol or 0.1 µg/ml
nocodazole) for 12-18 hours. When cells entered mitosis (8-10 hours after
release from the thymidine block), medium was supplemented with 10 mM HEPES
(pH 7.5), and phase-contrast images were taken every hour using an inverted
Nikon Diaphot microscope heated at 37°C and a 20x objective. At
least 50 mitotic cells were followed for 6 hours in each experiment.
Cell population dynamics and cell cycle/apoptosis quantification
Growth curves were plotted based on the number of viable cells, as
determined by Trypan blue exclusion (Sigma). Mitotic stages were scored
separately for cells staining negatively [Survivin()] or positively
[Survivin(+)] for Survivin. Both mitotic and apoptotic cells were scored based
on DAPI morphology and -tubulin immunostaining. As the precise timing
of depletion varied from one experiment to another (typically 36-72 hours),
representative graphs are shown to avoid blending of the data. All experiments
were performed at least three times and produced similar results.
FACS analysis
The apoptotic status of cells was assessed using TUNEL label and Annexin
V-FLUOS staining kits according to the manufacturer's guidelines
(www.biochem.roche.com).
Before TUNEL labeling, the total population (including non-adherent cells) was washed in PBS, fixed in 4% formaldehyde for 30 minutes at room temperature and permeabilized in 0.1% sodium citrate/Triton X-100 for 2 minutes on ice.
To assess ploidy, cells were washed in PBS and fixed for 1 hour in 70% ethanol at 4°C. After treatment with 50 µg/ml RNAse (Sigma) for 20 minutes at room temperature, cells were washed with PBS and incubated with 40 µg/ml propidium iodide (Sigma) for 30 minutes.
All samples were analysed using a fluorescence activated cell sorter (FACSCalibur, Becton Dickinson, Mountain View, CA) and Cell Quest software.
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Results |
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|
Following transfection with rhodamine-labelled control siRNA Survivin was
detected at the centromeres of prometaphase/metaphase cells
(Fig. 1C) and showed a typical
chromosome passenger pattern of localization at other stages of mitosis (data
not shown). In 50-75% (n=100) of prometaphase cells exposed to
specific siRNA, Survivin was not detectable at centromeres by 48-60 hours post
transfection (Fig. 1E). By
contrast, untransfected cells from this population exhibited a normal Survivin
distribution (Fig. 1D). We also
transfected cells that stably express Survivin-GFP with control or Survivin
siRNA. As shown in Fig. 2A, in
cells transfected with control siRNA, Survivin-GFP was localized at the
centromeres and was also diffuse in the cytoplasm as described previously
(Wheatley et al., 2001a). By
contrast, in cells exposed to Survivin siRNA, no GFP signal remained
(Fig. 2B). This result was
confirmed by immunoblot analysis using anti-GFP antibody (data not shown). We
conclude that Survivin is essential for the growth of HeLa cells and that the
protein can be depleted from cells by RNAi.
|
Staining of cells with a monoclonal antibody that detects microtubule
associated Survivin is unaffected by Survivin-specific RNAi
An ongoing vigorous debate in the field is whether human Survivin is a
microtubule associated protein, as originally described
(Li et al., 1998;
Li et al., 1999
), a chromosome
passenger protein, as subsequently claimed
(Skoufias et al., 2000
;
Wheatley et al., 2001a
;
Uren et al., 2000
) or whether
two Survivin pools of different magnitude exist within the cell
(Fortugno et al., 2002
). An
important reagent used in this debate has been the monoclonal antibody 8E2,
which helped to define Survivin originally
(Li et al., 1998
;
Li et al., 1999
) and which
recognizes a full-length Survivin fusion protein in vitro
(Wheatley et al., 2001a
).
A surprising result was obtained when we probed Survivin siRNA-treated
cells with the 8E2 monoclonal antibody or with a commercial polyclonal
antibody (NB500-201;
www.novusbiologicals.com)
reported to recognise all forms of Survivin
(Fortugno et al., 2002). In
cells transfected with control siRNA, the polyclonal antibody stained
centromeric Survivin (Fig. 2C
and ACA staining in Fig. 2C',
red), whereas 8E2 demarcated the mitotic spindle
(Fig. 2C,C', green). However,
in cells negative for Survivin, as detected with the commercial polyclonal
antibody (Fig. 2D, red), the
pattern of 8E2 localization remained unaltered
(Fig. 2D,D', green). Since we
have shown that 8E2 can recognise Survivin in vitro
(Wheatley et al., 2001a
), this
result strongly suggests that in vivo, 8E2 recognizes an epitope present on at
least one other microtubule-associated protein in addition to survivin.
Survivin depletion causes mislocalisation of the chromosomal
passenger proteins, Aurora-B and INCENP
It is now widely believed that INCENP and Aurora-B may act together with
Survivin throughout mitosis in a chromosomal passenger complex
(Wheatley et al., 2001a;
Bolton et al., 2002
;
Leverson et al., 2002
). As
predicted by this, at 60 hours after transfection with specific siRNA, cells
without detectable Survivin also lacked centromeric Aurora-B
(Fig. 3B). A diffuse pool of
Aurora-B and occasionally some residual staining of the chromosome arms was
observed (compare Fig.
3A" and Fig.
3B"). The availability of a mixed population of
Survivin-positive and Survivin-depleted cells following transfection enabled
us to use Survivin-positive cells as internal staining controls for
quantitative analysis, which revealed a linear correlation between levels of
Survivin and Aurora-B at the centromeres
(Fig. 3E). A similar
correlation was obtained between the levels of Aurora-B and INCENP at
centromeres following transfection with Survivin siRNA
(Fig. 3C,D,F). These data
demonstrate that both Aurora-B and INCENP require Survivin to accumulate at
centromeres during the early stages of mitosis.
|
Survivin depletion causes an accumulation of prometaphase cells with
defects in chromosome congression
To determine the effect of depletion of Survivin on the passage of cells
through mitosis, populations transfected with Survivin siRNA were stained for
Survivin, and the percentage of cells in each phase of mitosis was scored.
Survivin-positive cells (n=100 mitotics) maintained a wild-type
distribution of mitotic stages for the duration of the experiment
(Fig. 4A). In contrast, we
observed a decline in the percentage of Survivin-depleted mitotic cells in
metaphase, anaphase, telophase and cytokinesis from 36 hours post-transfection
(Fig. 4B; n=100
mitotics). Instead, we observed many prometaphase cells with bipolar spindles
but many maloriented chromosomes (Fig.
4D, arrows). Although cells with multipolar spindles were
occasionally seen, they were also present within the Survivin-positive
population (6% in Survivin-positive versus 4% in Survivin-depleted cells).
|
Live-cell imaging by time-lapse phase contrast microscopy confirmed that cells were delayed in mitosis following transfection with Survivin siRNA. Cells transfected with control or Survivin siRNA, were blocked in S-phase using 2 mM thymidine for 20 hours, then released into fresh medium and followed individually by time-lapse imaging (Fig. 4F). In the population transfected with control siRNA, 92% of mitotic cells (n=73) completed mitosis within 1 hour (Fig. 4G). In contrast, in the population transfected with specific siRNA, 53% of cells (n=93) remained in mitosis for longer than 1 hour (for an average of 1.7 hours in mitosis). Thus, repression of Survivin results in a delay in mitosis, and this correlates with difficulties in chromosome alignment at the metaphase plate.
Survivin depletion inhibits cytokinesis and causes a slight increase
in apoptosis
Previous studies in which Survivin function was affected by the expression
of dominant-negative mutants or treatment with antisense oligonucleotides have
shown an increase in apoptosis and multinucleation
(Li et al., 1999), albeit only
a moderate increase in some cases (Kallio
et al., 2001
). In the present study, time-lapse imaging revealed
that cells exposed to Survivin siRNA, after being delayed in mitosis for
longer than control cells (see above), either flattened in interphase after
failing cytokinesis or blebbed and took on an abnormal appearance. In
contrast, all cells in the control population exited mitosis normally
(Fig. 4F,G). We occasionally
observed a very low number of Survivin-depleted cells in anaphase and
telophase (Fig. 4B) but failed
to detect any Survivin-depleted cells in cytokinesis (data not shown).
Consistent with these observations, cultures treated with Survivin siRNA
showed a gradual increase in multinucleation, culminating in a four-fold
increase by 84 hours (Fig. 4E).
In addition, when DNA content was measured by FACS analysis of
propidium-iodide-stained cells at 84 hours, 43% of the Survivin
siRNA-transfected population had a DNA content of 4N or greater compared with
22% in the control population (data not shown). Together, these observations
reveal that Survivin-depleted cells eventually exit mitosis but generally fail
to complete cytokinesis.
To assess the apoptotic status of cells transfected with Survivin siRNA, we performed FACS analysis using TUNEL and Annexin V labelling and confirmed the results by examining the DNA morphology microscopically (data not shown). A similar trend was observed using both methods: a moderate increase in apoptosis was detected at 72 hours after transfection with Survivin siRNA (16% higher than controls using the TUNEL assay to detect DNA fragmentation; 7% higher than controls using the Annexin V assay to detect loss of membrane asymmetry).
Together, these observations confirm that Survivin is required for the efficient completion of cytokinesis, but its requirement for the prevention of apoptosis is minor.
Association of the spindle checkpoint protein BubR1 with kinetochores
is unstable in Survivin-depleted cells
The increase in multinucleation at later time points following transfection
with Survivin siRNA (Fig. 4E)
suggested that cells were eventually overriding the spindle checkpoint and
exiting mitosis despite the presence of maloriented chromosomes. We therefore
examined the status of the spindle checkpoint in cells depleted of Survivin
using antibodies against BubR1 and (in some experiments) Mad2. Normally, both
proteins are present on kinetochores as cells enter prometaphase and are part
of the constitutive cell cycle checkpoint that delays anaphase onset until all
chromosomes are properly aligned on the spindle
(Musacchio and Hardwick,
2002). Mad2 is lost from kinetochores as microtubules bind
(Waters et al., 1998
), and
kinetochore-associated BubR1 levels fall significantly as the spindle develops
tension on the kinetochore (Taylor et al.,
2001
; Skoufias et al.,
2001
).
When cells transfected with control siRNA were stained for BubR1, the
kinetochores of maloriented chromosomes stained brightly, whereas the
kinetochores of chromosomes aligned at the metaphase plate stained only very
faintly (Fig. 5A). In contrast,
in Survivin-depleted cells the BubR1 staining at kinetochores of lagging
chromosomes (quantified as described in Materials and Methods) varied widely
within individual cells (n=10), from negative to control levels
(Fig. 5B). The presence of
kinetochores with intense BubR1 staining suggests that the checkpoint pathway
that monitors spindle tension can be active in Survivin-depleted cells.
Therefore, Survivin is not essential for localization of BubR1 to
kinetochores. However, the simultaneous presence of BubR1-negative and
BubR1-positive kinetochores within the same cell implies that either (1)
certain kinetochores may be defective and unable to bind BubR1 or (2) BubR1
association with kinetochores may be unstable in the absence of Survivin
either it binds once, but then fails to re-bind if spindle tension is
lost, or it normally cycles on and off the kinetochore
(Howell et al., 2001), and in
the absence of Survivin tends to spend much more time in the off state.
|
To distinguish between these two possibilities, we treated cultures transfected with Survivin-siRNA with colcemid to depolymerize spindle microtubules. As shown in Fig. 5C, after 2 hours exposure to colcemid, both Survivin-positive and Survivin-depleted cells had BubR1 staining at all kinetochores. Therefore, kinetochores of chromosomes lacking detectable Survivin are capable of binding BubR1. This eliminates hypothesis one.
To test the stability of BubR1 localization to the kinetochores, cultures transfected with Survivin siRNA for 60 hours were exposed to colcemid for 3 hours, 6 hours or 12 hours, then fixed and stained for Survivin and BubR1. After 3 hours of colcemid treatment, BubR1 levels at kinetochores were similar for both Survivin-positive and Survivin-depleted cells (data not shown). However, by 12 hours in colcemid, BubR1 staining was absent from the majority of kinetochores in Survivin-depleted cells, although it remained readily detectable at kinetochores of Survivin-positive cells (Fig. 5D). Quantification of the fluorescence data revealed a linear correlation between the levels of Survivin at centromeres and BubR1 at kinetochores following 6 or 12 hours in colcemid (data not shown), indicating that Survivin is apparently required for the stable maintenance of BubR1 at kinetochores under conditions of protracted checkpoint activation.
Survivin is required for proper functioning of the spindle checkpoint
in the presence of taxol
The spindle assembly checkpoint has been proposed to be bipartite, with one
arm monitoring kinetochore occupancy with microtubules
(Waters et al., 1998) and the
other monitoring spindle tension exerted on the kinetochores
(Taylor et al., 2001
;
Skoufias et al., 2001
). The
abnormalities in BubR1 staining observed in Survivin-depleted cells led us to
test whether these cells were defective for one or both arms of this
checkpoint pathway.
Control or Survivin siRNA-transfected cells were blocked in S-phase and
released into fresh medium supplemented with 33 nM taxol for 18 hours. Taxol
dampens microtubule dynamics and effectively abolishes tension within the
spindle but does not necessarily prevent kinetochore-microtubule interactions
(Waters et al., 1998). Cells
entered mitosis
10 hours after release from the S-phase block and were
followed by time-lapse phase-contrast imaging. In the population transfected
with control siRNA, 99% of the cells that entered mitosis (n=101)
arrested there until the end of the experiment
(Fig. 6A,C). However, in the
population exposed to Survivin siRNA, 44% of the mitotics (n=103)
exited mitosis as judged by cell re-spreading and formation of multiple
micronuclei (Fig. 6A,C). That
these cells had exited mitosis was subsequently confirmed by staining with
DAPI (data not shown). By contrast, when cells were released from the S-phase
block into medium supplemented with 0.1 µg/ml nocodazole, a microtubule
depolymerizing agent, both control and SurvivinsiRNA-transfected populations
arrested efficiently in mitosis (Fig.
6B,C). These results show that repression of Survivin compromises
checkpoint function following exposure to taxol but not nocodazole.
|
To further characterise the checkpoint response after Survivin repression, live cells were followed in the presence of nocodazole or taxol and then subsequently fixed and immunostained for BubR1 and Mad2. Mitotic cells in cultures transfected with control siRNA showed a normal response to both drugs. Cells in taxol (kinetochores occupied with microtubules, but spindle tension defective) stained brightly for BubR1 at kinetochores, but only a few stained for Mad2 (most of the kinetochores are attached to microtubules) (Fig. 6D). Cells in nocodazole (kinetochores unoccupied and no tension) stained brightly for both BubR1 and Mad2 at kinetochores (Fig. 6F). In contrast, kinetochores of Survivin-depleted cells in taxol either completely lacked or showed a very decreased staining for both BubR1 and Mad2 (Fig. 6E). Furthermore, in the presence of nocodazole, Survivin-depleted cells also showed no or very low staining for BubR1, but Mad2 remained bright at the kinetochores (Fig. 6G).
In summary, these data, which are summarized in Table 1, indicate that Survivin is required for the function of the spindle checkpoint in response to taxol treatment but not in response to nocodazole.
|
![]() |
Discussion |
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Recently, microinjection of polyclonal antibodies that recognise all known
forms of Survivin into HeLa cells was reported to result in similar chromosome
congression defects (Giodini et al.,
2002). However, earlier studies had suggested that interference
with Survivin function compromised the ability of cells to form a normal
bipolar spindle. In HeLa cells (the cell type used here), a significant
increase in multipolar spindles was reported after exposure of cells to a
dominant-negative Survivin construct (C84A) or antisense oligonucleotides
(Li et al., 1999
) and, shown
more recently, after microinjection of Survivin antibodies
(Fortugno et al., 2002
).
Multipolar spindles were also observed after interference with Survivin
function in other systems, for example, early embryonic cells derived from
Survivin-knockout mice (Uren et al.,
2000
). It is worth noting that in the present study, although we
detected an approximately 10% increase in multinucleated cells over control
levels, no significant increase in the percentage of multipolar mitosis was
observed.
Time-lapse imaging revealed that transfection with Survivin siRNA caused a mitotic delay and failure of cytokinesis. The net result was a significant increase in the number of polyploid and multinucleated cells in the population. Furthermore, we found that levels of apoptosis rose only slightly, confirming that Survivin functions predominantly in mitotic progression as opposed to inhibition of cell death.
These observations confirm that Survivin is required for chromosome biorientation and completion of cytokinesis in HeLa cells. The reason for the defects in cytokinesis induced by repression of Survivin is not known, but one possibility is that Survivin-depleted cells exit mitosis with maloriented chromosomes and that these are trapped in the intercellular bridge, where they block the completion of abscission. Consistent with this view, we have observed that Survivin-depleted cells have defects in the spindle assembly checkpoint (discussed further below).
Survivin is not a mitotic spindle associated protein prior to
anaphase
Survivin was originally described as a mitotic-spindle-associated protein
(Li et al., 1998;
Li et al., 1999
). However we
and others have subsequently found that Survivin behaves like a chromosomal
passenger protein in human (Skoufias et
al., 2000
; Wheatley et al.,
2001a
; Jiang et al.,
2001
) and murine cells (Uren
et al., 2000
). Recent papers raised the possibility that there are
two populations of Survivin (Fortugno et
al., 2002
) or that different cell types had different
localizations of the protein (Jiang et
al., 2001
). At the centre of this controversy is a monoclonal
antibody, 8E2, which strongly stains the entire spindle in metaphase cells
(Fig. 2C). In this study we
found that the pattern of 8E2 staining remained unaltered when Survivin was
depleted from HeLa cells (Fig.
2D). Although this monoclonal antibody does recognise human
Survivin in vitro (Wheatley et al.,
2001a
), the persistence of spindle staining after depletion of all
known Survivin isoforms demonstrates that 8E2 is likely to also recognise an
additional unknown microtubule-associated protein in mitotic HeLa cells.
Mutual dependence of Survivin, Aurora-B and INCENP for localization
at centromeres
Aurora-B and INCENP fail to concentrate at centromeres in prometaphase
cells depleted of Survivin. Because a dominant-negative form of INCENP causes
the mislocalization of Aurora-B and Survivin in HeLa cells
(Adams et al., 2000;
Wheatley et al., 2001a
), it
appears that these three proteins are mutually dependent on each other for
localization to centromeres. In C. elegans, the homologues of
Survivin [BIR1 (Fraser et al.,
1999
)], Aurora-B (AIR-2) and INCENP (ICP1) also appear to interact
with one another, and the localization of AIR-2 is dependent upon ICP-1
(Kaitna et al., 2000
) and BIR1
(Speliotes et al., 2000
). In
addition, corroborating evidence regarding the relationship(s) between these
three chromosome passengers is also emerging in flies
(Adams et al., 2001c
), frog
eggs (Bolton et al., 2002
) and
yeast (Yoon and Carbon, 1999
;
Rajagopalan and Balasubramanian,
2002
; Cheeseman et al.,
2002
). One possible explanation for these data is that a stable
complex between Survivin, Aurora-B and INCENP is required to target this group
of proteins to the centromeres. However, the persistence of such a complex
throughout mitosis has yet to be demonstrated directly.
Survivin is required for the efficient localization of BubR1 to
kinetochores and a robust spindle checkpoint response in the presence of
taxol
As cells enter mitosis, kinetochores constitutively express signals that
delay the activation of the APC/C (anaphase-promoting complex/cyclosome) and
onset of anaphase (Pines and Rieder,
2001). These signals are gradually extinguished as sister
kinetochores capture microtubules from opposite spindle poles and tension
develops throughout the spindle. This signalling pathway, termed the spindle
assembly checkpoint, has been proposed to involve two separate components. The
BubR1 arm of the pathway has been suggested to monitor spindle tension
(Taylor et al., 2001
;
Skoufias et al., 2001
),
whereas the Mad2 pathway monitors microtubule attachment at the kinetochore
(Waters et al., 1998
).
However, a recent study has questioned whether these represent two completely
independent, parallel pathways or a single pathway that depends on both BubR1
and Mad2 (Shannon et al.,
2002
).
The present study has shown conclusively that Survivin-depleted cells
exhibit important differences from control cells in the function of their
spindle assembly checkpoint when exposed to agents that perturb microtubule
behaviour. We found that in Survivin-depleted cells, some kinetochores of
maloriented chromosomes expressed high levels of BubR1 protein, whereas
kinetochores of other maloriented chromosomes did not. Importantly,
kinetochores of Survivin-depleted chromosomes accumulated BubR1 normally
during a short colcemid (2 hours) treatment, indicating that Survivin is not
required for BubR1 binding to the kinetochore upon activation of the spindle
assembly checkpoint. Instead, we found that Survivin is required for the
maintenance of BubR1 at kinetochores during persistent activation of the
checkpoint. This is consistent with the results of a recent study
(Kallio et al., 2001) in which
it was reported that the phospho-epitope, 3F3/2, which identifies kinetochores
that are not under tension during prometaphase
(Nicklas et al., 1995
), was
precociously lost from centromeres when cells were microinjected with
Survivin-specific antibodies.
Normally, in the presence of taxol, kinetochores have elevated levels of
BubR1 and are negative for Mad2. In the presence of nocodazole both proteins
are normally present at elevated levels. Although recent experiments have
revealed that it is dangerous to draw conclusions about the activity of the
spindle assembly checkpoint based solely on the level of kinetochore staining
with anti-Mad2 (Martin-Lluesma et al.,
2002), in the case of Survivin siRNA treatment, the observed
pattern of kinetochore association of BubR1 and Mad2 is able to explain the
response of the cells to persistent checkpoint activation.
We have found that following Survivin RNAi, BubR1 accumulation at kinetochores is unstable in the presence of either taxol or nocodazole. Therefore, in the presence of taxol, kinetochores lack elevated levels of both BubR1 and Mad2. As a result, Survivin RNAi abrogates the ability of cells to sustain a prolonged mitotic arrest in the presence of taxol. In contrast, Survivin RNAi has no effect on the ability of kinetochores to retain high levels of Mad2 protein. Thus, in the presence of nocodazole or colcemid, kinetochores are BubR1 negative but Mad2 positive, and cells are able to sustain a prolonged mitotic arrest.
Kinetochores that express elevated levels of either BubR1 or Mad2 alone appear to be able to generate a `wait anaphase' signal and activate the spindle assembly checkpoint. The former are seen normally in the presence of taxol, and the latter are described here for the first time. It thus appears that either BubR1 or Mad2 and their downstream effectors are sufficient to activate the checkpoint.
We note that the major conclusions described above, and particularly the
requirement of Survivin for a robust checkpoint response to taxol but not
nocodazole, are consistent with the results of an independent study in which
Survivin silencing was achieved using a plasmid vector in several different
cell lines (Lens et al.,
2003).
Importantly, our data should not be interpreted as indicating that Survivin
is directly required for the detection of spindle tension. Rather, they
indicate that Survivin is required for stable maintenance of elevated levels
of BubR1 at kinetochores. The preferential sensitivity of the taxol-induced
checkpoint to loss of Survivin reflects more the behaviour of Mad2 than any
specific alteration in the behaviour of BubR1. The result may appear to be
selective for one arm of the checkpoint pathway, but the actual effect on
BubR1 is not selective. Thus, although the data presented here appear to
support the emerging view of a dual checkpoint pathway, where one arm detects
kinetochore occupancy, and the other detects spindle tension
(Taylor et al., 2001;
Skoufias et al., 2001
),
whether or not there is a dual input to the spindle assembly checkpoint
pathway remains an important question for future investigations.
![]() |
Concluding remarks |
---|
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---|
It is likely that one common thread underlying the multiple phenotypes seen
in the present study is the action of the Aurora-B kinase. The search for
relevant substrates of this kinase is ongoing and likely to yield many
candidates. However, in addition to targeting and activating Aurora-B, which
is becoming ever more recognised as an essential facilitator of mitotic events
(Adams et al., 2001a), it is
also possible that Survivin and INCENP have other roles in mitotic
progression. Thus, this remains an area of growing excitement and important
discovery.
![]() |
Acknowledgments |
---|
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References |
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Adams, R. R., Wheatley, S. P., Gouldsworthy, A. M., Kandels-Lewis, S. E., Carmena, M., Smythe, C., Gerloff, D. L. and Earnshaw, W. C. (2000). INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and the cleavage furrow. Curr. Biol. 10,1075 -1078.[CrossRef][Medline]
Adams, R. R., Carmena, M. and Earnshaw, W. C. (2001a). Chromosomal passengers and the (Aurora) ABCs of mitosis. Trends Cell Biol. 11,49 -54.[CrossRef][Medline]
Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M.
(2001b). Essential roles of Drosophila inner centromere
protein (INCENP) and Aurora-B in histone H3 phosphorylation, metaphase
chromosome alignment, kinetochore disjunction, and chromosome segregation.
J. Cell Biol. 153,865
-880.
Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M.
(2001c). Essential roles of Drosophila inner centromere
protein (INCENP) and Aurora-B in histone H3 phosphorylation, metaphase
chromosome alignment, kinetochore disjunction, and chromosome segregation.
J. Cell Biol. 153,865
-880.
Ainsztein, A. M., Kandels-Lewis, S. E., Mackay, A. M. and
Earnshaw, W. C. (1998). INCENP centromere and spindle
targeting: Identification of essential conserved motifs and involvement of
heterochromatin protein HP1. J. Cell Biol.
143,1763
-1774.
Altieri, D. C. (2001). The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol. Med. 7,542 -547.[CrossRef][Medline]
Altieri, D. C., Marchisio, P. C. and Marchisio, C. (1999). Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest. 79,1327 -1333.[Medline]
Ambrosini, G., Adida, C. and Altieri, D. C. (1997). A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat. Med. 3, 917-921.[Medline]
Andreassen, P. R., Palmer, D. K., Wener, M. H. and Margolis, R. L. (1991). Telophase disk: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. J. Cell Sci. 99,523 -534.[Abstract]
Biggins, S. and Murray, A. W. (2001). The
budding yeast protein kinase Ipl1/Aurora allows the absence of tension to
activate the spindle checkpoint. Genes Dev.
15,3118
-3129.
Biggins, S., Severin, F. F., Bhalla, N., Sassoon, I., Hyman, A.
A. and Murray, A. W. (1999). The conserved protein kinase
Ipl1 regulates microtubule binding to kinetochores in budding yeast.
Genes Dev. 13,532
-544.
Bishop, J. D. and Schumacher, J. M. (2002).
Phosphorylation of the carboxyl terminus of inner Centromere Protein (INCENP)
by the Aurora B kinase stimulates Aurora B kinase activity. J.
Biol. Chem. 277,27577
-27580.
Bolton, M. A., Lan, W., Powers, S. E., McCleland, M. L., Kuang, J. and Stukenberg, P. T. (2002). Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation. Mol. Biol. Cell (in press).
Cheeseman, I. M., Anderson, S., Jwa, M., Green, E. M., Kang, J.-S., Yates, J. R., Chan, C. S. M., Drubin, D. G. and Barnes, G. (2002). Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell 111,163 -172.[Medline]
Cutts, S. M., Fowler, K. J., Kile, B. T., Hii, L. L., O'Dowd, R.
A., Hudson, D. F., Saffery, R., Kalitsis, P., Earle, E. and Choo, K. H.
(1999). Defective chromosome segregation, microtubule bundling
and nuclear bridging in inner centromere protein gene (Incenp)-disrupted mice.
Hum. Mol. Genet. 8,1145
-1155.
Earnshaw, W. C. and Bernat, R. L. (1990). Chromosomal passengers: Towards an integrated view of mitosis. Chromosoma (Berl.). 100,139 -146.
Earnshaw, W. C. and Cooke, C. A. (1991). Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of three distinct substages of metaphase and early events in cleavage furrow formation. J. Cell Sci. 98,443 -461.[Abstract]
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411,494 -498.[CrossRef][Medline]
Fortugno, P., Wall, N. R., Giodini, A., O'Connor, D. S.,
Plescia, J., Padgett, K. M., Tognin, S., Marchisio, P. C. and Altieri, D.
C. (2002). Survivin exists in immunochemically distinct
subcellular pools and is involved in spindle microtubule function.
J. Cell Sci. 115,575
-585.
Fraser, A. G., James, C., Evan, G. I. and Hengartner, M. O. (1999). Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis. Curr. Biol. 9,292 -301.[CrossRef][Medline]
Giet, R. and Glover, D. M. (2001).
Drosophila Aurora B kinase is required for histone H3 phosphorylation
and condensin recruitment during chromosome condensation and to organize the
central spindle during cytokinesis. J. Cell Biol.
152,669
-681.
Giodini, A., Kallio, M. J., Wall, N. R., Gorbsky, G. J., Tognin,
S., Marchisio, P. C., Symons, M. and Altieri, D. C. (2002).
Regulation of microtubule stability and mitotic progression by survivin.
Cancer Res. 62,2462
-2467.
Howell, B. J., McEwen, B. F., Canman, J. C., Hoffman, D. B.,
Farrar, E. M., Rieder, C. L. and Salmon, E. D. (2001).
Cytoplasmic dynein/dynactin drives kinetochore protein transport to the
spindle poles and has a role in mitotic spindle checkpoint inactivation.
J. Cell Biol. 155,1159
-1172.
Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K., Grushcow, J. M., Brame, C. J., Caldwell, J. A., Hunt, D. F. et al. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102,279 -291.[Medline]
Jiang, X., Wilford, C., Duensing, S., Munger, K., Jones, G. and Jones, D. (2001). Participation of Survivin in mitotic and apoptotic activities of normal and tumor-derived cells. J. Cell. Biochem. 83,342 -354.[CrossRef][Medline]
Kaitna, S., Mendoza, M., Jantsch-Plunger, V. and Glotzer, M. (2000). Incenp and an Aurora-like kinase form a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. Curr. Biol. 10,1172 -1181.[CrossRef][Medline]
Kaitna, S., Pasierbek, P., Jantsch, M., Loidl, J. and Glotzer, M. (2002). The Aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous chromosomes during meiosis. Curr. Biol. 12,798 -812.[CrossRef][Medline]
Kallio, M. J., Nieminen, M. and Eriksson, J. E.
(2001). Human inhibitor of apoptosis protein (IAP) survivin
participates in regulation of chromosome segregation and mitotic exit.
Faseb J. 15,2721
-2723.
Kallio, M. J., McCleland, M. L., Stukenberg, P. T. and Gorbsky, G. J. (2002). Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. Curr. Biol. 12,900 -905.[CrossRef][Medline]
Kang, J., Cheeseman, I. M., Kallstrom, G., Velmurugan, S.,
Barnes, G. and Chan, C. S. (2001). Functional cooperation of
Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15
during chromosome segregation. J. Cell Biol.
155,763
-774.
Lens, S. M. A., Wolthuis, R. M. F., Klompmaker, R., Kauw, J., Agami, R., Brummelkamp, T., Kops, G. and Medema, R. H. (2003). Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension. EMBO J. (in press).
Leverson, J. D., Huang, H. K., Forsburg, S. L. and Hunter,
T. (2002). The Schizosaccharomyces pombe
Aurora-related kinase Ark1 interacts with the inner centromere protein Pic1
and mediates chromosome segregation and cytokinesis. Mol. Biol.
Cell 13,1132
-1143.
Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C. and Altieri, D. C. (1998). Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396,580 -584.[CrossRef][Medline]
Li, F., Ackermann, E. J., Bennett, C. F., Rothermel, A. L., Plescia, J., Tognin, S., Villa, A., Marchisio, P. C. and Altieri, D. C. (1999). Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat. Cell Biol. 1,461 -466.[CrossRef][Medline]
MacCallum, D. E., Losada, A., Kobayashi, R. and Hirano, T.
(2002). ISWI remodeling complexes in Xenopus egg
extracts: identification as major chromosomal components that are regulated by
INCENP-aurora B. Mol. Biol. Cell
13, 25-39.
Mackay, A. M., Ainsztein, A. M., Eckley, D. M. and Earnshaw, W.
C. (1998). A dominant mutant of inner centromere protein
(INCENP), a chromosomal protein, disrupts prometaphase congression and
cytokinesis. J. Cell Biol.
140,991
-1002.
Mahotka, C., Wenzel, M., Springer, E., Gabbert, H. E. and
Gerharz, C. D. (1999). Survivin-deltaEx3 and survivin-2B: two
novel splice variants of the apoptosis inhibitor survivin with different
antiapoptotic properties. Cancer Res.
59,6097
-6102.
Martin-Lluesma, S., Stucke, V. M. and Nigg, E. A.
(2002). Role of Hec1 in spindle checkpoint signaling and
kinetochore recruitment of Mad1/Mad2. Science
297,2267
-2270.
Morishita, J., Matsusaka, T., Goshima, G., Nakamura, T., Tatebe,
H. and Yanagida, M. (2001). Bir1/Cut17 moving from chromosome
to spindle upon the loss of cohesion is required for condensation, spindle
elongation and repair. Genes Cells
6, 743-763.
Murata-Hori, M. and Wang, Y. (2002). The Kinase activity of Aurora B is required for kinetochore-microtubule interactions during mitosis. Curr. Biol. 12,894 -899.[CrossRef][Medline]
Murata-Hori, M., Tatsuka, M. and Wang, Y. L.
(2002). Probing the dynamics and functions of Aurora B kinase in
living cells during mitosis and cytokinesis. Mol. Biol.
Cell 13,1099
-1108.
Murnion, M. E., Adams, R. A., Callister, D. M., Allis, C. D.,
Earnshaw, W. C. and Swedlow, J. R. (2001).
Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3
phosphorylation. J. Biol. Chem.
276,26656
-26665.
Musacchio, A. and Hardwick, K. G. (2002). The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell. Biol. 3, 731-741.[CrossRef][Medline]
Nicklas, R. B., Wards, S. C. and Gorbsky, G. J. (1995). Kinetochore chemistry is sensitive to tension and may link mitotic forces for a cell cycle checkpoint. J. Cell Biol. 130,929 -939.[Abstract]
Oegema, K., Desai, A., Rybina, S., Kirkham, M. and Hyman, A.
A. (2001). Functional analysis of kinetochore assembly in
Caenorhabditis elegans. J. Cell Biol.
153,1209
-1226.
Pines, J. and Rieder, C. L. (2001). Re-staging mitosis: a contemporary view of mitotic progression. Nat. Cell Biol. 3,E3 -E6.[CrossRef][Medline]
Rajagopalan, S. and Balasubramanian, M. K.
(2002). Schizosaccharomyces pombe Bir1p, a nuclear
protein that localizes to kinetochores and the spindle midzone, is essential
for chromosome condensation and spindle elongation during mitosis.
Genetics 160,445
-456.
Reed, J. C. and Bischoff, J. R. (2000). BIRinging chromosomes through cell division and survivin' the experience. Cell 102,545 -548.[Medline]
Rodriguez, J. A., Span, S. W., Ferreira, C. G., Kruyt, F. A. and Giaccone, G. (2002). CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin. Exp. Cell Res. 275,44 -53.[CrossRef][Medline]
Rogers, E., Bishop, J. D., Waddle, J. A., Schumacher, J. M. and
Lin, R. (2002). The aurora kinase AIR-2 functions in the
release of chromosome cohesion in Caenorhabditis elegans meiosis.
J. Cell Biol. 157,219
-229.
Schumacher, J. M., Golden, A. and Donovan, P. J.
(1998). AIR-2: An Aurora/Ipl1-related protein kinase associated
with chromosomes and midbody microtubules is required for polar body extrusion
and cytokinesis in Caenorhabditis elegans embryos. J. Cell
Biol. 143,1635
-1646.
Shannon, K. B., Canman, J. C. and Salmon, E. D.
(2002). Mad2 and BubR1 function in a single checkpoint pathway
that responds to a loss of tension. Mol. Biol. Cell
13,3706
-3719.
Silke, J. and Vaux, D. L. (2001). Two kinds of
BIR-containing protein inhibitors of apoptosis, or required for
mitosis. J. Cell Sci.
114,1821
-1827.
Skoufias, D. A., Mollinari, C., Lacroix, F. B. and Margolis, R.
L. (2000). Human Survivin is a kinetochore-associated
passenger protein. J. Cell Biol.
151,1575
-1582.
Skoufias, D. A., Andreassen, P. R., Lacroix, F. B., Wilson, L.
and Margolis, R. L. (2001). Mammalian mad2 and bub1/bubR1
recognize distinct spindle-attachment and kinetochore-tension checkpoints.
Proc. Natl. Acad. Sci. USA
98,4492
-4497.
Speliotes, E. K., Uren, A., Vaux, D. and Horvitz, H. R. (2000). The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone. Mol. Cell. 6,211 -223.[Medline]
Tanaka, T. U., Rachidi, N., Janke, C., Pereira, G., Galova, M., Schiebel, E., Stark, M. J. and Nasmyth, K. (2002). Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell 108,317 -329.[Medline]
Taylor, S. S., Hussein, D., Wang, Y., Elderkin, S. and Morrow, C. J. (2001). Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells. J. Cell Sci. 114,4385 -4395.[Medline]
Terada, Y. (2001). Role of chromosomal passenger complex in chromosome segregation and cytokinesis. Cell Struct. Funct. 26,653 -657.[CrossRef][Medline]
Terada, Y., Tatsuka, M., Suzuki, F., Yasuda, Y., Fujita, S. and
Otsu, M. (1998). AIM-1: a mammalian midbody-associated
protein required for cytokinesis. EMBO J.
17,667
-676.
Uren, A. G., Wong, L., Pakusch, M., Fowler, K. J., Burrows, F. J., Vaux, D. L. and Choo, K. H. A. (2000). Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr. Biol. 10,1319 -1328.[CrossRef][Medline]
Waters, J. C., Chen, R. H., Murray, A. W. and Salmon, E. D.
(1998). Localization of Mad2 to kinetochores depends on
microtubule attachment, not tension. J. Cell Biol.
141,1181
-1191.
Wheatley, S. P. and Wang, Y. (1996). Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells. J. Cell Biol. 135,981 -989.[Abstract]
Wheatley, S. P., Carvalho, A., Vagnarelli, P. and Earnshaw, W. C. (2001a). INCENP is required for proper targeting of survivin to the centromeres and the anaphase spindle during mitosis. Curr. Biol. 11,886 -890.[CrossRef][Medline]
Wheatley, S. P., Kandels-Lewis, S. E., Adams, R. R., Ainsztein, A. M. and Earnshaw, W. C. (2001b). INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow. Exp. Cell Res. 262,122 -127.[CrossRef][Medline]
Yoon, H. J. and Carbon, J. (1999).
Participation of Bir1p, a member of the inhibitor of apoptosis family, in
yeast chromosome segregation events. Proc. Natl. Acad. Sci.
USA 96,13208
-13213.