1 University of North Carolina, Department of Biology, 607 Fordham Hall,
CB#3280, Chapel Hill, NC 27599, USA
2 Harvard Medical School, Department of Cell Biology, Seely Mudd 529, 240
Longwood Avenue, Boston, MA 021 15, USA
3 Stanford University, Department of Biological Sciences, Gilbert Building, Room
345, Stanford, CA 94305-5020, USA
Author for correspondence (e-mail:
jccanman{at}email.unc.edu)
Accepted 16 July 2002
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Summary |
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Key words: INCENP, Spindle checkpoint, Mad2, Anaphase, 3F3/2, CENP-E, Dynein, BubR1, Microtubules, Mad1F10, Kinetochore, Centromere
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Introduction |
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Normally, as kinetochores interact with spindle microtubules in
prometaphase to form kinetochore microtubules, motor and checkpoint proteins
are depleted from kinetochores. Mad2 and cytoplasmic dynein are unique in that
they are substantially depleted by anaphase onset, while the other motor and
checkpoint proteins are only moderately depleted
(Hoffman et al., 2001;
Waters et al., 1998
). In
addition, kinetochore phosphorylation, as detected by 3F3/2 antibody to a
phosphoepitope, is turned off as kinetochores become under tension by the net
pulling forces of their sister kinetochores towards opposite poles
(Gorbsky and Ricketts, 1993
;
Nicklas et al., 1995
). These
reduced levels in kinetochore protein concentration and phosphorylation are
thought to be important for inactivating the spindle checkpoint and inducing
anaphase (Millband et al.,
2002
; Shah and Cleveland,
2000
).
Before anaphase, the centromeric region between sister kinetochores
contains high concentrations of a complex of `chromosome passenger' proteins,
including INCENP, aurora B kinase and survivin
(Adams et al., 2001). During
mitosis, these proteins leave the centromere and re-localize to the cell
cortex and the microtubule-rich midzone that forms between separating sister
chromatids in anaphase (for a review, see
Adams et al., 2001
). This
change in localization from the centromeres to the midzone complex is
essential for cytokinesis (Mackay et al.,
1998
).
By anaphase onset in mammalian PtK1 tissue cells, a substantial amount of
cyclin B1 is degraded indicating a substantial loss of Cdk1-cyclin B1 kinase
activity (Clute and Pines,
1999). Thus, it is possible that independently of microtubules,
either proteolysis or loss of Cdk1-cyclin B kinase activity could contribute
to kinetochore protein localization or tension-dependent kinetochore
phosphorylation. To address this issue, we induced anaphase in cells treated
with nocodazole to depolymerize all microtubules. We developed a new method to
induce anaphase onset in nocodazole-treated cells that does not require
microinjection of rabbit antibodies as was used previously to inhibit Mad2
function (Canman et al., 2000
;
Gorbsky et al., 1998
). Here we
show that microinjection of a bacterially expressed fragment of Mad1 protein,
GST-Mad1F10, induces anaphase with kinetics similar to the anti-Mad2 antibody
(Canman et al., 2000
). This
reagent allowed us to test, by using quantitative immunofluorescence
microscopy (Hoffman et al.,
2001
; King et al.,
2000
), whether anaphase onset or APC/C activation requires or
contributes to the depletion of kinetochore and centromere-binding proteins
independently of microtubules.
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Materials and Methods |
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GST alone and GST-Mad1F10 were purified by using GSTrap according to the manufacturer's instructions (Pharmacia Biotech). Purified protein was dialyzed for 24 hours at 4°C with 3 changes into HEK buffer (20 mM Hepes, 100 mM KCl, and 1 mM DTT, pH 7.7 with KOH). After dialysis, GST and GST-Mad1F10 were concentrated to 2 mg/ml by using a YM-30 Microcon centrifugal filter device (Millipore, Bedford, MA), aliquoted into 4 µl aliquots, drop frozen in liquid nitrogen, and stored at -80°C.
In vitro APC/C activation assays
Xenopus egg extracts that had been stably arrested in mitosis by
addition of a non-degradable cyclin B protein (cyclin B90) were made as
previously described (Desai et al.,
1999
). To assay for APC/C activity, a radioactive N-terminal
fragment of cyclin B (amino acids 1-102) was added. The kinetics of
degradation for radioactively labeled cyclin B was then measured in these
extracts by SDS-PAGE as described (Fang et
al., 1998
). When noted (Fig.
2, panel II), Mad2 protein was added at a concentration of 20
µM to arrest the extracts.
|
Tissue culture, microinjection and time-lapse microscopy
PtK1 cells were cultured as described
(Canman et al., 2000) with
minor adaptations. F-12 media was used (Sigma) with 10% FBS (Gibco) and
half-strength antibiotics (Sigma). Cells were fed every other day and split at
80% confluency. When cells were split, they were first washed with sterile PBS
(0.14 M NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 1.5 mM
KH2PO4; pH 7.2) and then washed twice with trypsin EDTA
(Sigma). The last trypsin was removed and the cells were allowed to detach at
37°C. Cells were suspended again in F-12 and split into flasks at a 1:3
dilution.
For microinjection experiments, PtK1 cells were grown on coverslips,
mounted into modified Rose chambers and microinjected as described previously
(Canman et al., 2000). Stage
temperature was maintained at
35°C by using an air curtain incubator
(Nevtek, Model ASI 400, Burnsville, VA). Time-lapse images were obtained every
30 seconds by using a MetaView image acquisition system (Universal Imaging,
Downingtown, PA) as described (Canman et
al., 2000
). Cells were microinjected with either 2 mg/ml purified
GST or 2 mg/ml GST-Mad1F10 in HEK buffer. Our microinjections typically
represent 20% of the cell volume making the intracellular concentration of
injected GST-Mad1F10 a maximum of 8 µM (assuming no protein loss or
denaturation during handling).
Immunofluorescence
For Mad2, BubR1, INCENP, ACA (anti-centromeric antigens), and cytoplasmic
dynein staining, cells were lysed in PHEM (54 mM Pipes, 22.5 mM Hepes, 10 mM
EGTA, 8 mM MgSO4, pH 7.0 with KOH) supplemented with 0.5% Triton
X-100 for 5 minutes at 37°C. Cells were fixed in 1% formaldehyde in PHEM
for 20 minutes at 37°C. For 3F3/2 staining, 100 nM microcystin (Sigma) was
added to the lysis buffer and cells were fixed as described above. Following
five 3 minute rinses in PHEM, cells were blocked in 6% heat-inactivated donkey
serum in PHEM at 25°C for 1 hour. Primary antibody dilutions were
performed in 6% boiled donkey serum in PHEM for 45 minutes at 25°C as
follows: 1:50 anti-Mad2; 1:500 BubR1 and 1:750 CENP-E (both generous gifts of
Tim Yen, Fox Chase Cancer Center, Philadelphia, PA); 1:200 cytoplasmic dynein
70.1 intermediate chain (Sigma); 1:5000 3F3/2 (a generous gift of Gary
Gorbsky, The University of Oklahoma Health Sciences Center, Oklahoma City,
OK); 1:1000 ACA serum (a generous gift of Kevin Sullivan, The Scripps Research
Institute, La Jolla, CA); and 1:1250 anti-INCENP (from A.S.). Following five 3
minute rinses in PHEM-T (PBS supplemented with 0.1% Triton X-100), cells were
incubated with appropriate secondary antibodies diluted into 6% heat
inactivated donkey serum in PHEM at 25°C for 40 minutes. Secondary
minimally crossed donkey antibodies conjugated to either Rhodamine Red X or
Cy2 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used at 1:100.
After two 3 minute rinses in PHEM-T, cells were incubated with DAPI for 5
minutes. Cells were then rinsed three times for 3 minutes in PHEM-T, rinsed
three times for 3 minutes in PHEM and mounted as described previously
(Canman et al., 2000).
Fluorescence microscopy
Images of fixed cells were obtained on a Nikon TE300 inverted microscope
(Nikon, Melville, NY) with an Orca I camera (Hamamatsu Photonics, Bridgewater,
NJ) by using a Nikon 100x 1.4 NA Plan Apochromat phase objective and a
Sutter filter wheel for selecting and shuttering of 360, 488 or 568 nm
wavelengths as described (Howell et al.,
2000). All images were acquired at 0.2 µm steps by using
MetaMorph software (Universal Imaging) controlling the Nikon TE300 focus
motor. Images were collected by using identical imaging settings. For each
group, control images were taken from the same coverslips as the injected
cells to control for handling differences. Image processing and figures were
made by using PhotoShop 5.5 (Adobe, San Jose, CA).
Fluorescence intensity measurements
Quantification of fluorescence intensities was performed as described in
detail (Hoffman et al., 2001;
King et al., 2000
) with the
following modifications. Computer generated 21x21 and 30x30 pixel
squares (for kinetochore proteins in order to include the entire kinetochore)
and 32x32 and 46x46 pixel squares (for INCENP to include the
entire centromeric region) were used to measure kinetochore/centromere and
background fluorescence (Hoffman et al.,
2001
). INCENP coverslips were co-stained for ACA to allow precise
identification of centromeres. For all other measurements, DAPI staining was
used to identify the centromeric region for intensity analysis by using
Metamorph software (Universal Imaging) and Microsoft Excel 2000 (Microsoft,
Redmond, WA).
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Results |
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GST-Mad1-F10 induces anaphase in early prometaphase and
nocodazole-blocked cells
To test the ability of GST-Mad1F10 to inhibit the spindle checkpoint
activity in mammalian tissue cells, we microinjected untreated and
nocodazole-treated (10 µM) prometaphase PtK1 cells with a needle
concentration of 2 mg/ml GST-Mad1F10 in HEK buffer. The untreated prometaphase
cells underwent precocious anaphase onset 15±2 minutes after
microinjection (n=12, Fig.
3, middle row). Following anaphase onset, all of these injected
cells exhibited chromosome segregation and cytokinesis with timing similar to
control cells (Fig. 3, top row
vs. middle row). The nocodazole-treated cells also underwent precocious
anaphase as seen by the separation of sister chromatids
(Fig. 3, bottom row). The delay
between microinjection and anaphase onset was about twice as long for
nocodazole-treated cells, taking 30±5 minutes after microinjection
(n=4, Fig. 3). These
times to anaphase onset in prometaphase and nocodazole-treated cells are
similar to those reported for microinjection of an anti-Mad2 antibody
(Canman et al., 2000;
Howell et al., 2000
). Also,
consistent with the anti-Mad2 antibody injection findings
(Canman et al., 2000
),
nocodazole-treated microinjected cells induced to enter anaphase in nocodazole
with GST-Mad1F10 underwent cortical contractions followed by cell
re-spreading, chromosome decondensation and nuclear envelope reformation;
features characteristic of the cytokinetic phase, or C-phase, of the cell
cycle (Fig. 3, bottom row)
(Canman et al., 2000
).
Microinjection of 2 mg/ml GST in HEK buffer alone did not trigger precocious
anaphase onset in prometaphase cells (n=10,
Fig. 3, top row) or in cells
treated with 10 µM nocodazole (n=5, data not shown). Thus
GST-Mad1F10, like anti-Mad2 antibody, appears to inactivate the checkpoint
without interfering with the mechanics of chromosome segregation or
cytokinesis.
|
Kinetochore cytoplasmic dynein, CENP-E, Mad2, 3F3/2, BubR1 and
centromere INCENP are not significantly reduced during anaphase in
nocodazole
To test how protein disassembly at kinetochores and centromeres by anaphase
depends on microtubules, we analyzed kinetochore protein localization under
four experimental conditions: (1) untreated prometaphase cells; (2) untreated
prometaphase cells induced into anaphase by GST-Mad1F10 microinjection; (3)
prometaphase cells treated with 10 µm nocodazole for 30 minutes to induce
depolymerization of all microtubules
(Canman et al., 2000); and (4)
nocodazole-treated prometaphase cells subsequently induced into anaphase by
GST-Mad1F10 microinjection. Individual cells induced into anaphase were
followed by time-lapse microscopy and fixed for immunofluorescence analysis
after sister chromatid separation. For untreated cells, we compared
fluorescence intensities of kinetochores/centromeres between unattached or
newly attached chromosomes near the spindle poles, which are expected to have
few or no kinetochore microtubules
(Hoffman et al., 2001
;
McEwen et al., 1997
), to the
values measured for kinetochores/centromeres after induction of precocious
anaphase. For cells treated with nocodazole, we compared changes in
fluorescence intensities for all kinetochores/centromeres in
nocodazole-treated cells before and after induction of anaphase by GST-Mad1F10
injection. It was not possible to compare the differences between the
measurements acquired for untreated cells with those acquired for
nocodazole-treated cells in these experiments as the experiments were done on
different days.
As expected (Gorbsky and Ricketts,
1993; Hoffman et al.,
2001
), antibodies to Mad2, 3F3/2, cytoplasmic dynein and BubR1
brightly stained unattached or newly attached kinetochores on PtK1 chromosomes
in prometaphase, but not the attached and tense kinetochores of chromosomes
aligned at the spindle equator (Fig.
4A-D, first rows). All kinetochores in nocodazole-treated cells
stained brightly for these kinetochore components
(Fig. 4A-D, third rows). By the
time untreated prometaphase cells were induced into anaphase, fluorescence was
greatly reduced at most kinetochores (Fig.
4A-D, second rows; Table
1A). The average values for Mad2, BubR1, 3F3/2 and dynein
fluorescence at untreated anaphase kinetochores decreased to -2%, 6%, 0% and
0% (respectively) of their average values in prometaphase
(Fig. 6;
Table 1). In contrast, when a
nocodazole-treated cell was induced to enter anaphase, Mad2, 3F3/2, dynein and
BubR1 were not substantially reduced (Fig.
4A-D, bottom rows; Table
1B). In the nocodazole-treated cells, average anaphase values for
Mad2, BubR1, 3F3/2, and dynein fluorescence at kinetochores after anaphase
induction were 39%, 84%, 77%, and 98%, respectively, of their average values
in prometaphase (Fig. 6;
Table 1). We also quantified
changes in kinetochore immunofluorescence for the microtubule motor, CENP-E.
Kinetochore-bound CENP-E is reduced by
50% upon microtubule attachment in
prometaphase (Hoffman et al.,
2001
), but remains at kinetochores in anaphase
(Cooke et al., 1997
;
Yen et al., 1991
). CENP-E did
not deplete from kinetochores in anaphase in the absence of microtubules
(Fig. 6; Table 1). This comparison
indicates that microtubules are required in anaphase for the production or
maintenance of reduced levels of Mad2, BubR1, dynein and CENP-E, and for the
loss of the phosphoepitope recognized by the 3F3/2 antibody at
kinetochores.
|
|
|
We next wanted to look at an inner-centromeric protein that is not depleted
from centromeres before anaphase onset. INCENP was a prime candidate
(Earnshaw and Cooke, 1991). In
PtK1 cells, INCENP localized to the centromeric regions of paired sister
chromatids in prometaphase (Fig.
5, top row) for both unaligned chromosomes and chromosomes aligned
at the spindle equator, and left the centromeres in anaphase [data not shown
(see also Adams et al., 2001
)].
Thus neither kinetochore microtubule formation nor the centromere tension at
aligned chromosomes (Waters et al.,
1998
) is sufficient to induce the normal depletion of INCENP from
the centromere in anaphase. When untreated prometaphase cells were induced to
enter anaphase by GST-Mad1F10 injection, INCENP left the centromeres and
localized to non-kinetochore microtubules around the kinetochore fibers and
also to the midzone microtubule complex that forms between separating
chromosomes as expected (Fig.
5, second row). In nocodazole, INCENP was associated with all
centromeres (Fig. 5, third
row). INCENP also remained at the centromeres when nocodazole-treated cells
were induced to enter anaphase by GST-Mad1F10 injection (Figs
5,
6;
Table 1). Interestingly, INCENP
levels in nocodazole-treated cells increased at centromeres in anaphase,
reaching an average 152% of prometaphase level
(Fig. 6;
Table 1). Thus, INCENP requires
both microtubules and the activation of anaphase to become depleted from
centromeres.
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Discussion |
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GST-Mad1F10 is a useful molecular tool for inducing anaphase, as it is not
expected to crosslink proteins at kinetochores as occurs with antibody-based
mechanisms for precocious anaphase induction
(Waters et al., 1998).
Therefore, GST-Mad1F10 should be a very useful tool for the analysis of
protein function in anaphase and cytokinesis, when functional disruption
before anaphase activates the spindle checkpoint (for a review, see
Canman et al., 2002
).
It is interesting to note that for GST-Mad1 F10 injected cells, as well as
for cells injected with anti-Mad2 antibodies (J.C.C. and E.D.S., unpublished)
(Canman et al., 2000), the time
from microinjection to anaphase onset was significantly longer for
nocodazole-treated cells (
22-30 minutes) than for normal prometaphase
cells (
9-15 minutes). This difference may be related to the strength of
the checkpoint signal generated by the much larger number of unattached (and
un-tense) kinetochores in nocodazole-treated cells that have high
concentrations of spindle checkpoint proteins
(Hoffman et al., 2001
).
APC/C activation does not require the depletion of checkpoint
proteins from kinetochores
Upon anaphase onset, the sister chromatids separate quickly and are moved
to separate poles. The current model to explain the swift shut off of
chromosome cohesion upon anaphase onset predicts a feedback loop, with APC/C
activity leading to a rapid depletion of checkpoint proteins from their sites
of activity at the kinetochores (for a review, see
Shah and Cleveland, 2000).
Here, however, we clearly showed that anaphase onset does not require the
depletion of Mad2, BubR1, dynein or CENP-E. We have also shown that APC/C
activity does not require de-phosphorylation of the 3F3/2 phosphoepitope (see
also below). Thus, if a feedback mechanism exists between anaphase onset and
spindle checkpoint de-activation, it is not required for the APC/C activity
needed to activate anaphase. Furthermore, anaphase onset can occur with
checkpoint proteins at the kinetochores so it is likely that inactivation of
the cytoplasmic pool of spindle checkpoint proteins is sufficient to induce
anaphase.
Microtubules are essential for maintaining or promoting protein
depletion at kinetochores, even in anaphase
A novel finding in this study is that even after anaphase is induced by
APC/C activity, microtubules are required for significant reductions at
kinetochores of Mad2, BubR1, the 3F3/2 antigen and dynein. In the
nocodazole-treated cells, BubR1 and dynein were only slightly reduced at
kinetochores after anaphase onset, while Mad2 was reduced by 61%
(Fig. 6,
Table 1). The decrease in
levels of Mad2 at kinetochores is probably the result of the sequestering of
Mad2 by the high concentration of GST-Mad1F10 used to induce anaphase.
Nevertheless, this 61% reduction is small relative to the 99+% reduction of
Mad2 for attached kinetochores at either metaphase
(Hoffman et al., 2001) or
anaphase (Fig. 6; Table 1). For Mad2, BubR1 and
dynein, kinetochore microtubule formation appears to be the dominant mechanism
inducing loss of these proteins from the kinetochore through
dynein/dynactin-driven transport of these proteins from kinetochores to the
poles along spindle microtubules (Hoffman
et al., 2001
; Howell et al.,
2001
; Wojcik et al.,
2001
). In contrast, un-tense kinetochores with a full complement
of kinetochore microtubules are brightly stained by 3F3/2 antibody
(Waters et al., 1998
)
indicating that the kinetochore phosphorylation recognized by this antibody is
mainly turned off by tension (Waters et
al., 1998
). How microtubule attachment or tension controls steady
state amounts of kinetochore motor and checkpoint protein concentrations or
kinetochore phosphorylation is still a significant unsolved problem
(Nicklas et al., 2001
).
Nevertheless, our results indicate that the molecular events activated by
APC/C activity at anaphase onset, such as proteolysis of securin and cyclin
B1, do not make a significant contribution in comparison with that made by
kinetochore microtubule formation and tension.
A previous study showed that when cells were induced into anaphase by
anti-Mad2 antibody injection and then subsequently released from nocodazole to
allow microtubule reassembly 20 minutes after anaphase onset, chromosomes were
able to move towards separated poles into two masses
(Canman et al., 2000). It was
not clear how anaphase kinetochores were able to undergo poleward movement so
long after anaphase onset. However, with the data from this paper, we can now
deduce that the chromosome poleward movement upon nocodazole washout in
anaphase was due to the retention of key kinetochore proteins that interact
with spindle microtubules (e.g. cytoplasmic dynein and CENP-E).
Evidence that the phosphoepitope recognized by 3F3/2 is not an
inhibitory phosphorylation(s) on APC/C components
3F3/2 staining also persisted at kinetochores after the activation of
anaphase in the absence of microtubules. This result indicated that loss of
the phosphorylation recognized by the 3F3/2 antibody is dependent on
microtubules, and independent of APC/C activation. If the 3F3/2 epitope
reflects an inhibitory phosphorylation on component(s) of the APC/C, one would
predict that the 3F3/2 antigen would disappear upon anaphase onset, to reflect
the activation of the APC/C. Therefore, it is unlikely that the
phosphoepitopes recognized by 3F3/2 antibodies are inhibitory sites on
kinetochore-bound APC/C components, as has been previously proposed
(Daum et al., 2000). It is
more likely that 3F3/2 antibodies recognize a phosphoepitope on a
tension-sensing component of the spindle checkpoint
(Campbell and Gorbsky, 1995
),
the dephosphorylation of which is not essential for anaphase onset as it
remains on kinetochores in anaphase in the absence of microtubules.
Both microtubules and anaphase onset are essential for INCENP
depletion at centromeres
Here we show that INCENP depletion from the centromeres in anaphase is a
microtubule-dependent process. The release of INCENP from centromeres is
important for proper INCENP localization to the midzone-microtubule complex
during anaphase and for the completion of cytokinesis
(Mackay et al., 1998). Unlike
the spindle checkpoint proteins, however, INCENP remains on kinetochore at
anaphase onset, even in the presence of microtubules (Figs
5,
6;
Table 1). Thus, both
microtubules and anaphase onset are required for INCENP to leave the
chromosomes.
Wheatley and colleagues have previously reported that INCENP moves to the
cell cortex in chicken tissue cells when they are treated with nocodazole
after the onset of anaphase (Wheatley et
al., 2001). In our hands, however, we do not see INCENP targeting
to the cell cortex in cells maintained in nocodazole both before and after
anaphase onset (Fig. 5, bottom
row, and data not shown). This difference is probably due to the fact that
without overcoming the spindle checkpoint, it was not possible to have precise
control over the timing of microtubule disruption relative to anaphase onset.
That is, INCENP may have already moved to the midzone-microtubule complex
prior to microtubule depolymerization in their experiments because they were
unable to treat with microtubule disrupting drugs prior to anaphase onset. It
is likely that the movement along non-kinetochore fibers towards the midzone
is key for proper positioning of INCENP and associated proteins [i.e. survivin
and aurora B (for a review, see Adams et
al., 2001
)].
In our studies, INCENP levels at centromeres increased after anaphase in
nocodazole. One interpretation of this finding is that in anaphase, binding of
INCENP to centromeres is promoted in order to supply INCENP at centromeres for
microtubule transport towards the plus ends of the microtubules in the midzone
complex at the equator of the cell. INCENP localization to the microtubule
midzone has been proposed to be essential for the completion of cytokinesis
(Kaitna et al., 2000;
Mackay et al., 1998
). MKLP1
(also called CHO1 and ZEN-4) is a kinesin-related protein required for
cytokinesis (Kuriyama et al.,
2002
; Powers et al.,
1998
; Raich et al.,
1998
) that moves along microtubules towards plus-ends and
localizes to the spindle midzone during cytokinesis
(Nislow et al., 1990
). This
motor has been shown to be required for the proper localization of another
passenger protein, aurora B (Severson et
al., 2000
). As aurora B has also been shown to bind to INCENP
(Adams et al., 2000
), perhaps
MKLP1 is the motor responsible for transporting INCENP from centromeres to the
midzone complex in anaphase. It will be interesting to visualize the movement
and dynamics of INCENP in vivo and determine whether the change in INCENP
localization from the centromeres to the midzone complex is dependent on
MKLP1.
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Acknowledgments |
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Footnotes |
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References |
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