Research Article
4033
Survivin dynamics increases at centromeres during
G2/M phase transition and is regulated by
microtubule-attachment and Aurora B kinase activity
Victoria A. Beardmore1,*,‡, Leena J. Ahonen2,*, Gary J. Gorbsky2 and Marko J. Kallio1,3,§
1University of Oklahoma Health Sciences Center, Department of Cell Biology, 940 Stanton L. Young Boulevard,
2Oklahoma Medical Research Foundation, Molecular, Cell and Developmental Biology Research Program, 825
Oklahoma City, OK 73104, USA
N.E. 13th Street, Oklahoma City,
OK 73104, USA
3VTT-Medical Biotechnology and University of Turku, P.O. Box 106, 20521 Turku, Finland
*Authors contributed equally to the work
‡Present address: MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
§Author for correspondence (e-mail: [email protected])
Accepted 1 April 2004
Journal of Cell Science 117, 4033-4042 Published by The Company of Biologists 2004
doi:10.1242/jcs.01242
Summary
The inhibitor of apoptosis protein survivin is implicated in
two key biological events: in the control of cell proliferation
and in the regulation of cell lifespan. Although the details
of mitotic roles of survivin are unclear, the protein appears
to modulate microtubule function and might participate in
regulating the spindle checkpoint. Survivin physically
associates with Aurora B, a serine-threonine kinase
involved in microtubule attachment to centromeres and
regulation of chromosome segregation. Here we have
examined the dynamics and localization of a survivin-GFP
chimera using high-resolution fluorescence microscopy and
photobleaching. Survivin forms a bi-partite structure at the
inner centromere that undergoes significant stretching
Introduction
Unbalanced chromosome segregation in mitosis contributes to
cancer malignancy. The pathways that regulate the fidelity of
chromosome segregation are incompletely understood. Survivin,
a human member of the inhibitor of apoptosis protein (IAP)
family, is undetectable in terminally differentiated adult tissues,
but is expressed in dividing cells (Chiou et al., 2003; Altieri,
2003). Survivin is highly overexpressed in transformed cell lines
and in human neoplasms of lung, colon, pancreas, prostate and
breast (Chiou et al., 2003; Altieri, 2003). Survivin is closely
related to the BIR motif proteins of budding yeast, fission yeast
(BIR1 and bir1, respectively), and Caenorhabditis elegans (BIR1 and BIR-2) (Reed and Bischoff, 2000; Uren et al., 1998). In
contrast to other IAPs, which have primary roles in protection
from apoptosis, members of this distinct subgroup of BIR motif
proteins have essential roles in spindle function and cell cleavage
(Li et al., 1998; Fraser et al., 1999; Giodini et al., 2002).
The mitotic localization of one subclass of survivin is
consistent with the proteins described as chromosomal
passenger proteins (Skoufias et al., 2000; Adams et al., 2001a).
Survivin physically interacts with two other passenger
proteins, Aurora B kinase and inner centromere protein
INCENP in vitro (Wheatley et al., 2001; Honda et al., 2003)
and in vivo (Bolton et al., 2002). Recently published data
suggest that vertebrate Aurora B and its yeast homolog Ipl1
during mitosis. Photobleaching experiments revealed
marked changes in rates of survivin turnover at
centromeres. These were regulated by stage of the cell
cycle, microtubule attachment, and Aurora B kinase
activity. We hypothesize that changes in the turnover
of survivin at centromeres influence the stability of
kinetochore-microtubule attachment and signaling of the
spindle checkpoint.
Movies available online
Key words: Survivin, Microtubules, Mitosis, Spindle checkpoint,
FRAP
are required for the release of inappropriate microtubulekinetochore interactions (Biggins and Murray, 2001; Tanaka et
al., 2002; Kallio et al., 2002b). Vertebrate Aurora B also has a
distinct role in direct signaling of the spindle checkpoint
(Kallio et al., 2002b). Cells in which survivin, Aurora B,
or INCENP are perturbed exhibit phenotypic similarities
suggesting that these proteins function in common pathways
(Uren et al., 2000; Adams et al., 2001a). Moreover, deletion of
one member from the complex results in mislocalization and
malfunction of the other two proteins (Speliotes et al., 2000;
Adams et al., 2001b; Wheatley et al., 2001).
Many of the regulatory proteins implicated in the control of
cell division associate with centromeres and centrosomes,
often at specific times in the cell cycle (reviewed by Musacchio
and Hardwick, 2002). To understand the mitotic functions of
survivin we examined dynamics of a survivin-GFP chimera at
different subcellular locations and at different cell-cycle phases
in HeLa and LLC-PK cells using high-resolution fluorescence
microscopy and fluorescence recovery after photobleaching
(FRAP).
Materials and Methods
Materials and cell culture
All chemicals were purchased from Sigma and tissue culture reagents
4034
Journal of Cell Science 117 (18)
from Gibco unless stated otherwise. Cell culture was performed as
described previously (Kallio et al., 2002b). For some experiments cells
were treated with nocodazole (8 µM) or taxol (3 µM) from 15 minutes
to 14 hours or with 2 µM ZM-447439 (a kind gift from Astra-Zeneca),
a small molecule inhibitor of Aurora B (Ditchfield et al., 2003), for 1060 minutes in the presence of 20 µM MG132, a proteasome inhibitor.
In order to synchronize cells at S phase, aphidicolin was used at 1
µg/ml for 16 hours after which the cells were released from the arrest
by extensive buffer washes. Cells growing on coverslips were fixed at
hourly time points (0-12 hours) after aphidicolin wash out and were
then processed for immunofluorescence.
Transfection and expression of survivin-GFP
Wild-type survivin was inserted into pEGFPc1 (Clontech
Laboratories). LLC-PK and HeLa cells were transiently transfected
with plasmids encoding full-length survivin-GFP or empty GFP
vector with Fugene 6 Reagent (Roche Diagnostics) according to the
manufacturer’s recommendation. At 24-72 hours after initiation of the
transfection protocol, the cells were fixed and processed for
microscopic analysis, subjected to biochemical studies, or live cell
video analysis. To assess the extent to which expression of survivinGFP increased the total pool of survivin, we compared the transfected
cells with non-transfected cells by quantitative immunofluorescence
with anti-survivin antibody. We measured the average integrated
intensity of total cellular survivin fluorescence from 10 non-tranfected
cells and from 10 transiently transfected cells at mitosis exhibiting a
low level of survivin-GFP fluorescence using the imaging system
described below.
Fixation and immunofluorescence
LLC-PK and HeLa cells expressing survivin-GFP were fixed with
2.5% formaldehyde in PHEM (60 mM Pipes, 25 mM Hepes, pH 6.9,
10 mM EDTA, 4 mM MgCl2) for 15 minutes. After washes with
MBST (10 mM Mops, 150 mM NaCl, pH 7.3, 0.05% Tween 20) DNA
was stained with DAPI and the cells on coverslips were mounted
in Vectashield (Vector Laboratories). For immunofluorescence, cells
on coverslips were extracted before fixation or simultaneously fixed
and extracted for 15 minutes in 0.25% CHAPS in PHEM containing
2.5% formaldehyde. Cells were labeled with antibodies against Crest
centromere marker, survivin, hCdc20, Aurora B, or tubulin as
previously described (Fortugno et al., 2002; Kallio et al., 2002a).
DNA was stained with DAPI. Imaging was performed using a Zeiss
Axioplan II microscope equipped with 63× (N.A. 1.4) and 100×
objectives (N.A. 1.4), a Hamamatsu Orca 2 camera (Hamamatsu
Photonics), and Metamorph imaging software (Universal Imaging).
Western blotting and immunoprecipitation
HeLa cells expressing full-length survivin-GFP or control GFP alone
were treated with nocodazole for 12 hours before harvest of mitotic
cells. Cell extracts and supernatants were prepared and used for
immunoprecipitation as described previously (Kallio et al., 1998). In
brief, for immunoprecipitation 5 µg of polyclonal anti-GFP antibody
(Abcam) was prebound to 25 µl of protein A beads for 2 hours at
+4°C. Immunoprecipitation was performed from 8×105 cells
overnight. For western blotting of HeLa and LLC-PK extracts,
monoclonal anti-survivin antibody 60.II was used at 2 µg/ml. For
western blotting of anti-GFP immunoprecipitations, monoclonal antihuman Aurora B antibody (anti-AIM1, Transduction Labs) was used
at 1 µg/ml.
Analysis of living cells, laser photobleaching and FRAP
For live cell experiments, cells were incubated in phenol red-free
DMEM supplemented with 10 mM Hepes, 10% fetal bovine serum,
and antibiotics (penicillin and streptomycin). The cells were analysed
using a Zeiss Axiovert 200M microscope equipped with 63× (N.A.
1.4) and 100× objectives (N.A. 1.4) and Hamamatsu Orca ER CCD
camera (Hamamatsu Photonics). Images were captured using
Metamorph software (Universal Imaging). The average signal
intensities of chromosome arm (n=5) and centromere (n=8) bound
survivin-GFP at different mitotic phases were analysed from live cell
sequences by measuring the integrated fluorescence intensities minus
the background per sample and time point.
Photobleaching experiments were performed using a Micropoint
Laser System (Phototonic Instruments) affixed to the epi-illumination
port of the microscope. Laser light was attenuated with a neutral
density filter to a level that did not affect cell viability or cell-cycle
progression. The laser was aligned to the target region using phase
contrast optics and a set of pre-bleach images was captured. A short
exposure of laser irradiation (5-10 pulses each 4 nanoseconds) was
used to reduce the fluorescence intensity of the target. Time-lapse
sequences (time intervals ranging from 1 frame/second to 1 frame
every 60 seconds) were captured using 30 to 500 millisecond exposure
times.
Statistical analysis of survivin-GFP dynamics at different
subcellular compartments
Measurement procedures were adapted from those reported
previously (Maddox et al., 2000; Howell et al., 2000; Kallio et al.,
2002a). A small region (Rtar) slightly larger than the target area was
marked and the integrated fluorescence intensity within the region
(Ftar) was logged into an Excel spreadsheet from each time point. The
background fluorescence was subtracted by placing a larger region
(Rback) over the smaller target area. Integrated fluorescence within the
Rback was measured (Fback). The following equation was used to
obtain integrated fluorescence intensity of a kinetochore minus the
background (Fint):
Fint =
Ftar – [(Fback – Ftar) 3 Rtar]
(Rback – Rtar)
.
The half-life of survivin-GFP fluorescence recovery was calculated
using the equation: t1/2=ln2/k. The constant, k, was derived from fitting
the data to a curve generated with the non-linear perturbation
relaxation equation within GraphPad Prism software. The percentage
of fluorescence recovery was calculated using the equation: 100%
[Fmax–F(0)]/[Fpre–F(0)]; where Fpre is the intensity of the region
before laser irradiation, Fmax is the intensity of the same region after
maximal recovery and F(0) is the intensity at the first time point after
photobleaching. Measurements were adjusted to control for any
overall decrease in cellular fluorescence caused by photobleaching or
imaging.
Results
Survivin accumulates near centromeres at early G2 and forms
a bi-partite flexible structure between sister kinetochores by
prophase. The subcellular localization of survivin has remained
controversial, with debate as to whether it is a chromosomal
passenger protein (Skoufias et al., 2000; Uren et al., 2000;
Wheatley et al., 2001), a microtubule-associated protein (Li et
al., 1998) or whether immunochemically distinct pools of
survivin exist (Fortugno et al., 2002). To resolve these conflicts,
we transiently transfected HeLa and LLC-PK cells with a
plasmid encoding full-length survivin-GFP chimera and
examined distribution of both exogenous and endogenous
survivin at different cell-cycle phases. The ectopically
expressed survivin-GFP was present throughout the cell cycle
Survivin dynamics
4035
Fig. 1. Survivin concentrates near centromeres in G2 and forms an extendable bipartite structure between the sister kinetochores in mitosis. (A) Accumulation of
survivin-GFP adjacent to centromeres of early G2 cells. See text for details. The
arrow and arrowheads denote the midbody and the spindle poles, respectively. The
merge shows overlay of survivin-GFP (green) and autoimmune anti-centromere
marker (red). The insets show higher magnification of selected areas. (B) Aurora B
does not co-localize with survivin-GFP foci (thin arrows) until M phase (short
arrows). (C) Survivin (red) undergoes extensive stretching between the sister
kinetochores (anti-hCdc20, green) in a manner dependent on microtubule attachment
and chromosome bipolarity. The arrows indicate a gap separating the two survivin
accumulations on chromosomes that are under robust tension. (a) prophase, (b)
unattached prometaphase, (c) attached prometaphase, (d) metaphase, (e) metaphaseanaphase transition. The numbers show the average length of the survivin segments
under each condition (n=10-20 chromosomes per each category). The three linescan
graphs show the average gray values for the above prometaphase (f), metaphase (g),
and metaphase-anaphase transition (h) chromosomes (red survivin; green kinetochores). (D) Aurora B kinase associates with survivin-GFP in
mitotic HeLa cells. Immunoprecipitations were performed with rabbit anti-GFP antibody utilizing HeLa cell populations expressing either
survivin-GFP or control GFP alone. The arrow denotes a ~42 kDa band detected with anti-hAurora B antibody and the asterisk the antibody
heavy chain at 55 kDa. IP, immunoprecipitate; S, supernatant. Bars = 10 µm (panels A,B) and 0.5 µm (panel C).
but its subcellular distribution appeared to be cell cycle
regulated. We investigated cell-cycle-dependent changes in
survivin localization. In G1 cells, identified by the presence of
a remnant midbody from the previous cytokinesis, most of
survivin-GFP was diffusely distributed in the cytosol and to a
lesser extent in the nucleus (Fig. 1A). We synchronized HeLa
cells expressing survivin-GFP at S phase by treatment with
aphidicolin and tracked changes in survivin localization with
progression through the cell cycle after release from the block.
In early S phase (Fig. 1A, time point 0 hours) the majority of
survivin-GFP was found within the nucleus where it remained
diffuse but was excluded from the nucleoli. Three hours after
the release from S phase block, faint traces of survivin-GFPpositive accumulations were observed in the nucleus, some of
which were adjacent to centromeres detected with human
autoimmune sera (Fig. 1A). Two hours later, all of the survivinGFP foci were in close proximity to centromeres indicating the
initial accumulation of the protein near the pre-kinetochores
of G2 cells (Fig. 1A). This result was confirmed by
immunofluorescence in HeLa (data not shown) and LLC-PK
cells utilizing monoclonal anti-survivin antibody (Fig. 2). In
prophase cells, survivin-GFP was found diffusely distributed
4036
Journal of Cell Science 117 (18)
in the cytosol and nucleus excluding the nucleoli, and
accumulated at centromeres with some label at chromosome
arms, especially during late prophase (Fig. 1A; time point +10
hours). After nuclear envelope breakdown (NEB), survivin
accumulated further at inner centromeres and chromosome
arms, and to some extent along the spindle microtubules (Fig.
1A; time point +11 hours, Fig. 2).
The other two chromosome passenger proteins, INCENP
and Aurora B, have not been reported to concentrate at
centromeres until prophase in Xenopus and porcine tissue
culture cells (Kallio et al., 2002b; MacKay et al., 1998)
although both proteins are found diffusely distributed in late
G2 nuclei. To examine the distribution of survivin and Aurora
B in human cells we synchronized HeLa cells expressing
survivin-GFP at S phase. The cells were fixed and processed
for immunofluorescence at 0, 5, 7, 9 and 10 hours after release
from the S phase block. No Aurora B was found in interphase
cells at time points between 5-10 hours (Fig. 1B). Only in M
phase cells did Aurora B co-localize with survivin-GFP at the
inner centromere (Fig. 1B) consistent with the idea that
survivin might recruit Aurora B to mitotic chromosomes.
In fixed mitotic HeLa and LLC-PK cells immunolabeled with
anti-survivin antibodies or expressing survivin-GFP, we noticed
that survivin accumulated in an oval-shaped region between the
sister kinetochores. This region changed morphology as
microtubules attached to the kinetochores and chromosomes
became bipolarly oriented. Owing to the larger size of porcine
chromosomes, we used LLC-PK cells for more detailed
analyses. We measured the average length of survivin labeling
between sister kinetochores at different stages of cell division
and in chromosomes that were either under tension from bipolar
attachment or were in a relaxed state (Fig. 1C). In prophase
cells, the length of survivin labeling between sister kinetochores
was relatively short (0.45±0.1 µm, n=20) and showed little
variation among different chromosomes. In prometaphase cells,
the length of survivin labeling varied considerably depending
on chromosome attachment and orientation. In unattached or
mono-oriented chromosomes the average length of survivin
signal was 0.3 µm (Fig. 1C panel b, n=10). In bi-oriented
chromosomes the distance increased to 0.79±0.13 µm (n=20,
Fig. 1C panel c). At metaphase, survivin showed an average
length of 1.01±0.16 µm (n=20, Fig. 1C panel d). In this highly
extended state the survivin at each centromere formed two
accumulations separated by a ~0.4 µm gap. At the metaphaseanaphase transition, this gap further extended to a width of ~0.7
µm and survivin showed its most lengthened arrangement
(1.77±0.31 µm, n=10, Fig. 1C panel e).
To determine whether survivin-GFP is incorporated
into the chromosome passenger protein complex, we
immunoprecipitated GFP-associated proteins from mitotic
HeLa cells expressing either full-length survivin-GFP or
control GFP alone. We found that survivin-GFP but not GFP
co-precipitated with a portion of the Aurora B kinase in the
cell extracts (Fig. 1D). This is in accordance with previously
published data (Wheatley et al., 2001).
From the localization data we conclude that among
those proteins that are not constitutive components of the
centromeres, survivin is one of the earliest G2/M phase
proteins to concentrate at the maturing centromere-kinetochore
complexes of interphase cells at early G2. Moreover, during
mitosis survivin forms two separate accumulations between the
Fig. 2. Indirect immunolocalization of endogenous survivin in LLCPK cells. (A) Survivin accumulates near the centromeres at G2 phase
(arrows). (B) In late prophase cells, survivin is between the sister
kinetochores (arrows). (C) During prometaphase, survivin changes
its morphology as microtubules attach to the kinetochores and
chromosomes become bipolarly oriented (the short arrow shows a
‘relaxed’ chromosome and the long arrow denotes a chromosome
under tension from both spindle poles). (D) Metaphase. (E) Early
anaphase. (F) Anaphase B. The arrowheads denote survivin strands
at the central spindle. (G) In telophase cells, survivin concentrates at
the cleavage furrow (arrowheads) before final accumulation at the
midbody (arrow). Merge images of survivin (red, detected with the
monoclonal anti-survivin antibody), kinetochores (green, detected
with anti-hCdc20 antibodies or Crest anti-centromere serum), and
DNA (blue, stained with DAPI) are shown. (H) The specificity of
monoclonal anti-survivin antibody. A single band corresponding to a
16 kDa protein was detected in interphase (I) and mitotic (M, 12 h
taxol treatment) HeLa and LLC-PK cells. Bars, 5 µm.
sister kinetochores, revealing that the inner centromere, like the
outer centromere and kinetochore, is arranged in a bi-partite
fashion. Survivin accumulations undergo notable stretching
after microtubule attachment and interkinetochore tension. We
speculate that changes in centromere structure upon
microtubule attachment and interkinetochore tension are
important in regulating structural arrangement of survivin and
its association with other proteins at inner centromeres. These
effects on survivin might, in turn, regulate the activities of
centromere-associated Aurora B kinase.
Dynamic redistribution of survivin during mitosis
To investigate the effects of survivin-GFP expression on cell
Survivin dynamics
4037
Fig. 3. Redistribution of survivin-GFP during cell division. (A) Timelapse sequence of an LLC-PK cell expressing survivin-GFP during
prophase, nuclear envelope breakdown (NEB) and prometaphase. The
amount of survivin-GFP at the inner centromeres increases
substantially around NEB (time point 0 minutes) and shows further
accumulation during prometaphase. The white circle denotes a
centromere that remained in focus throughout the observation period.
In contrast to inner centromere labeling, the intensity of chromosome arm labeling diminishes during prometaphase after peaking at NEB. A
video corresponding to the still images is available as supplementary information (see Movie 1, http://jcs.biologists.org/supplemental/).
(B) Changes in the average integrated fluorescence of chromosome arm (j, n=5) and inner centromere localized survivin-GFP (n, n=8) from
late prophase to onset of anaphase. (C) Co-localization of survivin-GFP with the spindle microtubules in anaphase cells is lost by early
telophase when survivin-GFP forms separate filamentous accumulations (arrows) that run between the microtubules of the central spindle. The
area inside the white boxes is shown in higher magnification. mts, microtubules; surv, survivin-GFP. The merge shows an overlay of DNA
(blue), survivin-GFP (green), and spindle microtubules (red). Bars = 10 µm (panel A) and 3 µm (panel C).
division we transiently transfected populations of HeLa cells
and monitored their progression through mitosis using phase
contrast and fluorescence time-lapse microscopy (data not
shown). We selected transfected cells exhibiting a low level of
GFP fluorescence for analysis. Transfected cells in mitosis
showed an average increase in survivin expression of 19.9%
(n=10) compared with endogenous survivin levels in nontransfected cells (n=10). A slight cell-cycle delay was observed
at metaphase where survivin-GFP transfected cells (n=30)
remained for an average of 43±39 minutes while GFP
transfected cells remained for 22±15 minutes (n=15).
To characterize the cell-cycle-regulated changes in
accumulation of survivin, we quantified the changes in
accumulation of survivin-GFP at different stages of mitosis.
Survivin first concentrates at chromosome arms and at
centromeres during late prophase. We measured the intensities
of the survivin-GFP pools at chromosome arms (n=5) and at
inner centromeres (n=8) in living LLC-PK cells as they
progressed from late prophase to early anaphase (Fig. 3A,B
and Movie 1, http://jcs.biologists.org/ supplemental/).
The average fluorescence intensity of survivin-GFP at both
the chromosome arm and the inner centromere increased
noticeably 2-3 minutes before NEB. Survivin at chromosome
arms achieved its maximum concentration ~2 minutes after
NEB and then faint traces diminished gradually to near
background levels during prometaphase (Fig. 3B, P<0.01). In
contrast, the average fluorescence intensity of survivin-GFP at
inner centromeres increased markedly through prometaphase
(P<0.01) and reached its maximum level at metaphase (Fig.
3A,B).
Another change in survivin distribution occurs at early
anaphase. Previous immunofluorescent studies suggest that the
protein moves from chromosomes to microtubules of the
central spindle at early anaphase and then concentrates to the
midbody at late telophase (Skoufias et al., 2000; Uren et al.,
2000; Wheatley et al., 2001). In contrast, recent live cell
experiments proposed a microtubule-independent translocation
of a survivin-dsRed chimera from chromosomes to the cytosol
at anaphase and then to the ends of polar microtubules by
early telophase (Temme et al., 2003). To investigate this, we
4038
Journal of Cell Science 117 (18)
Fig. 4. The turnover rate of survivin peaks at early M phase and is partially dependent on microtubulekinetochore attachment. (A-G) Target areas (indicated with white circles) in HeLa cells (panel A) or
LLC-PK cells (panels B-G) were photo-bleached and followed by fluorescence time-lapse microscopy. Pre-bleach and post-bleach images
representing partial and maximal recovery are shown. The insets show higher magnification views of the target area. At the end of each row are
corresponding graphs of survivin-GFP recovery (arrows indicate the pre-bleach fluorescence level of the target area). Proportions of
fluorescence recovery (recf) and half time of recovery (t1/2) are shown for each graph, as percentages. Supplemental Movies 2-6 corresponding
to the still images of panels A, B, C, E and F, respectively, are available online (http://jcs.biologists.org/supplemental/).
analyzed the distribution of endogenous survivin and survivinGFP in fixed HeLa and LLC-PK cells immunolabeled with
anti-tubulin antibodies. In these cells, survivin co-localized
with microtubules of the central spindle from early stages of
anaphase A to the start of anaphase B (Fig. 2E,F, Fig. 3C).
However, later in anaphase the accumulations of survivin in the
central spindle were displaced from microtubules and relocated
to narrow 3-6 µm long strands between the microtubule
bundles before finally co-localizing with the dense midbody in
late telophase (Fig. 2G, Fig. 3C). We conclude that survivin
Survivin dynamics
4039
Table 1. Dynamics of survivin at different cell cycle phases and subcellular locations in HeLa and LLC-PK cells
Cell cycle phase/location
Treatment
n
t1/2 (seconds)*
% Rec*
HeLa
G2 phase/centromere
Prometaphase/inner centromere
Prometaphase/inner centromere
Telophase/midbody
–
–
Nocodazole
–
5
10
10
5
1860±600‡
7.2±4.2
31.3±9.8
144±42
84±11
79±8
71±10
9±5
LLC-PK
G2 phase/centromere
Prophase/inner centromere
Prophase/chromosome arm
Metaphase/inner centromere
Prometaphase/inner centromere
Prometaphase/inner centromere
Prometaphase/inner centromere
Prometaphase/inner centromere
Metaphase/inner centromere
Anaphase/central spindle
Telophase/midbody
–
–
–
–
–
Nocodazole
ZM447439/MG132
MG132
Taxol
–
–
5
7
5
10
5
10
8
5
10
3
5
2940±480‡
7.2±4.4
6.8±3.1
5.3±3.5
4.6±1.1
74.2±18.1§
67.1±27.2
4.4±2.1
10.3±8.0
87±23
204±108¶
89±12
92±9
82±9
93±8
79±8
77±11
18±13
83±8
78±9
8±4
11±3
*Mean ± s.d.; ‡significantly (P<0.01) different from mitotic cells; §significantly (P<0.01) different from untreated and taxol-treated cells; ¶significantly
(P<0.01) different from interphase and early mitotic cells.
populations concentrated at chromosome arms and inner
centromeres experience dynamic changes in affinity during
early stages of mitosis resulting in reduction in chromosome
arm labeling and accumulation of the protein to inner
centromeres.
Turnover of inner centromere-bound survivin is cell-cycle
regulated
Changes in survivin concentration at mitotic organelles may be
related to changes in dynamic protein-protein interactions
such as the interaction between the passenger proteins. To
study survivin dynamics more directly we performed FRAP
experiments at different phases of cell cycle on HeLa and LLCPK cells expressing survivin-GFP. Our pull-down assays (Fig.
1D) and in vitro binding experiments performed by others
(Wheatley et al., 2001) suggest that tagging survivin with GFP
might affect the ability of the protein to interact fully with
Aurora B and INCENP. It is possible that less survivin-GFP
is in a complex with Aurora B and INCENP compared with
endogenous survivin in vivo. Thus our results regarding the
mobility of survivin-GFP might reflect to some extent the
behavior of unbound survivin. As a counterpoint, the observed
subcellular localization and dynamic translocation of survivinGFP are indistinguishable from those of the endogenous
protein. For this reason we believe that the behavior of
survivin-GFP reflects that of the endogenous protein in vivo.
In interphase cells at G2, we targeted the initial survivinGFP accumulation near the centromeres. The turnover was
slow at this point in both HeLa and LLC-PK cells showing
average half time for recovery of 1860±600 seconds and
2940±480 seconds, respectively (Fig. 4A, Table 1 and Movie
2, http://jcs.biologists.org/supplemental/). The total recovery
of fluorescence was high in both HeLa (84±11%) and LLC-PK
(89±12%) cells. After entry into M phase, the turnover rate of
survivin at inner centromeres increased sharply (P<0.01) in
both cell types investigated. We observed no significant
differences in the average recovery half times between
prophase, prometaphase and metaphase centromeres. Both cell
lines showed the same range of 3-12 seconds turnover at late
prophase and metaphase (Fig. 4B,C, Table 1, and Movies 3 and
4). The average turnover rates and the total recovery of
survivin-GFP fluorescence at chromosome arms were similar
to those of inner centromeres (Table 1). After the onset of
anaphase, survivin turnover in the central spindle (Fig. 4D) and
midbody became slow and limited in extent (Fig. 4E, Table 1,
and Movie 5). In conclusion, survivin turns over slowly near
centromeres of interphase cells, becomes highly dynamic on
chromosome arms and at centromeres during early phases of
mitosis, and becomes more stably associated with the central
spindle after the onset of anaphase.
Survivin dynamics at inner centromeres is partially
regulated by microtubule attachment
Because microtubule attachment and mechanical tension at
kinetochores regulate the spindle checkpoint and progression
through M phase, we sought to determine whether microtubule
attachment affects survivin dynamics at centromeres. We
treated survivin-GFP cells with nocodazole, which destabilizes
microtubules eliminating microtubule attachment and tension
or with taxol, a microtubule hyperstabilizing drug, which
maintains many microtubule-kinetochore attachments but
reduces inter-kinetochore tension. We then measured survivin
turnover using FRAP. Cells treated with nocodazole showed
significantly (P<0.01) slower survivin turnover compared with
that of normal cells or of cells treated with taxol in both cell
lines examined (Fig. 4F, Table 1). The average recovery half
time of survivin fluorescence was seven times longer
(74.2±18.1 seconds) in nocodazole treated LLC-PK cells (see
Movie 6, http://jcs.biologists.org/supplemental/) compared
with taxol-treated cells (10.3±8.0 seconds, Fig. 4G, Table 1).
These results are consistent with the notion that survivin
turnover at inner centromeres of prometaphase cells is
increased by microtubule occupancy at kinetochores but is
not markedly affected by tension. We recognize that
overexpression of survivin or the use of a chimeric protein
might influence survivin turnover. However, the consistent
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Journal of Cell Science 117 (18)
Fig. 5. Aurora B regulates survivin turnover at inner centromeres. (Top) FRAP analysis in an LLC-PK cell shows that survivin-GFP turnover at
metaphase centromeres occurs at its normally rapid rate in a cell treated with the proteaseome inhibitor MG132. (Bottom) In a cell pretreated
for 30 minutes with MG132 then treated for 10 minutes with ZM447439 in the continued presence of MG132, survivin-GFP at the inner
centromeres shows slow and limited turnover. Pre-bleach and post-bleach images representing partial and maximal recovery are shown. The
white circles denote the target area. The graphs show survivin-GFP recovery (arrows indicates the pre-bleach fluorescence level of the target
area). Proportion of fluorescence recovery (recf) and half time of recovery (t1/2) are shown as percentages. Movie 7 corresponding to the still
images of panel B is available online (http://jcs.biologists.org/supplemental/).
changes in dynamics seen with cell-cycle progression and
microtubule attachment suggest that the observations reflect
physiologically significant regulation of the survivin protein.
Aurora B kinase activity is required for maintenance of
survivin dynamics at inner centromeres
Aurora B has been proposed to modulate microtubulekinetochore interactions to promote proper bi-orientation of
chromosomes. The absence of normal Aurora B function leads
to defects in chromosome movement and alignment (Kallio et
al., 2002b; Ditchfield et al., 2003; Hauf et al., 2003). Activity
of Aurora B is also essential for spindle checkpoint signaling
as perturbation of its functions leads to rapid exit from M phase
even in presence of unaligned chromosomes (Kallio et al.,
2002b). To investigate how Aurora B kinase activity affects
survivin dynamics we applied ZM447439, a small molecule
inhibitor of Aurora B (Ditchfield et al., 2003), to LLC-PK cells
expressing survivin-GFP and performed time-lapse and FRAP
analysis. ZM447439 has been reported to inhibit Aurora A and
B with IC50 values of 110 and 130 nM, respectively, while the
majority of other protein kinases are not affected by the drug
(Ditchfield et al., 2003). We cannot formally exclude that
effects of ZM447439 on survivin behavior are due, at least
in part, to consequences of inhibition of other kinases,
particularly other Aurora kinases. However, ZM447439treated cells exhibit a very similar phenotype to Aurora B
RNAi- (Hauf et al., 2003; Adams et al., 2001b) but not to
Aurora A RNAi- (Hirota et al., 2003; Giet et al., 2002; Hannak
et al., 2001) treated cells. In our assays, we first added MG132,
a drug that blocks the onset of anaphase by inhibiting
proteasome activity, to prevent cells from exiting M phase
prematurely due to the inhibitory effects of ZM447439 on the
spindle checkpoint. In the presence of MG132 alone survivinGFP fluorescence and its average recovery half time at inner
centromeres was unaltered compared with untreated metaphase
cells (Table 1, Fig. 5). In contrast, in cells that were incubated
in 2 µM ZM447439 in the continued presence MG132,
survivin-GFP became stably associated with centromeres
showing very slow and limited recovery. The total recovery
was only 18±13%, and the average turnover time to achieve
this low extent of recovery was 67±27 seconds (Fig. 5,
Table 1 and Movie 7, http://jcs.biologists.org/supplemental/).
Consistent with previous observations (Ditchfield et al., 2003;
Hauf et al., 2003) we noticed that concentration of survivin was
lost from inner centromeres within 30 minutes after addition
of ZM447439 and MG132 (data not shown). These results are
consistent with a model in which the recruitment of survivin
to inner centromeres is dependent on Aurora B kinase activity.
Discussion
We extend previous observations of survivin behavior as a
chromosomal passenger protein that exhibits dynamic changes
in its localization, and turnover in a cell-cycle-dependent
manner. The ~20-fold increase in the turnover of survivin
at inner centromeres of prophase and prometaphase cells
compared with interphase and anaphase cells suggests an
active role for survivin dynamics in regulation of early mitotic
events. We hypothesize that changes in survivin dynamics at
centromeres might regulate Aurora B kinase activity, which,
in turn, governs kinetochore-microtubule interactions and
signaling of the spindle checkpoint. Rapid modulation of
Aurora B might be required for release of inappropriate
microtubule connections.
There is controversy about whether the spindle checkpoint is
initiated at kinetochores in response to the loss of microtubule
occupancy, the loss of mechanical tension, or both (reviewed by
Millband et al., 2002). We detect a slower turnover of survivin
in nocodazole-treated cells lacking microtubule-kinetochore
interactions compared with cells treated with taxol expected to
have a normal or near-normal number of microtubules bound to
kinetochores (Waters et al., 1996; McEwen et al., 1997) and
reduced tension (Waters et al., 1998). We propose that the
Survivin dynamics
longer residence time of survivin at the centromeres of
chromosomes lacking microtubule-kinetochore associations
increases Aurora B kinase activity and results in stronger
activation of the spindle checkpoint. This model is consistent
with the reports that small molecule inhibitors of Aurora kinases
more readily override the M phase arrest induced in mammalian
cells by taxol compared with the arrest induced by nocodazole
or other microtubule destabilizers (Ditchfield et al., 2003; Hauf
et al., 2003). Thus we suggest that one mechanism for dynamic
regulation of Aurora B kinase is modulation of the residence
time of survivin at centromeres, influenced by microtubule
occupancy at the kinetochores. We do not exclude the
possibility that mechanical tension might provide an additional
mechanism for regulating Aurora B activity.
Suppression of Aurora B activity by addition of a small
molecule inhibitor ZM447439 to the cells caused
hyperstabilation of survivin-GFP at inner centromeres. This
was accompanied with a gradual loss of survivin from this
location even in the presence of a proteasome inhibitor that
prevents cells from entering anaphase. This finding suggests
that Aurora B activity is required for maintenance of proper
survivin dynamics at inner centromeres. Further work will be
required to identify downstream targets of Aurora B kinase that
affect microtubule dynamics and cell-cycle regulation in M
phase.
We are grateful to Dario Altieri who provided anti-survivin
antibodies and survivin-GFP plasmids. We thank Astra-Zeneca for the
ZM447439 drug. This study was supported by grant R01-GM50412
from the National Institute of General Medical Sciences and from the
Presbyterian Health Foundation.
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