Timeless links replication termination to mitotic kinase activation.

Abstract

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

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Timeless Links Replication Termination to Mitotic Kinase
Activation
Jayaraju Dheekollu1, Andreas Wiedmer1, James Hayden1, David Speicher1, Anthony L. Gotter2, Tim
Yen3, Paul M. Lieberman1*
1The Wistar Institute, Philadelphia, Pennsylvania, United States of America, 2Merk Research Laboratories, West Point, Pennsylvania, United States of America, 3Fox Chase
Cancer Center, Philadelphia, Pennsylvania, United States of America
Abstract
The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely
understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin,
and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and
ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with
mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at
a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-
chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic
kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim
was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human
centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation
with termination of DNA replication.
Citation: Dheekollu J, Wiedmer A, Hayden J, Speicher D, Gotter AL, et al. (2011) Timeless Links Replication Termination to Mitotic Kinase Activation. PLoS
ONE 6(5): e19596. doi:10.1371/journal.pone.0019596
Editor: Beth A. Sullivan, Duke University, United States of America
Received January 31, 2011; Accepted April 1, 2011; Published May 6, 2011
Copyright: ? 2011 Dheekollu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding for this study was provided by National Institutes of Health (NIH) RO1 CA093606 to PML and the Wistar Institute Cancer Center Core Grant 5
P30 CA 010815-41. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: ALG is an employee of Merck. PML is a founder of Vironika, LLC. These competing interests do not alter the authors’ adherence to all the
PLoS ONE policies on sharing data and materials relating to this study. The other authors have declared that no competing interests exist.
* E-mail: Lieberman@wistar.org
Introduction
Cell cycle progression is monitored by a series of checkpoint
mechanisms that maintain genome integrity and cell viability [1].
Multiple checkpoint mechanisms have been defined for recogni-
tion and repair of DNA damage during interphase (e.g. G1/S and
intra-S phase checkpoints) and chromosome dynamics during
mitosis (e.g. spindle checkpoints) [2,3,4]. Relatively, little is know
about mechanisms that coordinate the terminal stages of DNA
replication with the entry into and progression through mitosis.
Completion of normal DNA synthesis involves post-replication
repair of small replication errors by translesional DNA polymer-
ases and rescue of collapsed replication forks and double strand
breaks by homologous recombination between sister chromatids.
Many of these events are monitored by the ATM-Chk2 and ATR-
Chk1 DNA damage checkpoint pathways, which regulate
replication fork progression by directly modifying factors like
PCNA and MCM subunits [2,5,6,7]. Progression through G2 and
mitosis requires the activation of a family of mitotic entry kinases,
which include cyclin-dependent kinase 1 (CDK1), Polo-like kinase
1 (Plk1), and Aurora kinases [1,8]. While these mitotic kinases can
be inhibited by DNA damage checkpoint kinases, the mechanisms
that promote their activation in response to normal termination of
DNA replication are not well characterized.
Completion of S phase requires the processing of numerous
replication structures including those formed by converging
replication forks and replication fork barriers [9,10,11,12]. Studies
from yeast and other model organisms have identified a set of
factors that regulate and monitor DNA replication during
termination or fork pausing [9,12,13]. The Swi1–Swi3 complex
from S. pombe has been implicated in recombination structure
formation at termination sites and programmed pause sites for
DNA polymerase [11,14]. The programmed pause sites at the
mating type switch locus and the ribosomal DNA repeats promote
recombination. Subsequent studies have shown that Swi1–Swi3
travel with the replication fork during S phase and prevent the
separation of the leading and lagging strand polymerases [15]. The
S. cerevisiae orthologues of Swi1–Swi3, Tof1-Csm3, have been
isolated in a stable replication pausing complex [16]. Genetic
analysis of Tof1-Csm3, as well as Swi1–Swi3, have also been
implicated in sister-chromatid cohesion [17]. It is not yet known
how these replication fork protection factors promote sister-
chromatid cohesion, an event associated with late G2 and early M
phase, nor how replication pausing is coupled to homologous
recombination (reviewed in [18]).
Timeless (Tim) and Tipin have been identified as the
mammalian orthologues of Swi1 and Swi3, respectively [19]. Like
their yeast counterparts, Tim and Tipin function in replication
fork protection and genome stability [19,20,21,22,23]. Tim and
Tipin form a stable complex that also includes Claspin, the
mammalian orthologue of the mediator of replication checkpoint
(Mrc1). Claspin is required for Chk1 and ATR activation during
replication fork arrest [20,24,25]. In one study, depletion of Tim
or Tipin resulted in reduced protein levels and cytoplasmic
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relocalization of Claspin [23]. Tim and Tipin associate with
components of the replication fork, including Pold, Pole, and
multiple MCM subunits [21,22,26]. In mouse embryo fibroblasts,
shRNA depletion of Tim produced a decrease in replication fork
progression and elevated sister chromatid exchanges, presumably
as a consequence of the increase in single strand DNA formation
and chromatid breaks [27]. These studies establish that Tim-
Tipin-Claspin function together as components of the mammalian
and yeast replisome that are required for maintaining replication
fork stability at programmed pause sites and during conditions of
DNA damage.
The entry into mitosis following DNA replication is regulated
through the interplay of CDK1-Cyclin B1, Plk1, and Aurora
kinases [28]. CDK1 is activated by the dual specificity phosphatase
CDC25 [29]. CDC25 activity can be amplified by a CDK1-
dependent interaction with Plk1 [30]. Plk1 can be activated by
Aurora A in G2, and this is controlled by the interaction of Aurora
A kinase with one of several regulatory proteins, including TPX2,
Ajuba, PAK1, Hef1, and hBora [8]. Plk1 can also interact with
components of the replication fork, including MCM7 [31] and
DDK [32]. The Xenopus paralogue, Plx, can bind and
phosphorylate Claspin during adaptation response to DNA
damage [33] and is required for chromosome DNA replication
especially under conditions of stress associated with DNA
polymerase inhibitor aphidicolin [34]. In a more recent study,
Plk1 was implicated in a G2 DNA damage response checkpoint
required for the stabilization of Claspin and dependent on the
ubiquitin ligase APC/Ccdhand the phosphatase Cdc14B, a protein
previously implicated in a later cell cycle control step during
mitotic exit [35].
In addition to their functions in regulating mitotic entry and G2
DNA damage checkpoint, Plk and Aurora kinases are also
components of the kinetochore that links the spindle microtubules
to the chromosomal centromere during mitosis [28]. One of the
major functions of Plk1 and Aurora B kinases at centromeres is the
phosphorylation of histone H3 variant CENP-A on serine 7
[36,37]. Aurora B kinase has also been implicated in the
phosphorylation of histone H3 S10 at numerous other chromo-
somal sites during mitosis [38,39]. Furthermore, Aurora B
phosphoryaltion of H3 S10 has been implicated in regulating
pericentric heterochromatin formation, which is essential for
centromere function during DNA replication, sister-chromatid
attachment, and kinetochore stability [37]. Centromeres contain
programmed replication fork barriers at the alpha satellite repeats
found in all mammalian centromeres [40]. Fork barriers, like those
found in centromeres and ribosomal DNA repeats, are thought to
promote formation of DNA recombination structures [12]. Yeast
orthologues of Tim and Tipin maintain replication fork stability at
fork barriers, but the precise mechanism of these proteins and the
function of the fork barriers remain enigmatic. In this study, we
show that Tim physically links mitotic entry kinases, Plk1 and
Aurora A, to a replication fork barrier within the centromeric
alpha satellite repeats in human cells. We also show that Tim is
required for the timely activation of mitotic kinases and
progression through mitosis.
Materials and Methods
Cell Culture and Cell Cycle Synchronization
HeLa, 293-T, and HCT116 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum,
glutamax, and antibiotics. For synchronization, cells were arrested
at the G1/S boundary by incubation in the presence of 2.0 mM
thymidine for 14–16 hrs twice with a 10 hr interval of growth
without the drug. After the second thymidine arrest, cells were
released into fresh DMEM media and harvested at the indicated
time intervals.
Antibodies
Rabbit anti-Tipin antibody and guinea pig anti-Tim antibody
were raised against human peptides and described previously
[19,21]. All antibodies were affinity-purified. Antibodies from
commercial sources were as follows: CDC2, PCNA, RPA34,
Cyclin B1, CDC25C (Santa Cruz Biotechnology); a-tubulin and
FLAG M2 (Sigma); c H2AX, CENPA p7 (Cell Signaling
Technology); Rabit-Tim (Bethyl Laboratories); Plk (Invitrogen);
H3PhosphoSer10 (Millipore); AuroraA, AuroraB, Cdh1, SMC5,
SMC6, MCM2, MCM3, MCM5, MCM7, BubR1 (Abcam).
siRNAs and Transfection
Transfection of small interfering RNA (siRNA) duplexes was
conducted by using Oligofectamine (Dharmacon, Inc), following
manufacturers specifications. All siRNA oligonucleotides were
purchased from Dharmacon. siRNA for Tim is 59-GUAGCUUA-
GUCCUUUCAAAdTdT-39. The sequences of control siRNA
and on-target smart pool siRNA for timeless were reported in
Dharmacon.
Metaphase spreads
Cells were transfected with siRNAs and after 48 hr post-
transfection, cells were treated with colcemid for 2.5 hrs. Cells
were collected and metaphase spreads were prepared as described
[41].
Flag purification
Stable cells were made by co-transfecting 293T cells with
pCMV-Flag-Timeless and puromycin resistant plasmids. Flag
purification and peptide elution has been described previously
[42].
Immunoprecipitation
Cells were extracted with lysis buffer (20 mM Tris-HCL,
pH 7.4, 1 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2, 150 mM
NaCl, 1 mM Na3VO4, 1 mM NaF, 20 mM sodium glycerophos-
phate, 5% glycerol, 0.5% TritonX100, 0.5% Nonidet P-40, 16
protease inhibitors (Simga), 16 Phosphatase inhibitors (Sigma), I
mM PMSF and 2 mM NEM). After rotation for 30 min at 4uC,
the lysate was centrifuged for 20 min at 15,0006 g, and the
supernatant was recovered. The cleared extracts were used for
immunoprecipitation with antibodies as indicated.
2d gel electrophoresis
Cells were synchronized and collected as described above. DNA
isolation and 2d gel electrophoresis were performed as described
previously [43]. The membranes were hybridized with32P labeled
D17Z1 a satellite specific probe [40].
Live cell imaging
GFP-H2B expressing Hela cells were transfected with siControl
or siTimeless siRNA and cells were synchronized by double
thymidine block and release. At 6 hr post-release, time-lapsed
microscopy was started with 5 min intervals.
Kinase assay
HCT116 cells were transfected with siControl or siTimeless
siRNA and synchronized by double thymidine block. Cells were
collected 6 hr after release from double thymidine block and lysed
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in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM
EDTA pH 8.0, 0.1 mM EGTA, 0.1% TritonX100, 0.5% NP-40,
1 mM PMSF, 1 mM NaF, 20 mM sodium pyrophosphate,
20 mM sodium glycerophosphate, 1 mM sodium vanadate, 16
proteinase inhibitor cocktail (Sigma) and, 16 phosphatase
inhibitor cocktail (Sigma)) on ice for 30 min with occasionally
agitation. Cell debris was removed by centrifuge at 12,000 g for
5 min at 4uC. Total 50 ug of protein lysate was used for
immunoprecipitation by incubating with 2 mg of rabbit anti-Plk,
anti-AuroraA, anti-AuroraB, and anti- IgG at 4uC for o/n
followed by protein A agarose conjugation at 4uC for 2 hours.
The beads were washed two times with lysis buffer and once with
kinase buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM
DTT and 0.01% NP40). The kinase reaction was performed at
30uC for 30 min in kinase buffer with or without 100 ng histone
H3 (Roche) as substrate and 10 mCi of32PcATP, 100 mM ATP.
The reaction was stopped by adding 26 Laemmli buffer and
boiled for 5 min before loading on a 4–20% SDS-PAGE. The gel
was then exposed to X-ray film.
FRET Assay
FRET assay to monitor Plk activity in vivo was performed
essentially as described previously [44].
Results
Mitotic kinases physically associate with Tim protein
complex
To investigate the molecular mechanisms through which Tim
regulates replication termination and cell cycle progression, we
generated a stable human cell line expressing FLAG-tagged Tim
protein. FLAG-Tim protein was affinity purified and compared to
a FLAG-vector control cell line for proteins that associate
specifically with Tim (Fig. 1A). Polypeptides enriched in Flag-
Tim samples were excised and analyzed by LC/MS/MS. Among
the most abundant polypeptides were Tim, Tipin, and Claspin,
which was expected from previous studies with mammalian, frog,
and yeast models of Tim (Fig. 1B). We also identified several
mitotic entry kinases, including CDK1, Plk1, Aurora A and
Aurora B as Tim-associated proteins and validated the specific
binding to Tim by Western blot analysis (Fig. 1B). In addition,
several subunits of the replicative helicase (MCM2, 3, 5, and 7),
and the structural maintenance of chromosome proteins (SMC5
and 6) were identified among the Tim-associated proteins by LC/
MS/MS and validated by Western blot (Fig. 1C). Other
replication proteins, including PCNA and RPA34, which had
been shown to interact with Tim in other studies [45], were not
found in our Tim-associated complex, suggesting that Tim may
form multiple and distinct protein complexes (Fig. 1C). Interac-
tions between endogenous Tim and Plk1 protein was demonstrat-
ed by co-immunoprecipitation from Raji nuclear extracts (Fig. S1).
Furthermore, the interaction between Tim and Plk1 was
insensitive to Dnase I treatment, indicating that the binding was
not mediated by non-specific DNA or chromatin linkages (Fig. S1).
To determine if Tim could interact directly with one of the
mitotic kinases, we purified His-tagged Tim protein from
baculovirus and GST-tagged Plk1 from E. coli. We compared
the ability of GST-Plk1 or GST to bind His-Tim or a control
protein His-Rap1 using a GST-pull down assay. We found that
GST-Plk1, but not GST alone could efficiently pull down His-
Tim, but not His-Rap1 (Fig. 1D, top panel). This suggests that
Tim and Plk1 polypeptides can interact independently of other
cellular proteins.
The Tim protein contains two highly conserved domains
spanning the amino terminus (aa 1–270) and the carboxy-terminus
(aa 750–1208). To determine if these domains were important for
interaction with Plk1, we generated a series of deletion mutations
in Tim. Tim deletion mutants were expressed as FLAG-tagged
proteins in 293 cells and assayed for their ability to coIP with
endogenous Plk1 (Fig. 1E). We found that Tim deletions D1–250
and D1000–1208 reduced their capacity to CoIP with Plk1.
Quantification of multiple IP experiments and quantification by
densitometry indicate that the Tim amino terminal domain (D1–
250) is largely responsible for Plk1 interaction (Fig. S2A).
Interestingly, FLAG-Tim (D1–250) and Tim (D250–500) trans-
fected cells had altered cell cycle profiles, suggesting the Tim
amino terminal region is important for both Plk1 binding and
proper execution of cell cycle events (Fig. S2B).
Mitotic colocalization of Tim and Plk1
TodetermineifTimcolocalizedwithPlk1invivo,weusedconfocal
microscopy and indirect immunofluorescence (IF) to analyze the
subcellular localization of endogenous Tim and Plk1 in HeLa cells
(Fig. 2). Since Plk1 has well-established roles in mitosis, we examined
colocalization between Plk1 and Tim throughout the mitotic cell
cycle. In S phase, the nuclear staining of Tim and Plk1 are mostly
diffuse, partially colocalized, with some accumulation at the nuclear
periphery.InG2cells,Plk1andTimconcentrateatthe centrosomes.
Inprophase,Plk1andTimlocalizeto the chromosomecongregation
centers. In prometaphase and metaphase cells, Tim and Plk1
concentrate at the centrosomes, as well as along the microtubule
network linking centrosomes to chromatin. In anaphase, Tim and
Plk1 colocalize strongly at the mid-body, but this colocalization
changes into discrete compartments by telophase and cytokinesis. A
similar pattern of colocalization was also observed between Tim and
Aurora A (Fig. S3). Tim antibody specificity was confirmed by
Western blotting and by IF experiments with Tim-targeted siRNA
depleted cells (Fig. S4). These data indicate that Tim partially
colocalizes with Plk1and AuroraA at multiplestages and subcellular
structures during G2 and mitosis.
Tim depletion causes metaphase chromosome
aberrations and mitotic catastrophe
The function of Tim in cell cycle control and chromosome
maintenance was investigated through siRNA and shRNA depletion.
Two different siRNA and a tet-inducible shRNA stable cell line were
examined for the effects of Timeless depletion in human cell lines.
siRNA depletion of Tim in HCT116 cells caused .90% depletion of
total Tim protein, as measured by Western blot (Fig. 3A). The effect
of siRNA depletion of Tim was first examined by cell cycle profile
using FACS analysis of propidium iodide stained cells, 24 hrs post-
transfection (Fig. 3B). We found that siTim depleted cells
accumulated in G2/M with a corresponding reduction in S and
G1 cells. Live cell imaging using GFP-tagged histone H2B cells
revealed that Tim transfected cells were able to enter mitosis, but
couldnot complete a normal M phase(Movie S1and S2). Analysis of
live cell images revealed several defects during mitosis, including
chromosome condensation defects and formation of lagging
chromosome structures (Fig. 3D and F). To further assess the defects
during M phase, we examined metaphase chromosomes after
colcemid treatment (Fig. 3C). Examination of metaphase spreads
revealed a striking reduction (,20 fold) in sister chromatid cohesion
(Fig. 3E). The failure to generate sister chromatid cohesion was also
observed in a stable 293 cell-line expressing an inducible shRNA that
efficiently depletes Tim (Fig. S5). This is consistent with findings from
Leman et al. showing that chromosome cohesion is compromised in
Tim depleted cells [46]. Bipolar spindle formation and microtubule
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assembly on condensed chromosomes were also disorganized in
siTim-depleted cells(Fig.3G).Althoughchromatincondensed during
metaphase, the formation of stable microtubules emerging from two
opposing poles was largely absent after Tim depletion (Table S1).
Taken together, these data indicate that Tim is required, directly or
indirectly, for multiple mitotic events, including chromosome
condensation, sister chromatid cohesion, bipolar microtubule
organization, and microtubule assembly on condensed chromatin.
Tim Depletion Alters Mitotic Entry Kinase Stability
The interaction of Tim with mitotic entry kinases provoked us
to examine the effect of siTim depletion on the cell cycle behavior
of these proteins. siControl or siTim depleted HCT116 cells were
synchronized in G1 by double thymidine block and release. The
cell cycle profile of these cells indicated that siControl cells pro-
gressed into S phase by 3 hrs, and G2/M by 6 hrs, and G1
reappearing by 9 and 12 hrs (Fig. 4A). siTim depleted cells had a
similar cell cycle profile, with the exception that many fewer cells
exit M phase after 9 and 12 hrs (Fig. 4B). Protein levels for several
cell cycle regulated proteins were examined by Western blot at 3,
6, 9, and 12 hrs after release (Fig. 4C). As expected, Western blot
with anti-Tim revealed a .90% reduction in Tim protein in siTim
transfected cells. Remarkably, we found a significant elevation of
Plk1, Aurora A and Aurora B, as well as Cyclin B1, at 9 and 12 hrs
post-release in the siTim transfected cells. As expected, CDK1
protein levels did not change. This abnormal accumulation in
Figure 1. Proteomic analysis of FLAG-Tim complex. A) FLAG-Tim expressing 293 cell lines was used to purify Tim complex using FLAG-agarose.
FLAG-vector control or FLAG-Tim proteins were visualized by SDS-PAGE and colloidal blue staining. B) Western blot analysis of FLAG purified proteins
from FLAG-Vector or FLAG-Tim expressing cells. Input represents 5% of the starting material from the FLAG-Tim stable cell line. Antibodies for FLAG,
Tipin, Claspin, CDK1, PLK1, Aurora B1, Aurora A1, or Actin are indicated. C) Same as in B, except with antibodies to SMC5, SMC6, MCM2, MCM3, MCM5,
MCM7, PCNA, and RPA34. D) Baculovirus expressed His-Tim or His-Rap1 were assayed for binding to purified GST or GST-Plk1 by GST-pull down assay.
Input and bound proteins were analyzed by Western immunoblot (IB) with anti-His antibody. GST-fusion proteins were visualized by Coomassie
staining of SDS-PAGE gels (lower panel). E) Western blot of immunoprecipitates with cells transfected with FLAG-vector, FLAG-Tim wt, or FLAG-Tim
deletion mutants (as indicated above each lane). Input is indicated. IPs were assayed by Western blot for Plk1 (top panel) or FLAG (lower panel).
Schematic of Tim protein showing that amino and carboxy-terminal conserved domains as pfam04821 and pfam05029, and summary of Plk1 binding
is indicated with purple rectangles.
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cyclin B1 and mitotic entry kinases in siTim treated cells is
consistent with a failure to complete M phase, which occurs in
siControl cells by 9 hrs post-release. We also observed that histone
H3 S10 phosphorylation, which is a mark for mitotic cells, was
significantly delayed in siTim treated cells relative to siControl.
Total histone H3 levels and other cell cycle regulatory proteins
(CDH1, Cdc25) were indistinguishable in the siControl compared
to the siTim depleted cells. Tim depletion did not lead to the
activation of the DNA damage checkpoint kinase Chk1, and only
activated Chk2 kinase at 12 hrs post-release from double-
thymidine block, reflecting a failure to exit mitosis (Fig. S6A).
Tim depletion did not activate mitotic checkpoint protein BubR1,
and prevented the normal activation of BubR1 after nocodazole
treatment [47] (Fig. S6B). These findings indicate that Tim de-
pletion does not induce an intra-S phase or mitotic checkpoint
response in HCT116 cells. Rather, Tim depletion leads to an
aberrant stabilization of the mitotic kinases Plk1, Aurora A and B
in M phase, and a delay in M phase-associated histone H3 S10
phosphorylation.
Cell cycle-dependent interaction of Tim with mitotic
kinases
To determine if the interaction between Tim and mitotic kinases
was cell cycle dependent, we examined the interactions at various
Figure 2. Colocalization of Tim and Plk1 during G2 and M phases. HeLa cells were synchronized by double thymidine block and release, and
then assayed by indirect immunofluorescences with antibodies to Plk1 (red), and Tim (green). DNA is stained with Dapi (blue) and merge images are
shown in the rightmost panel. Cell cycle stages are indicated to the left of each image.
doi:10.1371/journal.pone.0019596.g002
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