MITOTIC PROGRESSION BECOMES IRREVERSIBLE IN PROMETAPHASE AND
COLLAPSES WHEN WEE1 AND CDC25 ARE INHIBITED
Tamara A Potapova1,2, Sushama Sivakumar3, Jennifer N. Flynn1, Rong Li2 and
Gary J Gorbsky1,3
1Cell Cycle and Cancer Biology Research Program,
Oklahoma Medical Research Foundation,
825 NE 13th St, Oklahoma City, OK 73104 USA
2Stowers Institute for Medical Research
1000 E. 50th St. Kansas City MO 64110
3Department of Cell Biology, University of Oklahoma Health Sciences
Center, 1100 N. Lindsay, Oklahoma City, OK 73104
Tamara A. Potapova
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110
phone: (816) 926-4103
fax: (816) 926-4658
Running Head: Positive feedback on Cdk1 drives mitosis
Mitosis requires precise coordination of multiple global reorganizations of
the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary
upstream kinase that directs mitotic progression by phosphorylation of a large
number of substrate proteins. Cdk1 activation reaches the peak level due to
positive feedback mechanisms. By inhibiting Cdk chemically, we showed that in
prometaphase, when Cdk1 substrates
phosphorylation, cells become capable of proper M to G1 transition. We
interfered with the molecular components of the Cdk1-activating feedback
system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25
phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid
Cdk1 activation and morphologically normal mitotic entry, even in the absence of
G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1
inhibition prevented Cdk1/cyclin B kinase activation and full substrate
phosphorylation and induced a mitotic “collapse” - a terminal state characterized
by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This
was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These
findings suggest that the positive feedback in Cdk activation serves to overcome
the activity of Cdk-opposing phosphatases and thus sustains forward progression
The eukaryotic cell cycle is driven by the activities of cyclin-dependent
kinases (Cdks). Cdks belong to a family of heterodimeric Ser/Thr protein kinases,
consisting of two subunits: a catalytic subunit and an activating subunit termed a
cyclin. In budding and fission yeast a single Cdk associates with a number of
cyclins to drive the entire cell cycle. Metazoans express a number of Cdks.
Cdk1, activated by cyclin B, is the primary driver of mitosis, and it phosphorylates
a large number of substrates. In budding yeast, about 200 Cdk1 protein
substrates have been identified; however, the estimated number could be as high
as 500, or roughly 8% of the entire yeast proteome (Ubersax et al., 2003).
Analysis of human proteins associated with the mitotic spindle revealed a total of
more than 700 phosphorylated serine and threonine sites in 260 proteins
(Nousiainen et al., 2006). Most of these phospho-serines and phospho-
threonines were followed by proline residues, suggesting that they are
phosphorylated by Cdk1. Another recent large scale mass spectrometry study
evaluated total protein phosphorylation in mitotic HeLa cells and identified
phosphorylations on more than 3500 proteins (Dephoure et al., 2008). The
majority of these phosphorylation sites fit the Cdk consensus suggesting that all
these proteins may be Cdk1 substrates in human cells. Phosphorylation can
affect proteins in a number of ways; it can activate or inhibit them, alter binding to
other proteins, or change sub-cellular localization.
Cyclin B accumulates and binds to Cdk1 during S and G2 phases of the
cell cycle. However, the Cdk1/cyclin B complex is inhibited by phosphorylation on
inhibitory T14 and Y15 prior to mitotic entry. Two kinases are responsible for the
inhibitory phosphorylation: Wee1 and Myt1. Their action is opposed by a group of
approach the peak of their
dual specificity phosphatases termed Cdc25 phosphatases. In interphase, Wee1
and Myt1 are active, Cdc25 is inactive, and the Cdk activity is low. Wee1, Myt1
and Cdc25 are themselves Cdk1 substrates. Active Cdk1 phosphorylates and
inhibits Wee1 and Myt1 kinases, and phosphorylates and activates the Cdc25
phosphatases. These effects of active Cdk1 on Wee1/Myt1 and on the Cdc25
phosphatases comprise two positive feedback mechanisms, where active Cdk1
inhibits its inhibitors and activates its activator (Supplemental Figure 1A). These
feedback mechanisms can produce rapid autoamplification of Cdk1 activity
(Novak and Tyson, 1993; Tyson and Novak, 2001).
The activity of the Cdk1/cyclin B kinase is high until the mitotic spindle
checkpoint is satisfied, when cyclin B is targeted for degradation by an E3
ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (Glotzer et
al., 1991; Clute and Pines, 1999) associated with its activator Cdc20 (Kallio et al.,
1998). Importantly, active Cdk1 also activates its own inhibitor, the APC/C, by
phosphorylation (Hershko et al., 1994; Lahav-Baratz et al., 1995; Sudakin et al.,
1995; King et al., 1996). However, prior to anaphase onset, the degradation of
most APC/C-Cdc20 substrates is prevented by the mitotic spindle checkpoint.
The spindle checkpoint, which itself requires Cdk activity, prevents initiation of
cyclin B proteolysis until all chromosomes achieve stable bipolar attachment to
the mitotic spindle (Musacchio and Salmon, 2007). Then, the APC/C-Cdc20
inactivates Cdk1 by targeting cyclin B for degradation. In this manner Cdk1
activates its own inhibitor, the APC/C, establishing a negative feedback loop that
turns off Cdk1 allowing the cell to exit mitosis (Supplemental Figure 1).
Turning off Cdk1 allows dephosphorylation of substrates that were
phosphorylated in mitosis, and this dephosphorylation underlies mitotic exit. The
dephosphorylation of mitotic substrates is carried out by serine/threonine
phosphatases, whose identity and regulation are far less explored than that of
kinases. In yeast the primary phosphatase that catalyzes dephosphorylation of
Cdk1 substrates during mitotic exit is Cdc14 (Stegmeier and Amon, 2004). In
higher eukaryotes this role appears to be carried out by PP1 and PP2A
subfamilies of serine/threonine phosphatases (De Wulf et al., 2009). PP1 and
PP2A belong to the PPPs (phosphoprotein phosphatases) family (Andreeva and
Kutuzov, 2001). Members of PPP family are multimeric enzymes: PP1
holoenzymes consist of catalytic, regulatory and sometimes inhibitory subunits,
and PP2A holoenzymes consist of catalytic, scaffolding and regulatory subunits.
While there is little diversity among catalytic subunits, the repertoire of regulatory
subunits is very broad. Different combinations of catalytic and regulatory subunits
generate a large variety of phosphatase holoenzyme complexes. In the past,
phosphatases were often perceived as promiscuous, constitutively active
enzymes. More recent research indicates that at least some phosphatases are
very specific and their activity is tightly regulated, spatially and temporally
(Virshup and Shenolikar, 2009). Currently, much remains to be learned about
specificities and regulation of phosphatase holoenzymes in mitosis, but it is
becoming clear that that phosphatases participate in opposing kinases at all
stages of mitotic progression, from mitotic entry to mitotic exit (Bollen et al.,
Here we show that cells become capable of “forward” (M to G1) mitotic
progression after the prophase stage, in prometaphase and metaphase. In the
course of transition from prophase to prometaphase, phosphorylation of Cdk1
substrates increases sharply, reflecting the spike of Cdk1 activity in the cell.
Hence, cells become committed to forward mitotic around the peak of Cdk1
substrate phosphorylation. Interfering with the positive feedback mechanisms
that mediate rapid and complete activation of Cdk1 causes cells to fail mitosis, a
state we term “mitotic collapse”, where mitotic substrates became
dephosphorylated without cyclin B breakdown. This substrate dephosphorylation
depended on okadaic acid-sensitive phosphatases, suggesting that the biological
purpose of feedback-mediated Cdk activation may be to overcome the activity of
Cdk-opposing phosphatases and to sustain mitosis.
Cells commit to forward M to G1 transition at prometaphase.
APC/C-dependent proteolysis of mitotic regulators is the key element of
the “forward” (M to G1) mitotic transition (Sullivan and Morgan, 2007). To
determine when during mitosis inactivation of Cdk1 results in a “forward”
transition, cells were treated with the chemical Cdk inhibitor Flavopiridol at
different stages of mitosis. Flavopiridol inactivates Cdk1 and triggers rapid mitotic
exit at any point in mitosis. Importantly, Cdk inhibition allows APC/C-Cdc20 to
target its substrates for degradation before the spindle checkpoint is satisfied.
We have previously shown that Flavopiridol triggers degradation of the Cdk1
activator cyclin B in cells arrested in mitosis with nocodazole (Potapova et al.,
2006; Potapova et al., 2009). Depletion of Cdc20 by siRNA confirmed that
normal degradation of cyclin B and securin induced by chemical Cdk1 inhibitor
required normal levels of APC/C-Cdc20 but not APC/C-Cdh1 (Figure 1A)
We defined the point of commitment to “forward” mitotic transition as the
stage when APC/C-Cdc20 becomes proficient to process mitotic substrates in
response to Cdk inhibition. In other words, Cdk inhibitor was used as a tool to
determine when during mitosis APC/C-Cdc20 becomes capable of targeting its
substrates for destruction. We tested the proficiency of the APC/C-Cdc20 to
target endogenous cyclin B on by observing the ability of cells to re-enter mitosis
after washout of Cdk1 inhibitor Flavopiridol. Flavopiridol is a reversible Cdk
inhibitor. When it is washed out after induction of mitotic exit, cells can re-enter
mitosis if cyclin B is preserved (Potapova et al., 2006). However, turning off Cdk
activates Wee1 and Myt1 kinases that inhibit Cdk by phosphorylation. They can
lock Cdk in an inactive state even if cyclin B is preserved (Kapuy et al., 2009;
Potapova et al., 2009). To circumvent this feedback-mediated inhibition, we
treated the cells with PD0166285, a chemical inhibitor of Wee1/Myt1 kinases
(Wang et al., 2001; Li et al., 2002; Hashimoto et al., 2006). In these conditions,
the ability of cells to re-enter mitosis depended solely on the preservation of
cyclin B. Therefore, assaying reversibility gave us a tool to test APC/C-Cdc20
activation during mitotic exit induced by the Cdk inhibitor.
For these experiments, we imaged live Xenopus S3 cells expressing
alpha-tubulin tagged with GFP. Cells were treated with Flavopiridol and
PD0166285 at specific stages of mitosis from prophase to metaphase for one
hour, and then Flavopiridol was washed out. The results, summarized in table
1B, indicated that cells exited mitosis permanently only when Cdk was inhibited
after nuclear envelope breakdown.
If cells were treated with Cdk inhibitor in prophase, mitotic progression
stopped, chromosomes de-condensed, and cells became indistinguishable from
ordinary interphase cells. When Cdk inhibitor was washed out after 1 h, these
cells re-entered mitosis and were capable of normal mitotic progression (Figure
1C and Video 1). This result indicated that the cyclin B in these cells was
preserved. Thus, during prophase cells respond to Cdk1 inhibitor by retreating to
a G2-like state. This finding may be reminiscent of the observations on the
“anthephase checkpoint” – the ability of some cell lines to reversibly undo mitotic
entry when exposed to various stress factors in prophase (Matsusaka and Pines,
2004; Mikhailov et al., 2005)
In contrast when cells were treated with the Cdk inhibitor at any point in
prometaphase or metaphase, they underwent cytokinesis, de-condensed
chromosomes, re-formed nuclear envelopes and established interphase arrays of
microtubules. Washing out the inhibitor 1 hour after its addition did not result in
mitotic re-entry (Figure 1D and Video 2). Lack of mitotic entry was consistent with
the interpretation that that most cyclin B was degraded in these cells. Thus,
during prometaphase or metaphase, cells respond to Cdk1 inhibitor by advancing
to a G1-like state. Overall, Cdk inhibition in prophase results in “backtracking”
from M back to G2, whereas Cdk inhibition after prophase results in forward
The experiments above had the advantage of using endogenous cyclin B
to regulate Cdk1 activity and cell cycle responses, but did not allow us to assess
the dynamics of its degradation directly. To quantify the degradation of cyclin B in
living cells at different stages of mitosis we transfected Hela cells with plasmids
encoding human cyclin B fused to fluorescent proteins. Wild-type human cyclin
B1 fused with GFP was transiently transfected in HeLa cells stably expressing
histone H2B tagged with mCherry. Levels of cyclin B were monitored by time-
lapse fluorescence microscopy.
Cyclin B is cytoplasmic during interphase and rapidly translocates into the
nucleus in prophase (Pines and Hunter, 1991; Clute and Pines, 1999; Hagting et
al., 1999). After nuclear envelope breakdown, cyclin B disperses throughout the
cytoplasm with a propensity to accumulate on the mitotic spindle, chromosomes
and unattached kinetochores (for details see (Bentley et al., 2007)). In normal
cell cycle progression proteolysis of exogenously expressed, fluorescently
tagged cyclin B begins at metaphase, with most cyclin B being degraded before
the onset of anaphase (Clute and Pines, 1999). Consistent with previous reports,
in our experiments, the bulk of cyclin B-GFP disappears shortly before anaphase
onset (Figure 2A and Video 3).
In cells treated with the Cdk inhibitor in prophase, immediately after the
translocation of cyclin B-GFP in the nucleus, cyclin B breakdown was slow and
variable. Upon Flavopiridol addition, the fluorescent intensity of cyclin B1-GFP
decreased very slowly, dropping on average 30 – 35% after 1 hour (Figure 2B
and Video 4). This result supported the conclusion from mitotic reentry
experiments in Xenopus S3 cells that the APC/C-Cdc20 is incompletely
competent to target cyclin B for degradation during prophase. Also, when mitotic
progression stopped and the chromosomes de-condensed after Flavopiridol
addition, cyclin B translocated out of the nucleus in most cases. Our observation
that cyclin B-GFP is exported from the nucleus in response to Cdk inhibition in
prophase agrees with the report by Gavet and Pines, 2010.
In sharp contrast, Cdk inhibition in prometaphase and metaphase cells
resulted in proteolysis of most cyclin B (Figure 2 C and D and Videos 5 and 6).
However, the degradation kinetics varied depending on the stage of mitotic
progression. Metaphase cells degraded most of their cyclin B within 10 minutes
after Cdk inhibition, and most metaphase cells segregated chromatids.
Prometaphase cells degraded cyclin B more slowly, with most of their cyclin B
gone in 30 minutes. Prometaphase cells invariably failed to segregate
chromatids, resulting in chromosomes being trapped within the cleavage furrow –
the “cut” phenotype. Similar results were observed in cells transfected with cyclin
B1 tagged with DsRed (data not shown). These results are consistent with the
interpretation that the APC/C-Cdc20 becomes increasingly more competent for
ubiquitylation of cyclin B with progression through mitosis after prophase.
Together, these data suggest that Cdk inhibition after prophase results in
forward cell cycle progression. However, prometaphase cells exhibited slower
cyclin B breakdown and an inability to segregate chromosomes. This may be
attributed to a failure to fully activate the APC/C-Cdc20. The APC is
phosphorylated in mitosis on multiple sites primarily by Cdk1, but also by Plk1
and possibly other kinases (Steen et al., 2008). The exact functional significance
of each phosphorylation is not known, but replacing some of them with residues
that can not be phosphorylated hinders the catalytic activity of the complex
(Vodermaier and Peters, 2004). The functional studies indicate that the
phosphorylation of APC/C subunits promote binding of Cdc20 (Shteinberg et al.,
1999; Kramer et al., 2000; Rudner and Murray, 2000). Hence, reduction of the
APC/C phosphorylation in mitosis may hinder its ability to process substrates
whose degradation depends on APC/C-Cdc20. The indirect evidence that this
indeed may be the case comes from studies utilizing the Cdk1AF mutant, which
lacks inhibitory phosphorylation sites. Cdk1AF short-circuits the Wee1 and
Cdc25 feedback loops causing Cdk1 activity oscillate rapidly but with lower
amplitude. Importantly, this also leads to reduced APC/C activity (Pomerening et
al., 2005). All this, together with our results led us to hypothesize that the
amplitude of Cdk1 activity is the key determinant for the “forward” directionality of
mitotic progression. We next investigated the dynamics of Cdk activation during
mitotic entry by analyzing the phosphorylation of its substrates.
Cdk1 activity increases sharply during prophase and prometaphase.
It is well established that the activity of Cdk1/cyclin B complex is low in
interphase and high in mitosis, but the direct measurement of Cdk1/cyclin B
activation in intact individual cells has been a challenge. Work in the embryonic
Xenopus egg extract system showed that Cdk1 activation is rapid and complete
in response to the threshold concentration of its activator, cyclin B (Pomerening
et al., 2003; Sha et al., 2003). However, mitotic entry is a continuous process,
and we next explored when and how fast Cdk1 is activated in cells entering
mitosis. We measured the Cdk1 activity in individual cells by quantifying
immunofluorescence labeling of HeLa cells with three antibodies, MPM2, pS-Cdk
and phospho-Nucleolin that bind endogenous mitotic phosphoepitopes. The
fluorescent intensity of antibody labeling was measured at different stages of
mitotic progression - from prophase to metaphase. To precisely define mitotic
stage, cells were co-stained for DNA and Lamin B.
MPM-2 antibody recognizes a large number of proteins that are
phosphorylated in mitosis, predominantly by Cdk1 (Davis et al., 1983). MPM2
antibody stained brightly the nucleus and spindle poles in prophase. After nuclear
envelope breakdown, the labeling dispersed throughout cytoplasm with some
concentration at the mitotic spindle. Quantitative analysis of the integrated
intensity showed that the MPM2 signal sharply increased in prophase, but also
continued to rise during prometaphase (Figure 3A). Representative images are
shown in Supplemental Figure 3A.
Phospho-(Ser) CDKs Substrate antibody (pS-Cdk) is a commercially
available antibody (CellSignaling) that detects phosphorylated serine in a Cdk
substrate motif (K/R)(pS)PX(K/R). pS-Cdk antibody labeled prophase nuclei
similarly to MPM2, and then appeared dispersed throughout the cytoplasm in
prometaphase (Supplemental Figure 3B). Analysis of the pS-Cdk labeling also
indicated a steep rise in intensity during prophase. The fluorescent intensity
continued to increase in prometaphase, when the signal spread throughout the
cytoplasm (Figure 3B).
Phospho-nucleolin antibody recognizes the ribonuclear protein nucleolin at
a site phosphorylated specifically by Cdk1 (Dranovsky et al., 2001). This protein
localizes to the nucleoli of interphase cells and is dispersed throughout
cytoplasm in mitosis, with some concentration of protein enveloping condensed
chromosomes. Phospho-nucleolin antibody exclusively labels mitotic cells, and
co-localizes with the total nucleolin labeling (Supplemental Figure 3). Phospho-
nucleolin labeling serves as a reliable in vivo readout for Cdk1/Cyclin B activity
(Potapova et al., 2006; Potapova et al., 2009). Phosphorylated nucleolin
appeared at detectable levels in the nucleus in early prophase, when
chromosomes begin to condense. The nucleolus disassembles during prophase,
when many of its structural components become phosphorylated (reviewed in
(Boisvert et al., 2007)). Phosphorylation of nucleolin increased sharply and
rapidly, beginning from the onset of nucleoli disassembly in prophase and
continuing even after nucleoli were completely disassembled (for representative
images see Supplemental figure 2C). Similar to the other markers phospho-
nucleolin labeling increased sharply throughout prophase and prometaphase
(Figure 3C). Thus using these markers of endogenous Cdk1 phosphorylation
targets Cdk1 activity rises sharply in prophase and continues to rise after nuclear
From these experiments, we concluded that the bulk of Cdk activation
occurs in pro- and prometaphase. This is generally consistent with the previous
immunofluorescence studies (Lindqvist et al., 2007) and recent FRET analyses
(Gavet and Pines, 2010) As shown in figures 1 and 2, cells become irreversibly
committed to mitosis in prometaphase. Therefore, commitment to mitosis occurs
when the large part of Cdk substrates is phosphorylated (Figure 3B).
Mitotis fails in the absence of positive feedback during Cdk
Next, we investigated the relative importance of the timing of Cdk1/cyclin
B activation versus the feeedback-mediated dynamics of its activation. For this,
we evaluated the mitotic progression in cells entering mitosis prematurely and in
cells where the positive feedback of Cdk1 was reduced.
The Wee1/Myt1 inhibitor PD0166285 abrogates the G2 DNA damage
checkpoint and causes mitotic entry (Wang et al., 2001; Hashimoto et al., 2006).
Applying this drug to the asynchronous cultures of various cell lines led to the
emergence of a large number of mitotic cells. Presumably these were from the
G2 subpopulation. We used the Wee1/Myt1 inhibitor to stimulate premature
mitotic entry at the end of the S phase. For this, HeLa cells were synchronized by
double thymidine block, released, and treated with PD0166285 at the end of S
phase (see the diagram in Figure 4). After release from the second thymidine
block, HeLa cells are in S-phase for about six hours, and the subsequent G2
takes 2-6 hours. Mitotic entry typically begins at about 8 hours after release with
about a half of the cells being in mitosis by 10 hours. Addition of the Wee1/Myt1
inhibitor at the end of the S-phase (6 hours after second thymidine release)
completely overrode the G2 delay and triggered strikingly rapid and massive
mitotic entry (Figure 4A and Video 7). Most cells were able to build normal mitotic
spindles and align chromosomes at the metaphase plate (Figure 4A and 4C).
Anaphase was not visibly perturbed and chromosomes segregated after
complete alignment at the metaphase plate. This suggested that the mitotic
spindle checkpoint and the APC/C were functioning in cells that entered mitosis
Subsequent experiments addressed the ability of cells to progress through
mitosis when the positive dephosphorylation of Cdk1 on inhibitory T14 and Y15
by Cdc25, was compromised. Chemical inhibition of Cdc25 should slow down
activation of Cdk1. To accomplish this, we treated HeLa cells synchronized at the
end of S-phase with the Wee1/Myt1 inhibitor PD0166285 and the Cdc25 inhibitor
NSC663284 (Pu et al., 2002). The simultaneous inhibition of Wee1/Myt1 kinases
and Cdc25 phosphatases blocks both phosphorylation and dephosphorylation of
Cdk1 inhibitory residues. Surprisingly, many of the synchronized cells treated
with combination of Wee1/Myt1 and Cdc25 inhibitors entered prophase at nearly
the same time as cells treated with Wee1/Myt1 inhibitor alone (Figure 4B).
However, cells treated with Wee1/Myt1 and Cdc25 inhibitors remained in
prophase with condensed chromosomes 2-3 times longer than untreated cells or
cells treated with Wee1/Myt1 inhibitor alone. Rounding up of the cells,
characteristic for mitotic entry, was also slower. Most significantly, subsequent
mitotic progression was entirely perturbed. After prophase, cells treated with
Wee1/Myt1 and Cdc25 inhibitors failed to achieve a metaphase chromosome
alignment and did not segregate chromatids or undergo anaphase.
Approximately 1-2 hours later, the chromosomes partially de-condensed but
stayed in the middle of the cell. There was no concurrent blebbing of the cell
membrane or shrinkage of the cytoplasm characteristic of cell death. Most cells
did not flatten down and remained round (Figure 4B and Video 8). Cells
remained in this state for several hours before showing signs of apoptosis such
as membrane blebbing. Based on this morphology and biochemical analyses
reported below we termed this phenotype “mitotic collapse,” meaning an aborted
mitotic entry and failure to progress through mitosis.
In asynchronously growing cell cultures, simultaneous inhibition of
Wee1/Myt1 and Cdc25 also induced mitotic collapse in cells that entered mitosis
20-30 minutes after the addition of both inhibitors. In HeLa cells expressing
fluorescent mCherry - histone H2B and tubulin-GFP, prolonged prophase was
followed by extended prometaphase-like state. Then the mitotic spindle partially
disassembled and chromatin packed around the spindle poles (Supplemental
Figure 5A and Video 9). To rule out the possibility that this phenomenon may be
specific for HeLa cells, similar results were obtained with RPE-1 hTERT cells
stably expressing histone H2B-GFP (Supplemental figure 4B and Video 10).
Treatment with inhibitors did not affect the morphology or viability of cells that
remained in interphase during the experiment.
To examine the mitotic collapse phenotype in more detail, synchronized
HeLa cells were treated with a combination of Wee1/Myt1 and Cdc25 inhibitors
for 90 min and immunolabeled for alpha-tubulin and phospho-S10 histone H3 – a
commonly used early mitotic marker, phosphorylated by the mitotic kinase aurora
B (Wei et al., 1999). The labeling confirmed that the mitotic collapse phenotype
was characterized by a disorganized mitotic spindle and unaligned chromosomes
in most of the cells (Figure 4C). Interestingly, the phospho-histone H3 labeling
was notably reduced in some of these collapsing cells, suggesting that H3 may
be undergoing dephosphorylation.
To further characterize the effects of Wee1/Myt1 and Cdc25 inhibition,
cells were synchronized and treated with inhibitors as in previous experiments,
except that nocodazole was added to the medium to block cells from exiting
mitosis. Samples were collected from 6 to10 hours after second thymidine
release and analyzed by flow cytometry (Figure 5A) and Western blotting (Figure
For flow cytometry analysis, cells were fixed and stained with mitotic
marker antibody against phospho-histone H3 conjugated to CY5 fluorophore. In
untreated cells, mitotic entry began at about 8 hours after the second thymidine
release with more than half the cells entering mitosis by 10 hours (Figure 5A,
blue line). Addition of the Wee1/Myt inhibitor at the end of the S-phase triggered
a rapid increase in mitotic index that remained high throughout the experiment
(Figure 5A, green line). Cdc25 inhibitor by itself prevented mitotic entry (Figure
5A, brown line). When Wee1/Myt1 and the Cdc25 were simultaneously inhibited,
phospho-histone H3 increased during first two hours after the treatment, albeit
more slowly than in cells treated with Wee1/Myt1 inhibitor alone. However, after
two hours, the mitotic index dropped (Figure 5A, orange line). The loss of
phospho-histone H3 labeling indicated that cells co-treated with Wee1/Myt1 and
Cdc25 inhibitors were unable to stay in mitosis in nocodazole. The eventual
dephosphorylations of Cdk1 substrates nucleolin, mitotic phosphoepitopes
MPM2 and pS-Cdk were further confirmed by immunofluorescence experiments
(Supplemental figure 5). In cells that underwent mitotic collapse after treatment
with combination of Wee1/Myt1 and Cdc25 inhibitors (180 minutes after drug
addition), the fluorescent intensities of these markers plunged compared to cells
that remained arrested in mitosis in Wee1/Myt1 inhibitor alone.
This result was perplexing because the active spindle checkpoint triggered
by depolymerized microtubules should have prevented the activation of
APC/C/C-Cdc20 and mitotic exit. Moreover, the mitotic collapse phenotype
observed by live imaging was distinct from ordinary mitotic exit. This prompted us
to explore the mitotic collapse phenotype further by conducting a biochemical
analysis of cell cycle proteins in these cells. Consistent with the flow cytometry
data, Western blotting analysis showed that in cells co-treated with Wee1/Myt1
and Cdc25 inhibitors, phosphorylation of histone H3 was transient, while in cells
not treated with Cdc25 inhibitor it remained high. Nucleolin, a direct Cdk1
substrate, became dephosphorylated similarly to histone H3 (Figure 5B).
When cells treated with Wee1/Myt1 inhibitor but not treated with the
Cdc25 inhibitor were entering mitosis, the inhibitory residues T14 and Y15 on
Cdk1 became dephosphorylated, consistent with the activation of Cdk1. Wee1
and Myt1 acquired electrophoretic mobility shifts characteristic of phosphorylated
and inactive forms of these kinases (McGowan and Russell, 1995; Watanabe et
al., 1995; Booher et al., 1997; Kim et al., 2005). One of the Cdk-activating
phosphatases, Cdc25C, also shifted up, characteristic of its phosphorylated and
active form (Izumi and Maller, 1993; Hoffmann et al., 1994). The APC/C subunit
Cdc27 (APC3) also displayed a shift corresponding to its mitotically
phosphorylated form (Peters et al., 1996). Cyclin B1 levels were increasing
slightly, consistent with its accumulation in G2/M (Cogswell et al., 1995; Hwang
et al., 1995; Piaggio et al., 1995). Cyclin A2 levels dropped as cells accumulated
in mitosis, because cyclin A is targeted for degradation by the APC/C despite the
active mitotic checkpoint (den Elzen and Pines, 2001; Geley et al., 2001).
Because mitotic entry was more rapid and synchronous, these changes were
more pronounced in cells treated with Wee1/Myt1 inhibitor (Figure 5B, green
lanes), than in cells not treated with inhibitor (Figure 5B, blue lanes). .
When Wee1 and Myt1 were inhibited together with Cdc25 (Figure 5B,
orange lanes), inhibitory residues T14 and Y15 of Cdk1 remained
phosphorylated. Some reduction in phosphorylation of T14 and Y15 may be
attributed to incomplete inhibition of Cdc25C by NSC 663284, since this inhibitor
is most potent for Cdc25A (Pu et al., 2002). The weak phosphorylation of mitotic
markers and slight phosphorylation shifts of Wee1, Myt1, Cdc25 and Cdc27 at 1-
2 hours after drug addition in these cells may have been indicative of low Cdk1
activity, high Cdk-opposing phosphatase(s) activity, or both. One of the inhibitors
of Cdk-opposing phosphatases is Greatwall kinase (human MastL). MastL is a
Cdk1/cyclin B substrate, and it undergoes a mitotic phosphorylation shift that
may correspond to its activation (Burgess et al 2010.; Voets and Wolthuis 2010).
A portion of MastL protein showed a phosphorylation shift in cells that entered
mitosis, but not in cells undergoing mitotic collapse. This may hint that in the
absence of feedback-mediated activation of Cdk1, those phosphatases that are
inhibited through MastL remains active.
The most striking result of this experiment was that while mitotic
substrates became dephosphorylated 3-4 hours after the drug addition, cyclins A
and B were not degraded. Therefore, the dephosphorylation of mitotic substrates
in this case was not caused by inactivation of Cdk through proteolysis of cyclins,
as it is in normal mitotic exit. It also was not due to the increase of inhibitory
phosphorylation on Cdk1, because the Wee1 and Myt1 are inhibited by the
PD0166285. In fact, in vitro kinase assays of immunopurified Cdk1/cyclin B1
complex did not show a decrease in kinase activity as its substrate, Nucleolin,
became dephosphorylated (Figure 6A). Importantly, in cells that were already in
mitosis at the time of drug addition, simultaneous inhibition of both Wee1 and
Cdc25 did not cause mitotic substrate dephosphorylation (Figure 6B). Thus the
mitotic collapse phenotype may be interpreted as the inability to sustain mitotic
phosphorylation in the absence of the feedback-amplified activation of Cdk1
during mitotic entry.
The positive feedback loop in Cdk1 activation is required to
overcome Cdk-opposing phosphatases.
The mitotic collapse phenotype, observed in cells treated with both
Wee1/Myt1 and Cdc25 inhibitors, was accompanied by the dephosphorylation of
mitotic substrates but not cyclin proteolysis or Cdk1 inactivation by
phosphorylation. A phosphatase or phosphatases that oppose the action of
mitotic kinases were able to de-phosphorylate their substrates when the positive
feedback on Cdk1 was abrogated. This suggests that there may have been a
balance of phosphorylation and de-phosphorylation reactions that eventually
shifted toward dephosphorylation when the feedback-mediated Cdk activation
was prevented. Therefore, the activation of Cdk1 by positive feedback during
mitotic entry may be required to overcome the activity of Cdk-opposing
To test whether phosphatase activity played a direct role in the mitotic
collapse phenotype, we applied the phosphatase inhibitor, okadaic acid, at 1µM 1
h after the treatment of synchronized cells with Wee1/Myt1 and Cdc25 inhibitors,
before mitotic substrates became dephosphorylated. The addition of okadaic acid
prevented dephosphorylation of nucleolin and histone H3, consistent with the
involvement of PP1- or PP2A-like phosphatases to the mitotic collapse
phenotype (Figure 6C). Importantly, okadaic acid also increased the
phosphorylation of nucleolin, histone H3 and Cdc27 when the levels of
phosporylation of inhibitory Y15 residue of Cdk1 remained steady, providing
evidence for the counterbalance of the kinase and phosphatase activities in
mitosis. Unfortunately, because okadaic acid by itself induces strong
perturbations in cytoplasmic and nuclear morphology unrelated to the cell cycle,
we were not able to assess whether phosphatase inhibition could fully rescue the
mitotic collapse phenotype by morphological criteria.
These results indicated that blocking the activity of phosphatases allowed
mitotic substrates to remain phosphorylated when positive feedback of Cdk1
activation was suppressed. Failure to amplify Cdk1 activity through rapid
dephosphorylation of inhibitory residues leads to the mitotic collapse, which we
argue is a direct consequence of the inability to overcome Cdk-opposing
phosphatases. Together, these results highlight the importance of the feedback-
mediated Cdk1 activation for shifting the kinase-phosphatase balance toward
Mitotic progression requires a wave of Cdk1 activity that phosphorylates a
large number of substrates. However, the details of how this wave of
phosphorylation coordinates the precisely ordered physiological processes of
mitosis are incompletely understood. A particularly important issue that awaits
explanation is the relationship between mitotic kinases and their antagonistic
phosphatases. Here we show that cells become capable of the “forward” M to G1
cell cycle transition only after Cdk1 is fully activated. Under normal
circumstances, positive feedback-mediated Cdk1 activation may function to
overcome the activity of Cdk1-opposing phosphatases. This mode of Cdk
activation appears to be essential for maintaining the mitotic state and for the
proper ordering of mitotic events.
By chemically inhibiting Cdk1 at different stages of mitosis from prophase
to metaphase, we demonstrated that Cdk1 inhibition results in complete cyclin B
breakdown and irreversible cell division (“forward” mitotic transition) only if the
Cdk inhibitor was applied after prophase. Application of Cdk inhibitor in prophase
caused return to interphase without substantial cyclin B breakdown, and cells
could reenter mitosis when the Cdk inhibitor was removed. Thus Cdk inhibition in
prophase induces cells to retreat back to G2. Estimation of the Cdk1 activity at
different stages of mitotic progression by immunofluorescence analysis of the
phosphorylation of three mitotic substrates revealed that the rapid rise of Cdk1-
mediated phosphorylation occurs primarily during the short transition from
prophase to prometaphase. This is generally consistent with previous
immunofluorescence measurements by Lindqvist et.al., where Cdk activation
was assessed by measuring the dephosphorylation of the inhibitory Y15 on Cdk1
and phosphorylation of the Cdk1 substrate APC/C subunit Cdc27 (Lindqvist et
al., 2007). More recently, Gavet and Pines were able to measure the activity of
Cdk1/cyclin B complex in individual cells directly, by using a FRET biosensor
designed specifically for Cdk1/cyclin B1 kinase (Gavet and Pines 2010; Gavet
and Pines 2010). This elegant molecular tool utilized a short fragment of human
cyclinB1 harboring an autophosphorylation site. This biosensor exhibited a steep
increase in FRET signal during prophase and early prometaphase. Overall, this
trend was similar to the one observed in our immunofluorescence experiments.
Taken together, these data point toward the conclusion that the rapid increase of
Cdk1 activity in prometaphase determines the moment when cells become
committed to “forward” mitotic progression.
The primary indicator for “forward” mitotic progression in our studies was
proteolysis of cyclin B, which depends on the activation of APC/C-Cdc20.
APC/C-Cdc20 is itself a Cdk substrate that is heavily phosphorylated in mitosis
(Kraft et al., 2003; Herzog et al., 2005; Steen et al., 2008). Even though we did
not assess APC/C phosphorylation directly due to the lack of suitable
phosphoepitope antibodies, we anticipate the kinetics of APC/C phosphorylation
to be similar to that of the other mitotic substrates we did assess. Lindquist et.al.
performed quantitative analysis of mitotic phosphorylation of specific Cdk1 target
residues on one of the subunits of the APC/C – Cdc27/APC3 - T446 and S426.
Their study showed that the bulk of these residues became phosphorylated
during prophase and prometaphase (Lindqvist et al., 2007). In our study, live
imaging analysis of fluorescent cyclin B breakdown induced by Cdk inhibition
showed that functionally, APC/C-Cdc20 becomes progressively more efficient at
targeting cyclin B for degradation with advancing stages of mitosis. Therefore,
activation of Cdk1 is likely to be a determining factor for the ability of the APC/C-
Cdc20 to process mitotic substrates.
Our immunofluorescence analysis showed that the there is considerable
variability in final (metaphase) levels of Cdk1 activity from cell to cell. However,
this variability did not seem to impact mitotic progression. The final level of
Cdk1/cyclin B activity in the cell is likely determined by the amount of cyclin B
because Cdk1 was reported to be in vast excess over cyclins in cells (Arooz et
al., 2000). Several cyclin B knockdown studies reported a variety of relatively
minor mitotic perturbation in different cell lines, suggesting that overall mitotic
progression has room to be remarkably tolerant to reduction of cyclin B levels by
siRNA or shRNA (Yuan et al., 2004; Yuan et al., 2006; Bellanger et al., 2007;
Gong et al., 2007; Androic et al., 2008; Chen et al., 2008). Although the efficiency
of knockdown may partially explain the weak phenotype, this observation is also
consistent with the idea that the total level of Cdk1/cyclin B activity is less
important than the positive feedback-mediated rapidity of Cdk activation. For
instance, overexpression of the Cdk1-AF mutant, which lacks inhibitory
phosphorylation sites, causes a profound effect on cell cycle progression,
manifested by premature chromatin condensation, aberrant mitosis and
abbreviated cell cycles (Gavet and Pines 2010; Jin et al., 1996; Pomerening et
al., 2008). This phenotype was somewhat distinct from the mitotic collapse
phenotype, particularly in the aspect of persistent oscillations between mitotic
and interphase state that were not observed in our experiments. However, in the
above studies Cdk1-AF mutant was overexpressed above the endogenous wild
type Cdk1. Therefore a portion of Cdk1/cyclin B complex in these studies may
have been assembled with endogenous, wild-type Cdk1 that retained the ability
to be regulated by phosphorylation.
In this study, we used fast-acting chemical inhibitors to analyze the
importance of the switch-like activation of endogenous Cdk1 for the proper order
of mitotic progression. Inhibition of the Wee1 and Myt1 kinases in cells induced a
relatively normal mitosis in cells synchronized at the end of S phase, without
requiring a G2 stage. Ordinarily, during G2 cells grow and accumulate various
proteins including mitotic cyclins. In cells pushed into mitosis by the Wee1/Myt1
inhibitor, cyclin B1 did not accumulate to the level characteristic of cells that
entered mitosis without the inhibitor. Surprisingly, the amount of cyclin B present
by the end of the S phase in synchronized cells was sufficient for entry into
mitosis. Since inhibition of Wee1 and Myt1 kinases resulted in rapid
dephosphorylation of Cdk1 on inhibitory T14 and Y15, Cdk1 activation in these
cells was still rapid, even though their cyclin B levels were lower than in cells that
entered mitosis spontaneously. Nevertheless, these cells were able to progress
through mitosis, supporting the idea that for the proper order of mitotic events,
the final Cdk1 activity levels may be less critical than the feedback-mediated
dynamics of its activation.
Simultaneous inhibition of Wee1/Myt1 kinases and Cdc25 phosphatases
prevented both phosphorylation and dampened dephosphorylation of Cdk1 on
inhibitory T14 and Y15. Unexpectedly, this led to a sluggish mitotic entry followed
by dephosphorylation of mitotic substrates without cyclin B breakdown – a
phenotype that we termed “mitotic collapse.” The failure to degrade cyclin B likely
reflects insufficient activation of APC/C-Cdc20 by low levels of Cdk1 activity,
similar to the situation in prophase cells. The substrate dephosphorylation was
prevented by 1µM okadaic acid, indicating that the Cdk1 was actively
antagonized by phosphatase(s).
The possibility that the combination of Wee1 and Cdc25 inhibitors could
have some off-target effect that can influence phenotypic changes observed in
cells undergoing mitotic collapse cannot be completely excluded. This caveat is
intrinsic to any chemical inhibitor studies. However, it is highly unlikely that these
inhibitors can trigger the non-specific phosphatase activation, because
phosphorylation of Nucleolin and histone H3 was not lost in cells that were
already in mitosis at the time of drug addition (Figure 6B).
Historically, mitosis research has highlighted the mitotic kinases as key
regulators of cell division, while phosphatases have received much less attention.
However, it is becoming clear that the normal progression of mitosis is not only a
consequence of the change in activity of mitotic kinases, primarily Cdk1, but
require balanced actions of counteracting phosphatases (Trinkle-Mulcahy and
In budding yeast, the primary phosphatase opposing Cdk1 is Cdc14
(Visintin et al., 1998; Jaspersen et al., 1999), reviewed in (Stegmeier and Amon,
2004; Bosl and Li, 2005; Amon, 2008). However, in metazoans neither of the two
Cdc14 homologs, Cdc14A or Cdc14B, has been shown to counteract Cdk1
kinase during mitotic exit (Vazquez-Novelle et al., 2005; Berdougo et al., 2008).
Instead, in higher eukaryotes, the PP1 and PP2A families of protein
phosphatases, enzymes that can be inhibited by okadaic acid, appear to play
more important roles in mitotic entry and exit. In Xenopus egg extracts, depletion
studies have implicated both PP1 and PP2A in the dephosphorylation of Cdk1
substrates (Mochida et al., 2009; Wu et al., 2009). Interestingly, both PP1 and
PP2A phosphatases appear to be inhibited by high Cdk1 activity, constituting
another feedback mechanism where the Cdk1 kinase inactivates its antagonists,
shifting the balance toward mitotic phosphorylation (Figure 7A)
PP1 is phosphorylated by Cdk1 on the inhibitory T320 residue
(Dohadwala et al., 1994; Kwon et al., 1997; Wu et al., 2009). When Cdk1 is
inactivated during mitotic exit, PP1 activates itself by dephosphorylating this
T320 residue and another residue, T35 (likely a MAP kinase site), responsible for
the binding of the inhibitory protein I-1 (Wu et al., 2009). Another small protein
inhibitor of PP1 is the inhibitory protein 2 (I-2), which is also heavily
phosphorylated in mitosis (Li et al., 2007), and may be a Cdk1 substrate.
Therefore, the activation of Cdk1 may switch PP1 off, and inactivation of Cdk1
may switch PP1 on. Further experimental and modeling studies are needed to
evaluate the dynamics and robustness of this switch.
A similar mechanism of Cdk-dependent inhibition may exist for the PP2A.
The activity of PP2A–B55 delta is low when the Cdk1 is fully active in mitosis
(Mochida and Hunt, 2007). Unlike PP1, PP2A has not yet been shown to be
inhibited by Cdk1 phosphorylation directly. However, a kinase called Greatwall
(human MastL) has been shown to inhibit anti-mitotic phosphatases in the
Xenopus egg extract system (Castilho et al., 2009; Vigneron et al., 2009).
Greatwall kinase is a Cdk1/cyclin B substrate. Active Cdk1/cyclin B complex
phosphorylates and activates Greatwall which then inhibits PP2A and perhaps
other phosphatases, constituting another feedback loop that promotes mitotic
phosphorylation (Figure 7A).
Because the substrate of the human MastL kinase is not yet identified, we
were not able to assay its activity directly. By Western blotting, we observed a
phosphorylation shift during mitotic entry that was absent in mitotic collapse,
suggesting that MastL may be inactive in collapsed cells (Figure 5B). This may
partially explain the elevated phosphatase activity in these cells. MastL
knockdown was shown to cause defects in chromosome alignment and
segregation, and also incomplete cyclin B breakdown upon mitotic exit (Burgess
et al 2010.; Voets and Wolthuis 2010). However, strong MastL knockdown as
well as the Greatwall depletion in Xenopus egg extracts was reported to block
entry in mitosis. We attempted to override this block in MastL siRNA-treated
HeLa cells synchronized at the S/G2 border by treating them with the Wee1/Myt1
inhibitor PD0166285. The mitotic entry in this case was comparable in both
MastL siRNA and negative control siRNA-treated cells. The phenotype of MastL
knockdown cells that entered mitosis in Wee1 inhibitor was generally similar to
what has been reported previously (Supplemental figure 6 and Video 11),
although there was an increased incidence of mitotic cell death. We did not
observe defects reminiscent of mitotic collapse, which suggests that MastL may
be responsible for inhibition of some, but not all Cdk-opposing phosphatases
involved in generating mitotic collapse phenotype. Alternatively, the depletion of
MastL by siRNA may have been insufficient to fully release phosphatase
The phosphatase(s) responsible for the mitotic collapse phenotype in our
studies likely belonged to the PP2A family because the dephosphorylation of
mitotic substrates was prevented by 1µM okadaic acid. At this concentration PP1
is only partially inhibited (Bialojan and Takai, 1988). Okadaic acid not only
prevented the dephosphorylation of Cdk1 substrates, but also markedly
increased their phosphorylation (Figure 6C). Without okadaic acid, mitotic
phosphatases eventually overcame Cdk activity when it was not fueled by
positive feedback, resulting in mitotic collapse. One possible mechanism that
may aid somatic cells in countering phosphatase activity during mitotic entry is
spatial concentration of Cdk1 activity within the nucleus in early mitosis.
Cdk1/cyclin B complex translocates into the nucleus in prophase, and then
disperses throughout the cytoplasm after nuclear envelope breakdown (Pines
and Hunter, 1991; Hagting et al., 1998). It was recently confirmed that
translocation of Cdk1/cyclin B complex into the nucleus coincides with its
activation (Gavet and Pines 2010). Consistent with this, our immunolabeling
experiments show that the Cdk activity is concentrated in the nucleus in
prophase, and after nuclear envelope breakdown the cytoplasm fills with
phosphorylated Cdk1 substrates (Supplemental figures 2 A-C). Overall, it
appears that Cdk1 activity spikes around the time of the nuclear envelope
disassembly, when the activated Cdk/cyclin B complex spreads through the
cytoplasm. Therefore, it is possible that in the absence of the positive feedback,
active Cdk1 became too dilute in the cytoplasm when the nuclear envelope
disassembled or became permeable enough to permit the diffusion of
Cdk1/cyclin complexes out of the nucleus (Figure 7B). Under these
circumstances the concentration of the active kinase per unit of cytosol may have
fallen below the level that is needed to efficiently counteract Cdk-opposing
phosphatases and maintain mitosis.
The mitotic collapse phenotype that we observed was accompanied by
substrate dephosphorylation, but morphologically it was far from normal mitotic
exit. Mitotic exit, like mitotic entry, is a well-ordered sequence of events:
chromatid segregation is followed by cytokinesis, nuclear envelope re-assembly,
cytosceletal re-arrangements, etc. Whether this orderly progression requires a
particular sequence of dephosphorylation reactions is not known. However, our
results suggest that the proper interplay of kinase and phosphatase activities,
where feedback-mediated activation of Cdk first overcomes the activity of
phosphatases then is rapidly turned off, is essential for the normal mitotic entry
MATERIALS AND METHODS
Cell culture, plasmid and siRNA transfection. Xenopus S3 cells were
grown at 23ºC in 70% L-15 medium supplemented with 15% FBS. HeLa and
RPE1 cells (ATCC) were grown in DMEM with 10% FBS in 5% CO2 at 37ºC.
HeLa cells were transiently transfected using Fugene 6 or Fugene HD (Roche)
according to the manufacturer’s directions. Plasmid encoding the wild type
human cyclin B1-GFP was a generous gift from Dr. Randall King. Live imaging
experiments were conducted 24-48 hours following the transfection of cyclin B.
SiRNA targeting Cdc20 and Cdh1 were obtained from Dharmacon/Thermo
Scientific (ON-Target plus SMART pool #L-003225-00-0005 for Cdc20, #L-
015377-00-0005 for Cdh1 and #L-004020-00-0005 for MASTL). Hela cells were
transfected with the siRNAs using Lipofectamine RNAi (Invitrogen) according to
the manufacturer’s directions.
Chemical inhibitors. The Cdk inhibitor, Flavopiridol (Enzo Life Sciences)
was used at 10 µM. The proteasome inhibitor MG132 (Calbiochem) was used at
25µM. The Wee1/Myt1 inhibitor PD0166285 (Pfizer) was used at 0.5 µM. The
Cdc25 inhibitor NSC663284 (Sigma) was used at 25 µM. The other Cdc25
inhibitor, NSC95397 (Enzo Life Sciences) was used at 10-20 µM. Okadaic acid
(LLC Labs or Alexis Biochemicals) was used at 1 µM. Nocodazole (Sigma) was
used at 300ng/ml.
Drug treatments and Western blotting. For siRNA experiments, mitotic
Hela cells were collected by shake-off 24-48 hours after siRNA transfection
followed by a 3-4 hour nocodazole block. The mitotic cells were split into a
number of experimental groups and treated with Flavopiridol for indicated periods
of time. Cells were then pelleted by centrifugation and lysed in NuPAGE protein
sample buffer (Invitrogen) containing 50 mM DTT.
For synchronization experiments, Hela cells were grown in 35mm plates,
synchronized by double thymidine block and then treated as detailed in figure
legends. Each plate represented an experimental sample. Samples were
collected by trypsinization and lysed in NuPAGE buffer with 50 mM DTT.
Protein samples were separated by SDS-PAGE in 4% - 12% Bis-Tris gels
(Invitrogen), transferred to PVDF (Millipore) and blocked in 5% BSA. Primary
antibody against phospho-Nucleolin (pNucleolin) was a generous gift from Dr.
Peter Davies; cyclin A2 AT-10 antibody was a generous gift from Dr. Tim Hunt.
Cdh1, pT14Cdk1 and Nucleolin antibodies were from Abcam; cyclin B1 antibody
was from BD Biosciences; Cdc20(p55) antibody was a gift from Dr. Jasminder
Weinstein (Amgen), securin-1 antibody was from Zymed; pY15Cdk1, pS10
histone H3, Wee1, anti Myt1Cdc25C and Cdk1 antibodies were from Cell
Signaling. MastL antibody was from Abcam. Primary antibodies were detected
using horseradish-peroxidase conjugated IgG (Jackson ImmunoResearch) and
visualized using the West Pico Chemiluminescent kit (Pierce). For pNucleolin
and β-actin Western blots associated with Cdk1/cyclin B1 kinase assays in
Figure 6C, secondary antibodies used were labeled with Alexa-488 and Alexa-
568 (Invitrogen) and these membranes were scanned with a Typhoon 9400
Flow cytometry. For pS10 histone H3 analysis, cells were treated as
detailed in figure legends, trypsinized and fixed in 2% formaldehyde in PHEM
(60mM PIPES, 25Mm HEPES (pH 6.8), 10mM EDTA, 4mM MgCl2) for 15
minutes, then permeabilized with 90% methanol at -20ºC. Later cells were
washed three times with PBS, blocked with 5% BSA in PBS and labeled with
anti-pS10 Histone H3 antibody conjugated to Alexa Fluor 647 (Cell Signaling).
Analysis was carried out on a FACSCalibur flow cytometer (BD Biosciences).
Live imaging. Cells were grown either on 25 mm glass coverslips which
were inserted in an Attofluor culture chamber (Molecular Probes) before the
experiment, or in Lab-Tek Chambered Coverglass multi-well dishes. Xenopus S3
cells were imaged at room temperature in their normal growth medium. HeLa
cells were imaged in L-15 medium with 10% FBS at 37 ºC. Temperature was
maintained with an air curtain incubator (Nevtek) and an objective heater
(Bioptechs). Time-lapse phase contrast and fluorescent images were collected
using a Zeiss Axiovert 200M wide-field fluorescent microscope. The microscope
was equipped with Hamamatsu ORCA-ERG digital camera. A 40× Plan-Neofluar
oil immersion objective was used for most live imaging experiments. Drugs were
substituted by addition of concentrated stock solutions to the live imaging media
or by exchange of the media. Images were processed using the Metamorph
software (Molecular Devices).
Immunofluorescence. Hela cells were grown on glass coverslips and
treated as detailed in figure legends.
paraformaldehyde/PHEM solution containing 0.5% Triton X-100 for 15 minutes.
Coverslips were washed in PBST, blocked in 5%BSA/PBS, and incubated
overnight with primary antibodies. Samples were then incubated with secondary
antibodies for 2-3 hours, stained with DNA dye, DAPI, and mounted using
Vectashield (Vector Laboratories). For data displayed in Figure 3 and
Supplemental figures 2 and 5 the following antibodies were used: mouse MPM2
(Dako Corp.), rabbit pS-Cdk (Cell Signaling) or mouse IgM pNucleolin (a gift from
P. Davies). Each sample was co-incubated with an antibody against the Lamin
B1, either of mouse or of rabbit origin (both from Abcam). Secondary goat anti-
rabbit and goat anti-mouse or anti-mouse IgM antibodies were conjugated to Cy3
and FITC (Jakson Immunoresearch). DNA was stained with DAPI. The images
were acquired using Zeiss Axiovert 200M wide-field fluorescent microscope (40x
oil immersion objective) equipped with a Hamamatsu ORCA-ERG digital camera
and processed with MetaMorph (Molecular Devices).
For data displayed in Figure 4, cells were labeled with rat antibody against
tyrosinated alpha-tubulin (clone YL1/2, Abcam) followed by a secondary goat
anti-rat antibody conjugated to Cy3. Subsequently, cells were labeled with mouse
anti-pS10 Histone H3 antibody conjugated to Alexa Fluor 647 (Cell Signaling).
DNA was stained with Vybrant®DyeCycletm Green (Molecular Probes). For data
displayed in Supplemental figure 3, cells were first labeled with primary mouse
antibody against nucleolin (Abcam) and secondary goat anti-mouse antibody
conjugated to Cy5. Subsequently, cells were labeled with phospho-Nucleolin
mouse IgM antibody and the secondary antibody against mouse IgM conjugated
to Cy3. DNA was stained with Vybrant®DyeCycletm Green. Images from these
experiments were collected using a 63× PlanApochromat oil immersion objective
on a Zeiss AxioObserver equipped with a high-speed Yokogawa CSU 22
spinning disk confocal imaging system and a Hamamatsu ORCA-ERG digital
camera. Images were collected and processed with SlideBook software
(Intelligent Imaging Innovations).
Quantitative image analysis. To measure the fluorescent cyclin B1-GFP
degradation in living cells, time lapse images were collected at 1 minute intervals.
The region was drawn around each cell to be measured, and the identical region
was placed in an area without fluorescent objects to be used for background
subtraction. The net average fluorescent intensity of a pixel in the region of
interest was calculated for each time point. Because cells expressed different
levels of fluorescent cyclin B, the net average intensity values were normalized to
the initial (first time point) value that was designated as 1. Averages of
normalized intensity values of at least 5 identically treated cells were calculated
Cells were fixed in 2%
for each time point and plotted on a graph. For these experiments, all parameters
during image acquisition were the same.
To measure fluorescent intensities of MPM2, pS-Cdk and pNucleolin
antibody labeling, 1µm Z-stacks through cells of different stages of mitosis were
acquired. A region was drawn around each cell to be measured, and the same
size region was drawn in an area without fluorescent objects to be used for
background subtraction. The net integrated intensity for each cell was measured
at a single Z plane with highest integrated intensity values in the region of
interest (this was usually the plane with the best focus). The weak signal from
interphase cells was designated as 1, and the fluorescent intensity values at
each mitotic stage were normalized and plotted relative to interphase. Each bar
represents an average of 15-30 cells. The intensitiy of a signal from the control
slide labeled with secondary antibodies alone was comparable to the intensity of
the background in experimental samples.
Cdk1/Cyclin B1 kinase assays. Hela cells were grown in 60mm plates,
synchronized by double thymidine block, and then treated as detailed in figure
legend. Each plate represented an experimental sample. Samples were collected
by trypsinization and lysed in RIPA (150 mM NaCl, 0.5% sodium deoxycholate,
0.1% SDS, and 50 mM TrisHCl, pH 7.4, 1%NP-40) supplemented with 10mM
EGTA and HALT Protease and Phosphatase inhibitor cocktail (Pierce). A portion
of lysate was saved for the Western blotting analysis. Cdk1/cyclin B1 complex
was immunoprecipitad with cyclin B1 monoclonal antibody (BD Biosciences) on
protein A/G agarose resin (Pierce). For kinase reaction, immunoprecipitates were
incubated in kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-
glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2).
Each reaction contained 1-2 mg/ml Histone H1, 200μM ATP and 1μCi of
[γ32P]ATP. Reactions were incubated at 37˚C for 20 min, stopped by addition of
SDS sample buffer, and separated by SDS-PAGE in 4% - 12% Bis-Tris gels
(Invitrogen). The gel was exposed to a phosphor screen (Amersham), which was
then scanned with a Typhoon 9400 PhosphorImager (Amersham). The gel was
subsequently stained with Coomassie Blue.
We express our tremendous appreciation to Dr. Bélá Novak for critical reading of
the manuscript and for his astute comments and suggestions. We are grateful to
Drs. Jonathon Pines, Randall King, Peter Davies, and Osamu Hashimoto for
generously providing essential reagents. We thank Pfizer for providing
PD0166285. We are grateful to Todd Stukenberg, Jonathon Pines, Andrew
Murray, Peter Lénárt, Mark Terasaki and Boris Rubinstein for insightful
discussions. We thank the OMRF flow cytometry core facility for technical
assistance. We thank the members of the Gorbsky, Dresser and Li laboratories
for help and advice. Special thanks goes to Sreekumar Ramachandran for help
with kinase assays. The work in GJG’s laboratory was supported by Grant
2R01GM050412 from the National Institute of General Medical Sciences and by
the McCasland Foundation.
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Figure 1. Cells commit to forward mitotic progression in prometaphase.
(A) Chemical Cdk inhibition in mitosis induces cyclin B1 and securin degradation
that requires APC/C-Cdc20. HeLa cells were transfected with 50nM Cdc20
siRNA for 24 hours or with 50nM Cdh1 siRNA for 48 hours. Mitotic cells were
collected in nocodazole, treated with 10µM Flavopiridol for 30, 60 and 90
minutes, then lysed and processed for Western blotting. Depletion of Cdc20, but
not Cdh1, inhibits degradation of cyclin B and securin. (B) Summary of the live
imaging data from Xenopus S3 cells treated with Cdk inhibitor, Flavopiridol, and
Wee1/Myt1 inhibitor, PD 0166285 at different stages of the mitotic progression.
Flavopiridol was washed out 1 hour after addition. (C) A prophase Xenopus S3
cell expressing alpha tubulin-GFP was treated with Cdk inhibitor, Flavopiridol,
and Wee1/Myt1 inhibitor, PD 0166285. After treatment with Cdk inhibitor, mitotic
progression stopped, the chromosomes decondensed, and the cell returned to
an interphase morphology. Flavopiridol was washed out at 1 hour, and the cell
re-entered mitosis, indicating that Cdk1-activating cyclins were preserved. The
cell then progressed normally trough mitosis. (D) An early prometaphase
Xenopus S3 cell expressing alpha tubulin-GFP was treated with Flavopiridol and
PD 0166285. After treatment at time 0 the cell underwent cytokinesis without
chromosome segregation, the chromosomes decondensed, the nuclear envelope
reformed and an interphase array of microtubules appeared. Flavopiridol was
washed out at 1 hour. However, the cell did not reenter mitosis indicating that it
had advanced to a G1-like state. The complete timelapse sequences for A and B
are shown in Videos 1 and 2. Bar, 10µm
Figure 2. Cdk1 inhibition after prophase induces cyclin B degradation.
(A) HeLa cells expressing wild-type cyclin B1-GFP and histone H2b-mCherry
through normal mitosis. At left is an example of live cell imaging of a Hela cell
transiting mitosis. At right is plotted normalized cyclin B1-GFP intensity starting
30 minutes before anaphase onset for 5 cells. (B) Prophase, (C) prometaphase
and (D) metaphase HeLa cells expressing cyclin B1-GFP were treated with
Flavopiridol at time 0. Flavopiridol induced chromosome decondensation and
degradation of cyclin B. At left are examples of time-lapse imaging. On the right,
normalized fluorescent intensity of the GFP is plotted starting from the time of
Flavopiridol addition for 5 cells from each stage of mitosis. The rate of
Flavopiridol-induced cyclin B degradation increases with the stage of mitosis.
Complete timelapse sequences for A, B, C, and D are shown in Videos 3-6. Bar,
Figure 3. Cdk1 phosphoepitopes rise rapidly during early mitosis.
HeLa cells synchronized by double thymidine block were fixed 9 hours after the
release from the second thymidine block and immunolabeled with following
antibodies: MPM2 (A), pS-Cdk (B) and pNucleolin (C). The fluorescent intensities
were plotted according to mitotic stage. To assess the stage of mitosis precisely,
cells were co-stained with antibody against Lamin B and with DNA dye.
Integrated fluorescent intensity of a cell was measured at the brightest plane of a
z series taken at 1 µm intervals. Each bar on the graphs represents an average
of 15-30 cells for each stage. Error bars denote standard deviation.
Representative images of each stage are shown in Supplemental Figure 2.
(D) Diagram depicting relationship between Cdk substrate phosphorylation and
irreversible mitotic entry. Cells become committed to forward mitotic progression
in prometaphase, when Cdk substrates become phosphorylated.
Figure 4. Mitotic progression in cells synchronized at S/G2 and treated with
Wee1/Myt1 and Cdc25 inhibitors.
(A – B) HeLa cells stably expressing fluorescent histone H2B fused to GFP were
synchronized by the double thymidine block at the S/G2 border and treated with
the Wee1/Myt1 inhibitor, PD0166285, alone (A) or in combination with Cdc25
inhibitor, NSC663284 (B). While the Wee1/Myt1 inhibitor alone rapidly triggers
mitosis in the majority of cells, the combination of the Wee1/Myt1 and Cdc25
inhibitors results in slow mitotic entry followed by mitotic collapse. The complete
timelapse sequence is shown in Videos 7 and 8. Bar, 10µm. (C) Synchronized
HeLa cells were treated with the Wee1/Myt1 inhibitor, PD0166285, alone or in
combination with Cdc25 inhibitor, NSC663284, for 90 min. Cells were then fixed
and processed by immunofluorescence for alpha-tubulin and phosphorylated-
histone H3 on S10 (mitotic marker). Labeling shows disorganized mitotic spindle
and in some cells – reduced mitotic marker.
Figure 5. Inhibition of Wee1/Myt1 and Cdc25 in synchronized cells causes
(A) HeLa cells were synchronized at the S/G2 border after double thymidine
block and then treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25
inhibitor, NSC663284, and the combination of the two drugs. Nocodazole was
added to the medium to prevent mitotic exit. Cells were then collected at
indicated time points, fixed and stained with antibody to phospho-histone H3
(mitotic marker) conjugated with Cy5 and processed by flow cytometry. In cells
treated with vehicle only (DMSO, blue line), the mitotic index progressively
increased, with more than half the cells being in mitosis by the end of the
experiment. Cdc25 inhibitor, NSC663284, blocked mitotic entry (brown line).
Wee1 inhibitor, PD0166285, (green line) caused rapid mitotic entry during the
first hour after its addition. In cells treated with both PD0166285 and NSC663284
(orange line), the mitotic index first increased then fell. (B) HeLa cells were
treated as in (A), lysed and analyzed by SDS-PAGE. In cells not treated with
inhibitors (blue lanes), phosphorylations on histone H3 and nucleolin appeared
by 8 hours after second thymidine release and increased for the duration of the
experiment. Phosphorylation of Cdk1 on inhibitory T14 and Y15 decreased over
time, indicating the activation of the Cdk1/cyclin B complex. As cells were
entering mitosis, a portion of Wee1, Myt1 Cdc25C, Cdc27 and MastL acquired an
electrophoretic mobility shift. Cyclin B1 levels were increasing, and cyclin A2
levels dropped slightly as cells accumulated in mitosis. Inhibition of Wee1 and
Myt1 kinases with PD0166285 (green lanes) resulted in rapid phosphorylation of
Nucleolin and histone H3 that peaked 2 hours after the drug addition and
remained steadily high for the duration of the experiment. Cdk1 was rapidly
dephosphorylated on inhibitory T14 and Y15. Wee1, Myt1, Cdc25 and Cdc27
rapidly shifted up. By 1 hour after drug addition, Cyclin A2 was largely degraded
and cyclin B1 was stable. Inhibition of Wee1 and Myt1 together with Cdc25 by
addition of both PD0166285 and NSC 663284 (orange lanes) triggered the a
weak phosphorylation on Nucleolin and histone H3 that peaked at 1-2 hours and
disappeared at 3-4 hours after addition of the two drugs. Reduced mitotic
phosphorylation shifts of Wee1, Myt1, Cdc25 and Cdc27 indicated that these
proteins were not fully phosphorylated. Note that Cyclin B and most of the cyclin
A were not degraded in these cells. Panels on the right show quantifications of
indicated Western blots. All values were adjusted for loading and normalized to
the 4 hour time point of DMSO-treated cells.
Figure 6. Deposphorylation of mitotic substrates in “collapsed” cells is a
result of incomplete inhibition of Cdk-opposing phosphatases.
(A) Cdk1/cyclin B1 activity does not drop in mitotic collapse cells. HeLa
cells were synchronized at the S/G2 border and treated with the Wee1/Myt1
inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the
two in the presence of nocodazole. Cells were then collected at indicated time
points and lysed. An aliquot of the lysate was analyzed by Western blotting for
Nucleolin phosphorylation. β-actin served as a loading control. Cyclin B1/Cdk1
complex was immunoprecipitated from the rest of the lysate and subjected to an
in vitro kinase assay using histone H1 as a substrate. The kinase reaction
mixture was resolved by SDS-PAGE, and the gel was exposed to phosphor-
screen which was then scanned with phosphor-imager. For a control, samples
derived from the 4 hour time point of DMSO-treated cells were treated with Cdk
inhibitor (lane labeled “+Flavopiridol”), or processed omitting cyclin B1 antibody
from immunoprecipitation (lane labeled “mock”). The gel was subsequently
stained with Coomassie blue for loading. Panel on the right shows quantifications
of histone H1 phosphorylation normalized to the 4 hour time point of DMSO-
treated cells. An average of three independent assays is shown. Error bars
denote standard deviation. (B) Simultaneous inhibition of Wee1/Myt1 and Cdc25
in cells already in mitosis does not cause mitotic substrate dephosphorylation.
Mitotic HeLa cells were collected in nocodazole and then treated with Wee1/Myt1
and Cdc25 inhibitors for the indicated time, lysed and analyzed by Western
blotting. Mitotic substrates nucleolin and histone H3 remained phosphorylated
throughout the experiment. (C) The phosphatase inhibitor, okadaic acid, prevents
dephosphorylation of mitotic substrates in cells treated with a combination of
Wee1/Myt1 and Cdc25 inhibitors. HeLa cells were synchronized at the S/G2
border after double thymidine block and treated with the Wee1/Myt1 inhibitor,
PD0166285, and Cdc25 inhibitor, NSC663284, for the indicated time in the
presence or absence of okadaic acid. Addition of the okadaic acid resulted in
robust and sustained phosphorylation of mitotic substrates.
Figure 7. (A) Cdk substrate phosphorylation regulatory network. The
phosphorylation of mitotic substrates (enzymes and structural proteins) by
Cdk1/Cyclin B complex underlies mitotic entry. Cdk1/cyclin B is antagonized by
phosphatases PP1 and PP2A that dephosphorylate mitotic substrates. Wee1
kinase and Cdc25 phosphatases regulate Cdk1 activity: Wee1 inhibits Cdk1
(green inhibitory line) and Cdc25 activates it (blue arrow). Wee1 and Cdc25 are
themselves Cdk substrates. Cdk1 phosphorylates and inhibits Wee1, preventing
Wee1 from inactivating Cdk1. Also, Cdk1 phosphorylates and activates its
activator Cdc25. Active Cdk also inhibits antagonists – PP1 and PP2A by at least
two known mechanisms. First, Cdk1 can inhibit PP1 directly by phosphorylating
T320 residue on a catalytic subunit of the phosphatase (black inhibitory line).
Second, Cdk1 phosphorylates and activates the Greatwall/MastL kinase, which
inhibits PP2A and possibly PP1 by yet unidentified mechanisms (red inhibitory
line). Therefore, as Cdk activation is fueled by positive feedback, it also promotes
the inactivation of its antagonists, ensuring the stability of substrate
phosphorylation. (B) Failure to activate Cdk rapidly results in mitotic collapse
after nuclear envelope breakdown. The feedback-mediated activation of the
Cdk1/Cyclin B complex may be required to prevent the dilution of the kinase
activity throughout the cytoplasm when the nuclear envelope becomes
permeable. Cdk1 activity appears to spike around the time of the nuclear
envelope disassembly, when the activated Cdk/Cyclin B complex spreads
through the cytoplasm. In the absence of the positive feedback, active Cdk1
would be diluted in the cytoplasm when the nuclear envelope becomes
permeable. In the absence of positive feedback mechanisms the concentration of
the active kinase per unit of cytosol may fall below the level that is needed to
efficiently counteract Cdk-opposing phosphatases, which leads to the mitotic
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Regulation of the Cdk1/cyclin B complex by the
network of feedback-mediated mechanisms.
Cdk1/cyclin B complex phosphorylates and inhibits its own inhibitors
Wee1 and Myt1 (double-negative feedback loop), and phosphorylates and
activates its own activator Cdc25 (positive feedback loop). Cdk1 also activates its
own inhibitor APC/C-Cdc20 that targets Cdk1 activator cyclin B for degradation;
this builds a negative feedback loop that turns off the active Cdk1. The APC/C-
Cdc20 is inhibited by the mitotic spindle checkpoint that itself requires Cdk1
activity and prevents initiation of cyclin B proteolysis.
Supplemental Figure 2. Fluorescent intensities of phosphorylated Cdk1
substrates rise sharply in prophase and prometaphase. HeLa cells synchronized
by double thymidine block were fixed 9 hours after the second thymidine release
and immunolabeled with Cdk1 substrate antibodies: MPM2 (A), pS-Cdk (B) and
pNucleolin (C). Cells were co-labeled with antibodies against Lamin B. DNA was
stained with DAPI. Representative images of each mitotic stage are shown.
Immunofluorescence of Cdk substrates is depicted as intensity maps (scale is
shown on the right). Shown are the brightest Z planes that were used for the
integrated intensity quantification in Figure 3. Bar, 10µm
Supplemental Figure 3. Phospho-nucleolin labeling co-localizes with the
total nucleolin in mitosis. Asynchronously growing HeLa cells were fixed and
immunolabeled with antibodies to phospho-nucleolin and total nucleolin. In
interphase and very early prophase, before nucleoli disassemble, nucleolin is
primarily localized to the nucleoli, where it is not phosphorylated. As nucleoli
disassemble, nucleolin becomes phosphorylated and dispersed throughout the
cytoplasm with some concentration at the periphery of the chromosomes. Bar,
Supplemental Figure 4. Simultaneous chemical inhibition of Wee1, Myt1
and Cdc25 in asynchronously growing cells leads to mitotic collapse. (A)
Asynchronously growing RPE-1 cells stably expressing histone H2B fused to
GFP were treated with a combination of Wee1/Myt1 inhibitor, PD0166285, and
Cdc25 inhibitor, NSC663284, and followed by live cell imaging. The DNA
conformation of a typical cell that entered mitosis and underwent mitotic collapse
is shown. The complete timelapse sequence is shown in Video 10. (B)
Asynchronously growing HeLa cells stably expressing histone H2B fused to
mCherry and alpha-tubulin fused to GFP were treated with a combination of
Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, and
followed by live cell imaging. The morphology of a typical cell that entered mitosis
and underwent mitotic collapse is shown. Note that the interphase cell on the left
was not noticeably affected by this treatment. The complete time-lapse sequence
is shown in Video 9. Bar, 10µm.
Supplemental Figure 5. Simultaneous chemical inhibition of Wee1/ Myt1
and Cdc25 leads to the de-phosphorylation of mitotic substrates. (A) HeLa cells
were synchronized by the double thymidine block at the S/G2 border and treated
with the Wee1/Myt1 inhibitor PD0166285, alone and in combination with Cdc25
inhibitor NSC663284 for the indicated time. Nocodazole was added to the
medium. Cells were then fixed and labeled with MPM2, pS-Cdk and pNucleolin
antibodies. Comparison of average fluorescent intensities shows the decline of
all three markers 180min after the drug addition when both inhibitors were used.
(B) Representative images of cells treated with both inhibitors 90 and 180 min
after drug addition.
Supplemental Figure 6. MastL knockdown cells treated with Wee1/Myt1
inhibitor enter aberrant mitosis. HeLa cells stably expressing fluorescent histone
H2B fused to GFP were transfected with 100nM MastL siRNA or negative
siRNA, synchronized by the double thymidine block at the S/G2 border, treated
with the Wee1/Myt1 inhibitor PD0166285, and followed by live imaging. The
MastL siRNA - treated cells entered mitosis, but displayed defects in
chromosome alignment, segregation, and failed cytokinesis. The complete
timelapse sequence is shown in Video 11. Bar, 10µm. The table below
summarizes the incidence of mitotic defects observed in the experiment. After
the imaging, cells were lysed and analyzed for MastL protein levels by Western
blot (shown on the lower right). Quantification of a series of two-fold dilutions
showed an average 60% knockdown.
Video 1. Cdk1 inhibition in prophase causes return to interphase that is fully
reversible. This video shows Xenopus S3 cell that expresses alpha tubulin–GFP.
This cell was treated with the Cdk inhibitor, Flavopiridol, during prophase. The
Wee1/Myt1 inhibitor, PD 0166285, was added to prevent Cdk1 inhibition by
phosphorylation. After Flavopiridol addition, the cell stopped progression into
mitosis, the chromosomes de-condensed and the cell returned to an interphase
morphology. Flavopiridol was washed out at 1 hour, and the cell re-entered
mitosis and progressed through mitosis normally. Time is indicated as
hours:minutes after Flavopiridol addition. This video is the source for the images
shown in Figure 1C.
Video 2. Cdk1 inhibition in prometaphase causes irreversible mitotic exit. This
video shows a Xenopus S3 cell expressing alpha tubulin-GFP that was treated
with Flavopiridol in early prometaphase. The Wee1/Myt1 inhibitor PD 0166285
was added to prevent Cdk1 inhibition by phosphorylation. After treatment at time
0 the cell underwent cytokinesis without chromosome segregation, the
chromosomes decondensed, the nuclear envelope reformed and an interphase
array of microtubules appeared. Flavopiridol was washed out at 1 hour.
However, the cell did not reenter mitosis indicating that it had advanced to a G1-
like state. Time is indicated as hours:minutes after Flavopiridol addition. This
video is the source for the images shown in Figure 1D.
Video3. Normal mitosis in an untreated HeLa cell stably expressing histone H2B-
mCherry and transiently transfected with human cyclin B1-GFP. The video shows
a sequence of events from prophase to telophase. Time is indicated as
hours:minutes after initiation of imaging. This video is the source for the images
shown in Figure 2A.
Video 4 A HeLa cell expressing histone H2B-mCherry and human cyclin B1-GFP
was treated with Flavopiridol at prophase. The cell reverted to an interphase
morphology. Cyclin B-GFP persisted in the cell but was removed from the
nucleus. Time is indicated as hours:minutes after Flavopiridol addition. This
video is the source for the images in Figure 2B.
Video 5 A HeLa cell expressing histone H2B-mCherry and human cyclin B1-
GFP was treated with Flavopiridol at prometaphase. The cell underwent
cytokinesis without chromatid segregation. Cyclin B-GFP concentrated on the
spindle/chromosomes and dispersed in the cytoplasm disappeared gradually due
to degradation as the cell exited mitosis. Time is indicated as hours:minutes
after Flavopiridol addition. This video is the source for the images in Figure 2C.
Video 6. A HeLa cell expressing histone H2B-mCherry and human cyclin B1-
GFP was treated with Flavopiridol at metaphase. The cell segregates
chromatids and undergoes cytokinesis. Cyclin B-GFP concentrated on the
spindle/chromosomes and dispersed in the cytoplasm disappeared rapidly, much
of it prior to anaphase, due to degradation as the cell exited mitosis. Time is
indicated as hours:minutes after Flavopiridol addition. This video is the source for
the images in Figure 2D.
Video 7. Inhibition of Wee1/Myt1 at the end of S phase triggers rapid entry into
mitosis. This video shows HeLa cells stably expressing fluorescent histone H2B-
GFP that were synchronized by double thymidine block at the S/G2 border and
then treated with the Wee1/Myt1 inhibitor, PD0166285. Addition of the inhibitor
triggered mitotic entry. Many cells then divided normally. Time is indicated as
hours:minutes after the addition of Wee1/Myt1 inhibitor. This video is the source
for the images shown in Figure 4A.
Video 8. Inhibition of Wee1/Myt1 and Cdc25 at the end of S phase causes
mitotic collapase. This video shows HeLa cells stably expressing fluorescent
histone H2B-GFP that were synchronized by double thymidine block at the S/G2
border and treated with the combination of the Wee1/Myt1 inhibitor PD0166285
and Cdc25 inhibitor NSC663284. Addition of the two drugs triggered mitotic entry
followed by mitotic collapase. Time is indicated as hours:minutes after the
addition of Wee1/Myt1 and Cdc25 inhibitors. This video is the source for the
images shown in Figure 4B.
Video 9. Inhibition of Wee1/Myt1 together with Cdc25 in asynchronously growing
HeLa cells causes mitotic collapase. A HeLa cells stably expressing histone H2B
fused to mCherry and alpha-tubulin fused to GFP was treated with a combination
of Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, and
followed by live cell imaging. Drugs were added to the medium 30 minutes before
the initiation of imaging. Time is indicated as hours:minutes after the initiation of
imaging. This video is the source for the images shown in Supplemental Figure
Video 10. Inhibition of Wee1/Myt1 and Cdc25 in asynchronously growing RPE1
cells causes mitotic collapse. RPE-1 cell stably expressing histone H2B fused to
GFP was treated with a combination of Wee1/Myt1 inhibitor, PD0166285, and
Cdc25 inhibitor NSC663284, and followed by live cell imaging (phase contrast
and GFP). Drugs were added to the medium 20 minutes before the initiation of
imaging. Time is indicated as hours:minutes after the initiation of imaging. This
video is the source for the images shown in Supplemental Figure 4B.
Video 11. MastL knockdown causes defects in chromosome alignment,
segregation and cytokinesis in PD0166285-treated cells. This video shows H2B-
GFP HeLa cells that were treated with MastL siRNA, synchronized by double
thymidine block at the S/G2 border and then treated with the Wee1/Myt1
inhibitor, PD0166285. During mitotic entry stimulated by Wee1 inhibitor defects in
metaphase alignment and chromatid segregation were evident. One cell in the
field died. Time is indicated as hours:minutes after the addition of Wee1/Myt1
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inhibitor. This video is the source for the MastL siRNA images shown in
Supplemental figure 6.