Grapes(Chk1) prevents nuclear CDK1 activation by delaying cyclin B nuclear accumulation

ArticleinThe Journal of Cell Biology 183(1):63-75 · November 2008with36 Reads
Impact Factor: 9.83 · DOI: 10.1083/jcb.200801153 · Source: PubMed
Abstract

Entry into mitosis is characterized by a dramatic remodeling of nuclear and cytoplasmic compartments. These changes are driven by cyclin-dependent kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear CDK1 activities are coordinated is unclear. We injected cyclin B (CycB) into Drosophila melanogaster embryos during interphase of syncytial cycles and monitored effects on cytoplasmic and nuclear mitotic events. In untreated embryos or embryos arrested in interphase with a protein synthesis inhibitor, injection of CycB accelerates nuclear envelope breakdown and mitotic remodeling of the cytoskeleton. Upon activation of the Grapes(checkpoint kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, increased levels of CycB drives cytoplasmic but not nuclear mitotic events. Grp(Chk1) prevents nuclear CDK1 activation by delaying CycB nuclear accumulation through Wee1-dependent and independent mechanisms.

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JCB 63
Correspondence to Anne Royou: royou@biology.ucsc.edu
Abbreviations used in this paper: Aph, aphidicolin; AVD, average deviation;
Chk1, checkpoint kinase 1; CHX, cycloheximide; CycB, cyclin B; Grp, Grapes;
MTOC, microtubule-organizing center; NEB, nuclear envelope breakdown;
NEF, nuclear envelope formation; RLC, regulatory light chain.
The online version of this article contains supplemental material.
Introduction
Entry into mitosis requires a dramatic reorganization of nuclear
and cytoplasmic compartments. These changes are driven by
the activity of CDK1 associated with mitotic cyclins, notably
cyclin B (CycB; Morgan, 2006 ). Activation of CDK1 requires
suf cient levels of CycB and the removal of CDK1 inhibitory
phosphorylation. CDK1 phosphorylation is controlled by con-
served kinases Wee1 and Myt1 and by the phosphatase Cdc25.
The rapid onset of CDK1 activation at the end of G2 is driven
by inactivation of Wee1 and activation of Cdc25. Active CDK1
contributes to these changes in Wee1 and Cdc25 activity, thus
establishing a positive feedback loop that drives cells into mito-
sis ( Ferrell, 2002 ; Morgan, 2006 ).
In spite of these insights, little is known about the mecha-
nisms by which nuclear and cytoplasmic CDK1 activities are
coordinated. Resolving this issue requires determining whether
nuclear and cytoplasmic CDK1 pools are differentially regu-
lated. Support for a differential regulation comes from the  nd-
ing that subcellular localization of CycB plays a role in regulating
CDK1 activity. In vertebrates, CycB is predominantly cytoplas-
mic at interphase because of Crm1-mediated nuclear exclusion
( Hagting et al., 1998 ; Yang et al., 1998 ). At prophase, CDK1 CycB
is activated in the cytoplasm before its entry into the nucleus
( De Souza et al., 2000 ; Jackman et al., 2003 ), and the abrupt
CDK1 CycB nuclear translocation is triggered by the phosphory-
lation of CycB on its cytoplasmic retention signal ( Ookata et al.,
1993 ; Pines and Hunter, 1994 ; Li et al., 1997 ; Hagting et al.,
1999 ; Takizawa and Morgan, 2000 ).
Controlling the subcellular localization of CycB may also
be involved in checkpoint function. Un-replicated or damaged
DNA results in the activation of the conserved S-phase check-
point kinase 1 (Chk1), which inhibits Cdc25 and activates Wee1
( Furnari et al., 1997 ; Peng et al., 1997 ; Sanchez et al., 1997 ; Lee
et al., 2001 ). Consequently, the S-phase checkpoint delays the
cell cycle in interphase by Chk1-mediated inhibition of CDK1
( Walworth, 2001 ; Melo and Toczyski, 2002 ). However, expression
of CDK1
AF
, a version of CDK1 lacking the phosphorylation
inhibitory sites, only partially bypasses the interphase arrest
induced upon DNA damage ( Jin et al., 1996 ). This arrest is fully
bypassed by coexpressing CDK1
AF
and nuclear-targeted CycB
( Heald et al., 1993 ; Jin et al., 1998 ). These studies imply that
prevention of CycB nuclear localization is one of the mecha-
nisms by which the S-phase checkpoint delays nuclear entry
into mitosis. Thus, the coordination of cytoplasmic and nuclear
mitotic entry likely involves the control of CycB subcellular local-
ization as well as the CDK1 phosphorylation state.
The late syncytial nuclear cycles of the Drosophila mela-
nogaster embryo are regulated by levels of CycB and S-phase
E
ntry into mitosis is characterized by a dramatic
remodeling of nuclear and cytoplasmic compart-
ments. These changes are driven by cyclin-dependent
kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear
CDK1 activities are coordinated is unclear. We injected
cyclin B (CycB) into Drosophila melanogaster embryos
during interphase of syncytial cycles and monitored ef-
fects on cytoplasmic and nuclear mitotic events. In un-
treated embryos or embryos arrested in interphase with a
protein synthesis inhibitor, injection of CycB accelerates
nuclear envelope breakdown and mitotic remodeling of
the cytoskeleton. Upon activation of the Grapes(checkpoint
kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, in-
creased levels of CycB drives cytoplasmic but not nuclear
mitotic events. Grp(Chk1) prevents nuclear CDK1 acti-
vation by delaying CycB nuclear accumulation through
Wee1-dependent and independent mechanisms.
Grapes(Chk1) prevents nuclear CDK1 activation
by delaying cyclin B nuclear accumulation
Anne Royou , Derek McCusker , Douglas R. Kellogg , and William Sullivan
Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064
© 2008 Royou et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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JCB • VOLUME 183 • NUMBER 1 • 2008 64
To determine whether new rounds of CycB synthesis are
required for each mitotic cycle, we investigated the effect of
injecting CycB after treatment with the protein synthesis inhibitor
cycloheximide (CHX). Injection of CHX at metaphase induced
a cytoplasmic and nuclear interphase arrest in the next cycle
( Fig. 1 C , left). Exogenous CycB locally overcame the CHX-
induced interphase arrest as indicated by NEB and rapid spindle
formation ( Fig. 1 C , right; and Video 1, available at http://www
.jcb.org/cgi/content/full/jcb.200801153/DC1). The data suggest
that new rounds of CycB synthesis are necessary to drive the
next mitotic cycle. We determined by single embryo Western
that the amount of injected CycB was equivalent to the total
level of endogenous CycB at metaphase of cycle 13. Taking into
account the limited diffusion of the injected CycB, we estimate
that the 65 μ M of exogenous CycB at the center of the gradient
was less than  vefold the level of endogenous CycB at mitosis
(see Materials and methods).
Injection of CycB prematurely drives the
cytoplasm into mitosis
We next determined whether injected CycB prematurely drives
the cytoplasm into mitosis. We addressed this issue by examin-
ing the dynamics of three cytoplasmic proteins fused with GFP
that undergo dramatic changes in subcellular distribution during
the cortical cycles: nonmuscle myosin II regulatory light chain
([RLC]; RLC-GFP; Fig. 2, A and D ; Royou et al., 2004 ), moesin
(GFP-moesin; Fig. 2 B and Video 2, available at http://www.jcb
.org/cgi/content/full/jcb.200801153/DC1; Edwards et al., 1997 ),
and Nuclear fallout (GFP-Nuf; Fig. 2 C and Video 3; Riggs
et al., 2007 ).
In all embryos, CycB was injected at the onset of cycle 13
interphase. We monitored the timing of cytoplasmic marker
dynamics and NEB in areas close to and distant from the site of
injection. We will refer to these as injected and control areas,
respectively. In control areas, RLC-GFP concentrated at the
cortex throughout interphase and dispersed just before NEB.
This dispersion occurred 12 min after the onset of interphase
( n = 23, average deviation [AVD] = 1.8; Fig. 2, A and D ; and
see Fig. S1 for quanti cation of the RLC-GFP signal, available
at http://www.jcb.org/cgi/content/full/jcb.200801153/DC1).
In CycB-injected areas, RLC-GFP dispersed prematurely, 4 min
after interphase onset ( n = 23, AVD = 1.8; Fig. 2, A and D , blue
outlines). Interestingly, in both areas, RLC-GFP disappearance
always preceded NEB ( Fig. 2, A and D , orange outlines). A pre-
vious study has shown that RLC-GFP dispersion is driven by
CDK1 activity ( Royou et al., 2002 ). Therefore, our results indi-
cate that high levels of CycB promote premature cytoplasmic as
well as nuclear CDK1 activation.
GFP-moesin, a marker for F-actin, reorganizes from caps
at interphase into furrows in mitosis ( Edwards et al., 1997 ).
By focusing 5 μ m below the plasma membrane, only furrows
that have extended to this depth are visualized ( Fig. 2 B ).
In control and CycB-injected regions, F-actin furrows pro-
gressed to this depth at 11.5 min. and 3.5 min after injection,
respectively ( Fig. 2 B , blue outlines). These experiments indi-
cate that exogenous CycB drives premature reorganization of
the actin cytoskeleton.
checkpoint activity ( Edgar et al., 1994 ; Fogarty et al., 1997 ; Sibon
et al., 1997 ; Stif er et al., 1999 ; Price et al., 2000 ; Stumpff
et al., 2004 ; Crest et al., 2007 ). These cycles provide an excel-
lent system to address the role of CycB subcellular localization
in driving cytoplasmic and nuclear mitotic events during normal
and S-phase checkpoint activated conditions. We addressed this
question by taking advantage of our ability to inject functional
CycB in the syncytial embryo at precise times during the cell
cycle and to monitor its effects on multiple cytoplasmic and nu-
clear events. Increasing the level of CycB during early inter-
phase of cycle 13 induces premature nuclear envelope breakdown
(NEB) and the reorganization of the cytoskeleton. Upon activa-
tion of the S-phase checkpoint, increased levels of CycB drives
cytoplasmic but not nuclear mitotic events. We demonstrate that
the S-phase checkpoint protects the nucleus from active cyto-
plasmic CDK1 CycB via two distinct mechanisms involving
Grapes(Chk1)-dependent control of CycB nuclear localization
and Wee1-dependent inhibition of nuclear CDK1.
Results
Injection of CycB prematurely drives NEB
and spindle assembly
We injected recombinant CycB N-terminal GST fusion protein
into living Drosophila embryos at precise times during inter-
phase of the syncytial cycle 13. GST-CycB is able to induce
CDK1 phosphorylation on T161 and promote its kinase activity
in vitro ( Edgar et al., 1994 ). We will refer to the recombinant
protein as CycB.
Nuclear CDK1 activity is thought to promote chromosome
condensation and NEB ( Lamb et al., 1990 ; Peter et al., 1990 ;
Enoch et al., 1991 ). Therefore, nuclear mitotic events were de-
ned by chromosome condensation (monitored with GFP-H2Av)
and NEB (monitored by nuclear infusion of injected rhodamine-
conjugated tubulin). The central regions of embryos were injected
with either 71 μ M GST or 65 μ M CycB at the onset of interphase
of nuclear cycle 13. After GST injection, NEB occurred almost
synchronously throughout the embryo at 14.3 min after injection,
and the chromosomes entered anaphase shortly after NEB ( Fig.
1 A , GST). After CycB injection, the timing of NEB was greatly
accelerated near the site of injection compared with NEB in areas
more distant from the site of injection ( Fig. 1 A , CycB, white out-
lines). Premature NEB occurs as a wave probably caused by the
slow diffusion of CycB from the injection site. These observa-
tions indicate that CycB injection at 65 μ M is suf cient to induce
a local activation of CDK1, which triggers premature NEB and
spindle assembly. This result is consistent with work demonstrating
that exogenous CycB promotes premature disassembly of the nu-
clear pore complex in syncytial Drosophila embryos ( Onischenko
et al., 2005 ). Although CycB induced premature NEB, it did
not promote premature chromosome condensation before NEB.
At NEB, the level of chromosome condensation was much greater
in the control embryo than in the CycB-injected embryo ( Fig. 1 B ,
top row). In most cases, once NEB occurred in the area near the
site of CycB injection, the chromosomes rapidly condensed and,
by metaphase, achieved the same state of condensation as control
embryos ( Fig. 1 B , bottom row).
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65COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
raises the possibility that cytoplasmic and nuclear CDK1 activ-
ities are differentially regulated by CycB levels. To explore this
possibility, we monitored nuclear (NEB) and cytoplasmic (myosin
dispersion) mitotic events after injecting CycB at a lower concen-
tration of 32 μ M ( Fig. 2 D ). In six out of eight injected embryos,
32- μ M CycB dilution induced cytoplasmic and nuclear CDK1
activation at a similar rate as 65 μ M CycB. However, in two em-
bryos, although CycB induced premature myosin dispersion, it
did not induce premature NEB ( Fig. 2 D ). This uncoupling be-
tween the cytoplasmic and nuclear CDK1 activation rate suggests
that different mechanisms regulate these two activities.
To further examine independent activation of cytoplasmic
and nuclear CDK1, we took advantage of the prolonged inter-
phase of nuclear cycle 14. During this cycle, the nuclei arrest in
interphase while the cytoskeleton reorganizes to form furrows
that elongate and encompass each nucleus in a process known
To con rm that the effect of CycB was not con ned to
actin and myosin, we monitored the effect of CycB injection
on Nuf, a Rab11 effector that exhibits a cell cycle dependent
association with the microtubule- organizing center (MTOC;
Fig. 2 C ; Riggs et al., 2003; Cao et al., 2008 ). We  nd that GFP-
Nuf is concentrated at the MTOC through interphase of cycle 13
in the control area. Upon CycB injection, Nuf prematurely
disperses from the MTOC ( Fig. 2 C , blue outlines). Collec-
tively, these observations indicate that high levels of CycB in-
duce local cytoplasmic CDK1 activation and trigger remodeling
of the cytoskeleton.
CycB drives cytoplasmic events
independently of the nuclear cycle
In the aforementioned studies, we observed that cytoplasmic
mitotic events precede nuclear mitotic events. This observation
Figure 1. CycB drives premature NEB and overrides CHX-induced interphase arrest. (A) Injection of CycB induces premature NEB. GFP-H2Av (cyan) em-
bryos were injected with rhodamine-tubulin (red) and with either GST or CycB at the onset of cycle 13 interphase. The schematic describes the injection and
imaging sequence. Insets are enlarged images of the noninjected areas (white boxes) and the CycB-injected areas (blue boxes). The GST and CycB-injected
embryos are representative of four injected embryos for each protein. The nuclear cell cycle stages of the noninjected and CycB-injected areas (white out-
lines) are indicated at the top left and the bottom of the images, respectively. NEB is detected when rhodamine-tubulin invades the nucleoplasm (compare
left inset with right inset, second row). Time is given in minutes/seconds. Arrowheads mark the site of injection. Bars, 20 μ m. (B) Injection of CycB does
not promote premature chromosome condensation before NEB. GFP-H2Av (cyan) embryos were injected with rhodamine-tubulin (red) followed by GST or
CycB injection at the beginning of cycle 13 interphase. The top two rows show the state of chromosome condensation at NEB, which was determined by
the nucleoplasm being fi lled with rhodamine-tubulin. The bottom two rows show the state of chromosome condensation at metaphase. Bars, 5 μ m.
(C) CycB injections overcome the CHX-induced cytoplasmic and nuclear interphase arrest. GFP-H2Av (cyan) embryos were injected with rhodamine-tubulin
(red) followed by CHX at mitosis. GST or CycB was injected at the onset of the following interphase. The schematic describes the injection and imaging
sequence. Arrowheads mark the site of injection. CycB induced NEB, chromosome condensation, and mitotic spindle formation (white outlines; see Video 1,
available at http://www.jcb.org/cgi/content/full/jcb.200801153/DC1). The areas outlined in gray in the third row are enlarged in the bottom row.
Time is given in minutes/seconds. Bars, 20 μ m. (D) Single embryo Western blot of uninjected and CycB-injected embryos arrested at mitosis of cycle 13
with colchicine. Anti-CycB antibodies were used to detect endogenous and injected CycB. Anti-GFP was used to detect a marker for loading controls.
The injected embryo extract reveals an additional 78-kD molecular mass GST-CycB band.
Page 3
JCB • VOLUME 183 • NUMBER 1 • 2008 66
at http://www.jcb.org/cgi/content/full/jcb.200801153/DC1).
However, the timing of NEB was not coordinated with the timing
of myosin dispersion. Although CycB injection induced myosin
dispersion promptly (4.6 ± 0.6, n = 5; Fig. 3, A and C ), NEB
was delayed and occurred 5.5 min after myosin had dispersed
(10.1 ± 2.1, n = 5; Fig. 3, A and C ). This uncoupling between
the cytoplasmic and nuclear cycle was even more dramatic
when CycB was injected 6 9 min after the onset of cellulariza-
tion. Injection of CycB at this later time failed to induce NEB
( Fig. 3, B and C ), yet the cytoplasm entered mitosis rapidly
after CycB injection ( Fig. 3, B and C, blue outlines ; and Fig. S2).
CycB also induced the reorganization of GFP-moesin into
as cellularization. The mechanisms that trigger cellularization
are not understood. To determine whether CycB is limiting for
driving CDK1 activation at cycle 14, we injected CycB at differ-
ent times during cellularization and monitored cytoplasmic and
nuclear CDK1 activation. Cytoplasmic CDK1 activation was
monitored by examining RLC-GFP, GFP-moesin, and GFP-Nuf
dynamics ( Fig. 3 ). Nuclear CDK1 activity was measured by
monitoring NEB (when the cytoplasmic markers invade the
nucleoplasm). When injected early during cellularization (3 5 min
after nuclear envelope formation [NEF]), CycB triggered both
cytoplasmic and nuclear entry into mitosis ( Fig. 3, A and C,
blue and orange outlines, respectively ; and Video 4, available
Figure 2. CycB triggers reorganization of the
cytoskeleton. (A C) RLC-GFP (A), GFP-moesin
(B; see Video 2, available at http://www.jcb
.org/cgi/content/full/jcb.200801153/DC1),
and GFP-Nuf (C; see Video 3) embryos were in-
jected with CycB at early interphase of cycle 13.
The schematic describes the injection and
imaging sequence. The timing of CycB injec-
tion in GFP-Nuf was performed at a time when
Nuf was concentrated at the MTOC (midinter-
phase). Arrowheads mark the sites of injec-
tion. Top row insets are enlarged images of
the control area, which is distant from the site
of injection (white boxes), and bottom row in-
sets are enlarged images of the CycB-injected
area (blue boxes). The RLC-GFP, GFP-moesin,
and GFP-Nuf embryos are representative of
15, 4, and 3 injected embryos, respectively.
NEB is determined when the GFP markers fi ll
the nucleoplasm. The blue and orange outlines
mark the areas where CycB has an effect on
the cytoplasm and the nucleus, respectively.
Time is given in minutes/seconds. Bars: (pan-
els) 20 μ m; (insets) 10 μ m. (D) The timing of
cytoplasmic and nuclear CDK1 activation
after CycB injection was monitored with RLC-
GFP embryos. CycB was injected at 65 or
32 μ M at interphase onset, and the embryo was
examined within 30 s after injection. The tim-
ing of cytoplasmic CDK1 activation was de-
termined when RLC-GFP disappeared from the
focal plane. The timing of nuclear CDK1 ac-
tivation was determined when RLC-GFP fi lled
the nucleoplasm (NEB). For each embryo, we
monitored the timing of cytoplasmic and nu-
clear CDK1 activation in control and injected
areas. Data are represented as mean ± AVD.
n, number of embryos injected.
Page 4
67COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
mutants and normal embryos, we injected the embryos simulta-
neously with CHX and Aph. This provides time to perform the
injections in the exceedingly short cell cycles of the checkpoint-
compromised embryos. In wild-type embryos, the results are
the same for injecting Aph alone or a combined injection of Aph
and CHX (unpublished data). For both a wild-type and a grp
mutant, only the combined injections are described.
In all experiments presented in Fig. 4 , CHX and Aph were
injected together at mitosis of cycle 12 in embryos expressing
RLC-GFP, which allowed us to simultaneously monitor cyto-
plasmic CDK1 activity (myosin dispersion) and nuclear CDK1
activity (NEB). We injected CycB at two different time points
during the next interphase, as represented by the schematic in
Fig. 4 . We injected CycB at the onset of interphase (early injec-
tion) and 10 min later (late injection). Injection of CycB during
early interphase overcame the CHX + Aph induced interphase
arrest ( Fig. 4 A ). The timing of cytoplasmic and nuclear CDK1
activation was similar to embryos in which only CycB was in-
jected ( Fig. 4 D ). The results were strikingly different when
CycB was injected 10 min after the onset of interphase. NEB
occurred in only 3 out of 15 embryos ( Fig. 4, B and D ; and
Fig. S1). Furthermore, NEB was delayed relative to the timing
of cytoplasmic CDK1 activation (9.7 ± 1.9 min vs. 3.8 ± 0.2 min,
n = 3). CDK1 was activated in the cytoplasm but not in the
nucleus in 7 out of 15 embryos ( Fig. 4, B and D ; and Fig. S1).
furrows and the dispersion of Nuf from the MTOC ( Fig. 3 B,
blue outlines; and Video 5). However, there were no cases in
which NEB occurred. CycB injected 15 min after the onset of
cellularization had no effect on the progression of nuclear
or cytoplasmic events ( n = 6; unpublished data). These experi-
ments show that increasing the level of CycB is suf cient to in-
duce an additional syncytial cycle. They demonstrate that CycB
remains limiting for cytoplasmic and, to a lesser extent, nuclear
CDK1 activation during early cellularization. Furthermore, they
provide evidence that CDK1 can be activated in the cytoplasm
independently of its activation in the nucleus.
The Grapes (Grp)-dependent S-phase
checkpoint protects the nucleus from
cytoplasmic CDK1 activity
We next addressed the effects of the S-phase checkpoint activity on
cytoplasmic and nuclear CDK1 activation. To do so, we observed
the effects of CycB injections in syncytial embryos arrested in
interphase by injecting the DNA replication inhibitor aphidicolin
(Aph). When Aph is injected during mitosis of cycle 12, it activates
the S-phase checkpoint and induces a prolonged cytoplasmic and
nuclear interphase arrest at the next cycle (unpublished data).
We next addressed whether CycB can overcome the
S-phase checkpoint induced cytoplasmic and nuclear interphase
arrest. To perform this analysis in checkpoint-compromised
Figure 3. CycB drives cytoplasmic mitotic
events independently of the nuclear cycle
during cellularization. (A) CycB is limiting for
nuclear and cytoplasmic CDK1 activity when
injected at the onset of cycle 14 interphase.
An RLC-GFP embryo was injected with CycB
at cycle 14, 3 min after NEF. The arrowhead
marks the site of injection. At the site of injec-
tion, RLC-GFP disappeared from the cortex
5 min after injection (blue outlines). NEB oc-
curred 5 min later (orange outline; see Video 4,
available at http://www.jcb.org/cgi/content/
full/jcb.200801153/DC1). Time is given in
minutes/seconds. Bar, 20 μ m. (B) CycB drives
the cytoplasm but not the nuclei into mitosis
when injected later during interphase of cycle
14. RLC-GFP, GFP-moesin (see Video 5), and
GFP-Nuf embryos were injected with CycB at
cycle 14, 8 min after NEF. Arrowheads mark
the sites of injection, and blue outlines mark
the areas where the cytoplasm entered mitosis.
Time is given in minutes/seconds. Bars, 20 μ m.
(C) The timing of cytoplasmic and nuclear
CDK1 activation was determined as described in
Fig. 2 . Data are represented as mean ± AVD.
n, number of embryos injected.
Page 5
JCB • VOLUME 183 • NUMBER 1 • 2008 68
sistent premature cytoplasmic and nuclear entry into mitosis at
the site of injection ( Fig. 4, C and D ).
These results support our previous conclusion that cyto-
plasmic CDK1 activity is independent of its activity in the
nucleus. In addition, they suggest that the S-phase checkpoint
is more effective at inhibiting CDK1 in the nucleus than in
the cytoplasm.
CycB has a dynamic localization
through the syncytial cycle and associates
with kinetochores before NEB and
during mitosis
Given that the nucleus can be protected from active cytoplasmic
CDK1 CycB by the Grp-dependent S-phase checkpoint, this
raises the possibility that the checkpoint operates by regulating
CycB nuclear import ( Jin et al., 1998 ). To pursue this idea, we
Finally, in 5 out of 15 embryos, neither cytoplasmic nor nuclear
CDK1 activity was detected ( Fig. 4 B ).
An explanation for the different results upon early and late
injection of CycB into CHX + Aph arrested embryos is that the
S-phase checkpoint is activated in late but not in early injected
embryos. We propose this idea because live analysis using a
Grp-GFP fusion protein reveals that Grp is dispersed in the
cytoplasm during mitosis and accumulates in the nucleus upon
NEF (Fig. S3, available at http://www.jcb.org/cgi/content/full/
jcb.200801153/DC1). Thus, suf cient time after NEF is re-
quired to establish a functional S-phase checkpoint. To test this
hypothesis, we performed the same experiments in the S-phase
checkpoint – compromised mutant grp ( Fig. 4 C ). As with wild-
type embryos, CHX + Aph arrested the nuclei and cytoplasm
of grp embryos in interphase (unpublished data). In contrast to
wild type, CycB injected late into grp embryos triggered con-
Figure 4. CycB injections overcome the S-phase checkpoint induced cytoplasmic cell cycle arrest, and to a lesser extent, nuclear cell cycle arrest. (A C)
Wild-type (WT, A B ) or grp mutant embryos (C) expressing RLC-GFP were injected with CHX and Aph together (CHX + Aph) at metaphase of cycle 12.
The embryos were injected with CycB at the onset of interphase (A, early CycB injection) or 10 min after the onset of interphase (B C, late CycB injection).
The schematic describes the injection and imaging sequence. Arrowheads mark the sites of injection. Blue outlines mark areas with cytoplasmic CDK1
activity, and orange outlines mark areas where NEB occurs. Time is given in minutes/seconds. Bars, 20 μ m. (D) Timing of cytoplasmic and nuclear CDK1
activation was measured as described in Fig. 2 . Data are represented as mean ± AVD. n, number of embryos injected.
Page 6
69COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
we injected CycB-R after CHX + Aph treatment into wild-
type and grp mutant embryos expressing RLC-GFP ( Fig. 6 ).
We assayed three different CycB-R concentrations (100, 56,
and 13 μ M). We monitored the induction of cytoplasmic and
nuclear CDK1 activity by observing cortical myosin dispersion
and NEB, respectively. We also monitored the timing of nuclear
CycB-R localization. Embryos were injected with CHX + Aph
in mitosis of cycle 12 followed by injection of CycB-R 10 min
after the onset of cycle 13 interphase ( Fig. 6 , schematic).
In wild-type embryos injected with 100 μ M CycB-R, the cyto-
plasm entered mitosis, as indicated by the disappearance of
the RLC-GFP signal from the cortex ( Fig. 6, A and F , blue out-
lines; Fig. S1; and Video 6, available at http://www.jcb.org/cgi/
content/full/jcb.200801153/DC1). However, up to 15 min after
CycB-R injection, we did not observe NEB or CycB-R localiza-
tion in the nucleus ( Fig. 6, A and F ; and Video 6). In contrast,
the same injection in grp mutant embryos provoked a rapid
CycB-R nuclear import concomitant with cytoplasmic entry
into mitosis ( Fig. 6, B and F , blue outlines; and Video 7) and
was immediately followed by NEB ( Fig. 6, B and F , orange out-
lines; and Video 7).
Injection of 56 or 13 μ M CycB-R into wild-type embryos
previously treated with CHX + Aph did not trigger cytoplasmic
and nuclear entry into mitosis for up to 15 min ( Fig. 6, C, G,
and H ). The  rst sign of CycB-R in nuclei (appearance of CycB-R
puncta in at least three nuclei) was detected at a mean of 8.9 min
for both CycB-R concentrations ( Fig. 6, C and I , red arrow).
Once in the nucleus, the CycB-R signal at the kinetochore in-
creased progressively. This observation rules out the possibility
that the S-phase checkpoint is preventing CycB nuclear accu-
mulation by increasing the rate of nuclear CycB degradation
( Fig. 6 C , compare the second row with the third row). In contrast,
injection of 56 or 13 μ M CycB-R in the grp mutant triggered
analyzed the subcellular localization of CycB in living embryos
by injecting rhodamine-labeled CycB (CycB-R).
We injected a low concentration of 13 μ M CycB-R at the
onset of interphase of cycles 12 and 13 in embryos expressing
RLC-GFP ( Fig. 5 A ), the kinase Polo fused with GFP (GFP-Polo;
Fig. 5 B ; Moutinho-Santos et al., 1999 ), or the centromere-
associated protein Cid fused with GFP (GFP-Cid; Fig. 5 C ; Schuh
et al., 2007 ). At this concentration, CycB-R did not induce prema-
ture entry into mitosis in  ve out of six wild-type embryos. CycB-R
accumulates at the centrosome and is excluded from the nucleus
during interphase, in agreement with our previous observations
( Huang and Raff, 1999 ). During early prophase, before NEB,
CycB-R maintains its association with the centrosome and accu-
mulates in discrete puncta within the nucleus, indicating that the
recombinant CycB used in these experiments is ef ciently im-
ported into the nucleus ( Fig. 5 A , arrows). After NEB, CycB-R is
primarily associated with the bipolar spindle, consistent with ob-
servations in other cell types ( Pines and Hunter, 1991 ; Yang et al.,
1998 ). In addition, a strong signal was also detected on the meta-
phase plate ( Fig. 5 ). CycB-R colocalized with kinetochore GFP-
Polo before NEB and throughout mitosis ( Fig. 5 B , arrows).
The CycB-R puncta in the nucleus before NEB colocalized with
GFP-Cid ( Fig. 5 C , arrows). This colocalization is observed during
mitosis until late anaphase. Collectively, these observations suggest
an association of CycB-R with the kinetochore that is initiated be-
fore NEB. The speci c localization of CycB-R into kinetochores
before NEB provides a means to monitor CycB nuclear import.
The S-phase checkpoint prevents CycB
nuclear accumulation by maintaining CDK1
in an inactive state
To determine whether the S-phase checkpoint prevents nuclear
CDK1 activation by controlling CycB subcellular localization,
Figure 5. CycB localizes to the kinetochores before NEB. RLC-GFP (A, cyan), GFP-Polo (B, cyan), and GFP-Cid (C, cyan) embryos were injected with
13 μ M CycB-R (red) at interphase of cycle 12 (A) or 13 (B and C). Arrows highlight the punctate nuclear localization of CycB-R (A) and its colocalization
with GFP-Polo (B) and GFP-Cid (C) before NEB. Time is given in minutes/seconds. Bars, 5 μ m.
Page 7
JCB • VOLUME 183 • NUMBER 1 • 2008 70
a CDK1 inhibitor ( Lee et al., 2001 ). Moreover, in mammalian
cultured cells, Wee1 can protect the nucleus from active cyto-
plasmic CDK1 ( Heald et al., 1993 ).
To test whether a threshold of nuclear CDK1 activity is
required to promote CycB nuclear accumulation, we performed
the same experiment as described in the previous paragraph
in wee1 mutant embryos. We monitored the accumulation of
CycB-R in the nucleus and its effect on cytoplasmic and nuclear
CDK1 activation. The injection of CHX + Aph during the previous
mitosis induced an interphase arrest for at least 10 min in a wee1
background. Injection of 100, 56, or 13 μ M CycB-R provoked a
rapid cytoplasmic and nuclear CDK1 activation ( Fig. 6, E H ,
cytoplasmic and nuclear entry into mitosis ( Fig. 6, D, H, and I ,
blue and orange outlines, respectively). The  rst sign of CycB-R
nuclear accumulation before NEB was detected at 2.2 min and
3.7 min after injection of 56 μ M and 13 μ M CycB-R, respec-
tively ( Fig. 6, D and I , red arrow).
These data indicate that the Grp S-phase checkpoint pre-
vents mitotic entry by delaying nuclear CycB accumulation.
It may be that a threshold of nuclear CDK1 activity is required
to trigger CycB import. Activation of the S-phase checkpoint
may prevent nuclear CDK1 from reaching this threshold. Sup-
port for this idea comes from experiments in Xenopus laevis ,
demonstrating that Chk1 phosphorylates and activates Wee1,
Figure 6. The S-phase checkpoint delays nuclear CycB accumulation and CDK1 activation by a Grp and Wee1-dependent mechanism. (A E) Wild-type
(A and C), grp (B and D), and wee1 (E) embryos expressing RLC-GFP (cyan) were injected with a mix of Aph and CHX (CHX + Aph) at metaphase of cycle 12.
10 min after the onset of the next interphase, they were injected with CycB-R (red) at the specifi ed concentrations. The schematic describes the injection
and imaging sequence. Time is given in minutes/seconds (see Videos 6, 7, and 8 for A, B, and E, respectively, available at http://www.jcb.org/cgi/
content/full/jcb.200801153/DC1). The right column of each panel is an enlargement of the area outlined with the white box shown on the fi rst image of
the left column. However, for A and B, the areas are at a focal plane 2 μ m above the focal plane shown in the left columns. Blue outlines mark the areas
with cytoplasmic CDK1 activity, and orange outlines mark the areas undergoing NEB. (C and D) Red arrows point to the nuclear localization of CycB-R.
n, number of embryos injected. Bars: (left columns) 20 μ m; (right columns) 5 μ m. (F H) 100 (F), 56 (G), and 13 μ M (H) CycB-R was injected 10 min after
the onset of interphase in CHX + Aph treated embryos. The timing of cytoplasmic and nuclear CDK1 activation was determined as described in Fig. 2 .
(I) The timing of CycB-R accumulation in nuclei was determined when CycB-R appeared at kinetochores in at least three nuclei. (F I) Data are represented
as mean ± AVD. n, number of embryos injected.
Page 8
71COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
2006 ) and Roscovitine ( Meijer et al., 1997 ). We performed
the same experiments as described in Fig. 6 , monitoring CycB
nuclear import in wild-type, grp , and wee1 embryos but with
CDK1 maintained inactive through injection of CDK1 inhibi-
tors ( Fig. 7 ). In grp and wee1 mutant embryos treated with
CHX + Aph, CycB-R triggered rapid myosin dispersion from
the cortex and NEB ( Fig. 6, D H ). In contrast, the addition of
Roscovitine or RO-3306 before CycB-R injection prevented
both myosin dispersion and NEB ( Fig. 7, B and C ). This demon-
strates that these compounds ef ciently inhibit cytoplasmic and
nuclear CDK1 activity.
In wild-type embryos treated with CHX + Aph and RO-
3306, no nuclear CycB-R signal was detected in three out of
seven embryos for up to 15 min. The  rst signs of CycB-R in
the nuclei were detected at a mean of 12.9 min ( n = 7; Fig. 7 D ).
A similar result was obtained with Roscovitine; no nuclear
CycB-R signal was detected in 5 out of 10 embryos for up to
15 min (unpublished data). In the other  ve injected embryos,
CycB-R started accumulating in the nucleus at a mean of 12.2 min
( Fig. 7, A and D ). In grp mutant embryos injected with CHX +
blue and orange outlines, respectively; and Video 8, available
at http://www.jcb.org/cgi/content/full/jcb.200801153/DC1).
In each experiment, CycB-R nuclear import always occurred
< 3 min after injection ( Fig. 6 I ).
Collectively, these data reveal that high nuclear CDK1 ac-
tivity correlates with rapid CycB nuclear import, and low nu-
clear CDK1 activity correlates with delayed CycB nuclear
import ( Fig. 7 E ). The S-phase checkpoint, acting through Grp
and Wee1, inhibits nuclear CDK1 activity and prevents CycB
nuclear accumulation.
Grp also delays CycB nuclear accumulation
via a mechanism independent of Wee1 and
the state of nuclear CDK1 activity
To determine whether the S-phase checkpoint in uences CycB
nuclear import independently of the state of nuclear CDK1
activity, we monitored CycB-R dynamics in wild-type, grp ,
and wee1 mutant embryos under conditions in which CDK1 is
maintained inactive. This was achieved through injecting dis-
tinct, small molecule CDK1 inhibitors RO-3306 ( Vassilev et al.,
Figure 7. Grp prevents CycB nuclear accumulation by a Wee1-independent mechanism. (A C) Wild-type (A), grp (B), and wee1 (C) mutant embryos ex-
pressing RLC-GFP (cyan) were injected with a mix of Aph and CHX (CHX + Aph) at metaphase of cycle 12. 5 8 min after the onset of the next interphase,
they were injected with 10 mM of the CDK1 inhibitor Roscovitine followed by 13 μ M CycB-R (see Videos 9 and 10 for B and C, available at http://www
.jcb.org/cgi/content/full/jcb.200801153/DC1). The schematic describes the injection and imaging sequence. The right column of each panel is an en-
largement of the area outlined by the white box in the fi rst images of the left column. Red arrows point to the nuclear localization of CycB-R. Time is given
in minutes/seconds. Bars: (left) 20 μ m; (right) 5 μ m. (D) Histograms indicate the time until CycB-R starts accumulating in at least three nuclei in embryos
treated with CHX + Aph and the CDK1 inhibitors Roscovitine or RO-3306. The embryos injected with RO-3306 and Roscovitine were subsequently injected
with 56 μ M and 13 μ M, respectively. Data are represented as mean ± AVD. n, number of embryos injected. *, only 5 out of 10 wild-type embryos injected
with Roscovitine showed nuclear CycB-R accumulation before 15 min, which is the time limit of each video for the Roscovitine assay. Therefore, the mean
timing of nuclear CycB-R import represents only half of the embryos injected. (E) The table summarizes the results in Figs. 6 and 7 . (F) The schematic pre-
sents a model for the control of CycB nuclear import. CycB nuclear accumulation is inhibited by Grp and activated by nuclear CDK1 activity, which is itself
inhibited by Grp and Wee1.
Page 9
JCB • VOLUME 183 • NUMBER 1 • 2008 72
knockdown of the three mitotic cyclins with RNAi blocks the
syncytial cycles but does not affect the timing of cellularization
( McCleland and O Farrell, 2008 ). Both  ndings are compat-
ible with the idea that low levels of CycB are required for the
correct timing of cellularization. Earlier work demonstrated
that inhibition of zygotic transcription also prevents cellu-
larization and drives an additional round of syncytial mitosis
( Edgar et al., 1986 ). Collectively, these results  t a model in
which cellularization is triggered by thresholds of high lev-
els of as yet unknown zygotic gene products and low levels
of CycB. Although injecting CycB within the  rst 9 min of
cycle 14 prevents cellularization, injecting CycB after this
time point has no effect on cellularization. This suggests a nar-
row time window in which the embryo is not fully committed
to cellularization.
CycB injections uncouple the cytoplasmic
and nuclear cycles
Our results also demonstrate that CDK1 can be activated in the
cytoplasm independent of its activation in the nucleus. The pro-
longed interphase of nuclear cycle 14 provided us with an op-
portunity to determine the effects of CycB injections later in
interphase. Early injections of CycB (0 5 min after NEF) pre-
maturely drive nuclear and cytoplasmic events. However, CycB
injections later in interphase (6 9 min after NEF) drive the
cytoplasm but not the nuclei into mitosis. Speci cally, we ob-
serve myosin dispersion while the nuclear envelope remains in-
tact. Given that myosin dispersion requires high CDK1 activity
( Royou et al., 2002 ), these studies indicate that CDK1 is acti-
vated in the cytoplasm but not in the nucleus. This result is con-
sistent with the observation that the cytoplasm continues to
cycle in enucleated frog embryos ( Wasserman and Smith, 1978 ).
It is also in accord with studies in mammalian cultured cells and
Xenopus egg extracts, indicating that the CDK1 CycB complex
is activated in the cytoplasm before its nuclear activation ( Heald
et al., 1993 ; Jin et al., 1996 ; De Souza et al., 2000 ; Draviam et al.,
2001 ; Jackman et al., 2003 ).
The S-phase checkpoint component Grp
prevents nuclear CDK1 activation
In performing CycB injection in embryos treated with Aph
and CHX, we observed that the effects of CycB on nuclear and
cytoplasmic CDK1 activation varied depending on the timing
of injection. CycB injections during early interphase drive both
cytoplasmic and nuclear CDK1 activation, whereas later injec-
tions induced only cytoplasmic CDK1 activation. Moreover, this
nuclear protection from premature cytoplasmic CDK1 activity
upon late CycB injection depends on the S-phase checkpoint
component Grp. Our observation that Grp must be imported into
the nucleus at the beginning of interphase raises the possibility
that time is required for suf cient quantities of Grp to accu-
mulate in the nucleus and create a functional S-phase check-
point. Injection of CycB induces mitosis in both the nuclei and
cytoplasm early in interphase because the S-phase checkpoint
is not fully established. However, later in interphase, Grp has
accumulated suf ciently in the nucleus to completely prohibit
nuclear CDK1 activation.
Aph and either RO-3306 or Roscovitine, no sign of cytoplasmic
and nuclear CDK1 activity was observed. In contrast to wild-
type embryos, CycB-R rapidly accumulated in the nucleus in
all injected embryos. The  rst signs of CycB-R in the nucleus
were detected at a mean of 5.5 min and 5.8 min in Roscovitine-
and RO-3306 injected embryos, respectively ( Fig. 7, B and D ;
and Video 9, available at http://www.jcb.org/cgi/content/full/
jcb.200801153/DC1). In contrast, in the wee1 mutant, no nu-
clear CycB-R was detected in any Roscovitine- or RO-3306
injected embryos for up to 15 min ( Fig. 7, C and D ; and Video 10).
These data reveal that in addition to its role in maintaining
nuclear CDK1 in an inactive state, Grp prevents nuclear accu-
mulation of CycB through a separate mechanism not shared by
Wee1 ( Fig. 7 F ).
Discussion
Increased levels of CycB induce premature
cytoplasmic and nuclear CDK1 activation
In this study, we directly address the role of CycB in driving the
syncytial divisions in the Drosophila embryo by demonstrating
that increased levels of CycB are suf cient to trigger nuclear and
cytoplasmic mitotic entry. Unlike more conventional cell cycles,
the maternal pool of CycB is not fully degraded during these
syncytial mitotic divisions; therefore, it was not clear whether
CycB was limiting for CDK1 activation ( Edgar et al., 1994 ).
Our  nding that inhibition of protein synthesis induced a prolonged
interphase in wild-type and S-phase checkpoint compromised
embryos indicates that new rounds of protein synthesis are
required to drive the late syncytial cycles. Of more signi cance
is the fact that injection of CycB into these interphase-arrested
embryos ef ciently drives cytoplasmic and nuclear entry into
mitosis. Our data imply that, even though CycB is not fully de-
graded at mitosis, new rounds of CycB synthesis are required
to trigger the next mitosis. Support for this idea comes from
the fact that increasing the maternal copies of CycB affects the
timing of entry into mitosis during the late syncytial cycles
( Ji et al., 2004 ). It may also be that the pool of endogenous
CycB is maintained in a form that prevents CDK1 activation
through posttranslational modi cations and that the exogenous
CycB lacks these modi cations.
Careful analysis of the timing of cytoplasmic and nuclear
mitotic events in the last syncytial cycle reveals that initiation of
chromosome condensation and reorganization of the cytoskele-
ton occur before NEB. We have found that, even in the absence
of protein synthesis, exogenous CycB can ef ciently coordinate
the reorganization of the cytoskeleton with NEB. However, ex-
ogenous CycB does not preserve the order of the nuclear mitotic
events because the chromosomes did not condense before NEB.
There are three maternal mitotic cyclins in a Drosophila em-
bryo, cyclin A, B, and B3, with overlapping functions ( Jacobs
et al., 1998 ). One interesting idea is that the proper ratio of the
three mitotic cyclins is required to maintain the order of the nu-
clear events.
We have observed that increased levels of CycB at the
onset of cellularization during cycle 14 drive an additional
round of syncytial mitosis. A recent study has shown that the
Page 10
73COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
Grp also delays CycB nuclear accumulation
by a mechanism independent of Wee1
Signi cantly, our observation that Grp-mediated inhibition of
CycB nuclear import is independent of both Wee1 and the state
of nuclear CDK1 activity reveals an additional mechanism by
which the S-phase checkpoint prevents nuclear CDK1 activation.
Monitoring CycB nuclear import in Aph and CHX-treated wild-
type wee1 and grp mutant embryos in which CDK1 is main-
tained in an inactive state reveals that CycB is rapidly imported
into the nucleus only in the grp mutant. These results indicate
a novel mechanism by which the S-phase checkpoint prevents
mitotic entry: a Grp-dependent but Wee1-independent mecha-
nism that prevents CycB nuclear accumulation. A satisfying
aspect of this result is that it readily explains why bypassing
interphase arrest caused by DNA damage not only requires a
constitutively active form of CDK1 but also nuclear localized
CycB ( Jin et al., 1998 ). It would be of interest to determine
whether Grp inhibits nuclear import of other cyclins as well.
The observation that CycB rapidly accumulates in the
nucleus in the wee1 mutant where Grp is fully active contrasts
with our results that Grp delays CycB nuclear import. One idea
is that, in the absence of wee1 , the active form of nuclear CDK1
associated with basal levels of CycB promotes CycB (and more
likely the CDK1 CycB complex) nuclear import, which in turn
increases nuclear CDK1 activity. This triggers a positive feed-
back loop that ef ciently bypasses the Grp inhibitory signal.
Model for the regulation of cytoplasmic and
nuclear CDK1 activity
This study provides a model to explain the differential regu-
lation of cytoplasmic and nuclear CDK1 in a normal cycle.
We speculate that, at interphase, the slow rate of CycB synthesis
in the cytoplasm provides suf cient time for cytoplasmic CDK1
inhibitors, such as Wee1 and Myt1, to prevent cytoplasmic
CDK1 activation ( Price et al., 2000 ; Stumpff et al., 2004 ; Jin
et al., 2005 ). However, nuclear CDK1 inhibition is maintained
by controlling CycB nuclear accumulation. At interphase, the
rate of CycB nuclear translocation is low because of inhibition
of nuclear CDK1 by Wee1 and Grp-dependent inhibition of
CycB transport. Upon completion of S phase, Grp and Wee1
activity decreases. This triggers cytoplasmic CDK1 activation
and initiation of cytoplasmic mitosis. This decrease in Grp and
Wee1 activity also allows the abrupt CycB nuclear translocation
catalyzed by nuclear CDK1 activity, thus increasing CDK1 ac-
tivity to a level suf cient for triggering NEB.
Materials and methods
Drosophila stocks
The fl y stocks GFP-H2Av ( Clarkson and Saint, 1999 ), GFP-moesin ( Edwards
et al., 1997 ), RLC-GFP ( Royou et al., 2004 ), GFP-Nuf ( Riggs et al., 2007 ),
GFP-Polo ( Moutinho-Santos et al., 1999 ), and GFP-Cid ( Schuh et al., 2007 )
were used in these studies. The grp embryos were derived from a female
homozygote for the grp
1
null allele ( Fogarty et al., 1994 ). The wee1 em-
bryos were derived from females carrying the null allele d wee1
ES1
over
Df(2L) dwee1
wo5
defi ciency ( Price et al., 2000 ).
GFP-Grp cloning
GFP-Grp bearing transgenics were constructed by ligating a full-length grp
cDNA into pEGFP-C3 (Clontech Laboratories, Inc.) upstream of the GFP
How does Grp protect the nucleus from active cytoplas-
mic CDK1? In response to damaged or incompletely replicated
DNA, Chk1 phosphorylates Cdc25, altering its relative import/
export rates such that it remains cytoplasmic ( Furnari et al., 1997 ;
Peng et al., 1997 ; Sanchez et al., 1997 ; Takizawa and Morgan,
2000 ). Additionally, Chk1 has been shown to phosphorylate and
activate Wee1 ( Lee et al., 2001 ). Although these  ndings remain
to be demonstrated in Drosophila , it provides an explanation for the
Grp-dependent protection of the nucleus from active cytoplas-
mic CDK1. Grp maintains the inhibitory tyrosine phosphory-
lation on nuclear CDK1 via activation of Wee1 and exclusion
of Cdc25 from the nucleus. Support for this idea comes from a
study in mammalian cultured cells in which overexpession of
Wee1 prevents NEB in the presence of high cytoplasmic CDK1
activity ( Heald et al., 1993 ). The fact that increased levels of
cytoplasmic CycB overrides the cytoplasmic but not the nuclear
S-phase checkpoint inhibitory signal suggests that the S-phase
checkpoint is more ef cient at inhibiting CDK1 in the nucleus.
One idea is that the level of S-phase checkpoint activity is higher
in the nucleus than in the cytoplasm. This notion is supported
by experiments in binucleate yeast and sea urchin embryos in
which one of the two nuclei has altered DNA. Entry into mito-
sis is delayed in the nucleus with altered DNA with respect to
the other normal nucleus and the common cytoplasm ( Sluder
et al., 1995 ; Demeter et al., 2000 ). In addition, our observa-
tions, as well as others , that Grp and Wee1 are predominantly
nuclear during S phase support this idea ( Heald et al., 1993 ;
Purdy et al., 2005 ).
Grp and Wee1 delay CycB
nuclear accumulation
The subcellular localization of CycB plays an important role
in regulating nuclear CDK1 activity ( Takizawa and Morgan,
2000 ). Speci cally, it has been shown in mammalian cultured
cells that CycB is predominantly cytoplasmic during the G2
arrest caused by DNA damage, and a nuclear-targeted CycB can
partially bypass this arrest ( Smeets et al., 1994 ; Jin et al., 1998 ).
To test the hypothesis that the S-phase checkpoint affects CycB
subcellular localization, we injected CycB-R and monitored its
dynamics after inhibition of DNA replication in wild-type em-
bryos and embryos lacking Grp or Wee1. In a normal cycle, we
observed that CycB-R is imported into the nucleus and forms
discrete puncta, which colocalize with GFP-Polo and GFP-Cid,
suggesting an association of CycB-R with kinetochores before
NEB. This provides an excellent means to monitor CycB nu-
clear accumulation.
In embryos with high S-phase checkpoint activity, CycB
nuclear accumulation was impaired. In contrast, in grp mutant
embryos, CycB rapidly accumulated in the nucleus. One expla-
nation is that in the absence of Grp, Wee1 activity is low, result-
ing in high nuclear CDK1 activity, which in turn promotes CycB
import. Consistent with this hypothesis, we also found that CycB-R
is rapidly imported into the nucleus and promotes NEB in wee1
mutant embryos. Interestingly, the observation that high levels
of CycB ef ciently triggered cytoplasmic CDK1 activation but
failed to localize in the nucleus reveals that cytoplasmic CDK1
activity is not suf cient to promote CycB nuclear import.
Page 11
JCB • VOLUME 183 • NUMBER 1 • 2008 74
until they reached interphase of cycle 13. They were injected with 0.5 mM
colchicine and monitored until they reached metaphase. Each embryo was
either collected and boiled in 10 μ l of sample buffer or injected with 65 μ M
CycB and immediately collected and boiled in 10 μ l of sample buffer.
The samples were loaded on a 10% acrylamide gel and transferred onto
the nylon membrane, and the membrane was probed with anti-CycB to re-
veal endogenous CycB and GST-CycB. Membranes were also probed with
anti-GFP (Roche) to reveal RLC-GFP as a loading control. As expected, the
injected embryo extract reveals an additional higher molecular weight
GST-CycB band ( Fig. 1 D , injected CycB). The intensities of the endog-
enous and injected CycB bands were estimated to be equivalent. Because
CycB is injected at the center of the embryo lengthwise and diffuses very
slowly through a fi fth of the embryo, we estimated that, at the center of the
gradient, the injected CycB is < 10-fold the amount of endogenous CycB at
mitosis of cycle 13.
Online supplemental material
Fig. S1 shows RLC-GFP signal intensities over time in control and CycB-
injected areas of embryos previously injected or not injected with CHX
and Aph. Fig. S2 shows a saggital view of an embryo expressing RLC-
GFP injected with CycB 8 min after the onset of cellularization. Fig. S3
shows the dynamics of Grp-GFP from cycle 13 through cycle 14 in an
embryo previously injected with rhodamine-tubulin. Video 1 corresponds
to the embryo presented in Fig. 1 C . Video 2 corresponds to the embryo
presented in Fig. 2 B . Video 3 corresponds to the embryo presented in
Fig. 2 C . Video 4 corresponds to the embryo presented in Fig. 3 A . Video 5
corresponds to the embryo presented in Fig. 3 B . Video 6 corresponds
to the embryo presented in Fig. 6 A . Video 7 corresponds to the embryo
presented in Fig. 6 B . Video 8 corresponds to the embryo presented
in Fig. 6 E . Video 9 corresponds to the embryo presented in Fig. 7 B ,
where only CycB-R is visualized. Video 10 corresponds to the embryo
presented in Fig. 7 C , where only CycB-R is visualized. Online sup-
plemental material is available at http://www.jcb.org/cgi/content/full/
jcb.200801153/DC1.
We thank K. Yu (Exploratorium, San Francisco, CA) for the GFP-Grp construct
and transgenic stock, S. Campbell (University of Alberta, Edmonton, Canada)
and E. Homola for the Cy5-histone and critical reading of the manuscript, and
M. McCleland and P. O Farrell for helpful discussions.
A. Royou and W. Sullivan were supported by grants from the California
Institute for Regenerative Medicine and the National Institutes of Health
(GM046409), respectively. D. McCusker and D. Kellogg were supported by
a grant from the National Institutes of Health (GM69602).
Submitted: 24 January 2008
Accepted: 3 September 2008
References
Cao , J. , R. Albertson , B. Riggs , C.M. Field , and W. Sullivan . 2008 . Nuf, a Rab11
effector, maintains cytokinetic furrow integrity by promoting local actin
polymerization. J. Cell Biol. 182 : 301 – 313 .
Clarkson , M. , and R. Saint . 1999 . A His2AvDGFP fusion gene complements
a lethal His2AvD mutant allele and provides an in vivo marker for
Drosophila chromosome behavior. DNA Cell Biol. 18 : 457 – 462 .
Crest , J. , N. Oxnard , J.Y. Ji , and G. Schubiger . 2007 . Onset of the DNA replica-
tion checkpoint in the early Drosophila embryo. Genetics . 175 : 567 – 584 .
De Souza , C.P. , K.A. Ellem , and B.G. Gabrielli . 2000 . Centrosomal and cyto-
plasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events. Exp.
Cell Res. 257 : 11 – 21 .
Demeter , J. , S.E. Lee , J.E. Haber , and T. Stearns . 2000 . The DNA damage check-
point signal in budding yeast is nuclear limited. Mol. Cell . 6 : 487 – 492 .
Draviam , V.M. , S. Orrechia , M. Lowe , R. Pardi , and J. Pines . 2001 . The localiza-
tion of human cyclins B1 and B2 determines CDK1 substrate speci city
and neither enzyme requires MEK to disassemble the Golgi apparatus.
J. Cell Biol. 152 : 945 – 958 .
Edgar , B.A. , C.P. Kiehle , and G. Schubiger . 1986 . Cell cycle control by the nucleo-
cytoplasmic ratio in early Drosophila development. Cell . 44 : 365 – 372 .
Edgar , B.A. , F. Sprenger , R.J. Duronio , P. Leopold , and P.H. O ’ Farrell . 1994 .
Distinct molecular mechanism regulate cell cycle timing at successive
stages of Drosophila embryogenesis. Genes Dev. 8 : 440 – 452 .
Edwards , K.A. , M. Demsky , R.A. Montague , N. Weymouth , and D.P. Kiehart .
1997 . GFP-moesin illuminates actin cytoskeleton dynamics in living
tissue and demonstrates cell shape changes during morphogenesis in
Drosophila . Dev. Biol. 191 : 103 – 117 .
construct. The GFP-Grp subclone was ligated into the Germ-10 transforma-
tion vector. Transgenics were generated by standard methods.
CycB purifi cation
Two independent GST-CycB purifi cations were performed and produced
identical results. CycB cDNA was cloned at its N terminal to GST in pGEX-1
vector. The recombinant protein was overexpressed and purifi ed from
4 liters of XL-10 bacteria culture (Stratagene) grown in 2XYT (tryptone, yeast
extract, and NaCl). The culture was grown at RT until it reached an OD of
0.8. The cells were induced with 100 μ M IPTG for 12 h at RT. The cells were
harvested by spinning them for 5 min at 5,000 rpm. The pellet was ground
in liquid nitrogen for 15 min. The powder was dissolved in 5 vol PBS that
contained 0.5% Tween 20, 1 mM PMSF, and 1 M NaCl. The solution was
sonicated three times for 20 s. 10 mM DTT was added to the solution before
its ultracentrifugation at 40,000 rpm for 1 h in a 60.2 Ti rotor (Beckman
Coulter). The supernatant was loaded onto a 9-ml glutathione-agarose col-
umn (Sigma-Aldrich) for a period of 2 4 h. The column was extensively
washed with PBS, pH 7.4, 1 M NaCl, 0.05% Tween 20, and 0.5 mM DTT
and rinsed with a 1-vol column with wash buffer without Tween 20. The pro-
tein was eluted with 50 mM Tris, pH 8.1, containing 500 mM KCl and
5 mM of reduced glutathione. The fractions were pooled and dialyzed ex-
tensively into 50 mM Hepes, pH 7.6, 500 mM KCl, and 30% glycerol.
In all of the experiments shown in Figs. 1 4 , CycB was injected at a concen-
tration of 65 μ M. A recent study used such purifi ed recombinant GST-CycB
and has demonstrated that it retains its ability to bind and activate CDK1 in
vitro ( Edgar et al., 1994 ). Another recent study has shown that GST-CycB is
resistant to degradation in Xenopus embryo extracts ( Su et al., 1998 ). Our
studies have focused on mitotic entry, not exit, so the fact that it is not de-
graded should not affect the interpretation of our experiments.
CycB labeling
CycB was coupled with NHS-rhodamine (Thermo Fisher Scientifi c) in a 1:4
ratio. The solution was mixed gently and sat at RT for 75 min. The solution
was loaded onto a 3-ml Sephadex G-25 column (GE Healthcare). The col-
umn was washed with 50 mM Hepes, pH 7.6, 500 mM KCl, and 30%
glycerol. The absorbance of the fractions was measured with a NanoDrop
(Eppendorf). The stochiometry of the labeling was calculated as 0.6 mol
NHS-rhodamine per mole of CycB.
Microscopy
Microscopy was performed at RT with an inverted microscope (DMIRB;
Leitz) equipped with a scanning laser confocal imaging system (TCS NT;
Leica). Images were acquired with a 63 × lens and confocal software (ver-
sion 2.61; Leica). All embryos were monitored at three different z positions
every 30 s for various amounts of time. Each image in all fi gures represents
a single focal plane.
Microinjection
The embryos were prepared for microinjection as follows. They were decho-
rionated by hand on a double-sided sticky tape and aligned on a coverslip
covered with glue. They were dehydrated and covered with halocarbon oil.
The embryos were injected at the onset of interphase 2 min after NEF with
either dialyzed buffer (50 mM Hepes, pH 7.6, 500 mM KCl, and 30%
glycerol), 71 μ M GST, or 65, 32, 13, and 6.5 μ M CycB. The embryos were
injected at the center lengthwise and one third heightwise toward the surface.
The same hole was used for double and triple injections. The embryos were
monitored within 30 s after injection. Injection of CycB at 13 and 6.5 μ M
did not have a signifi cant effect on timing of cytoplasmic and nuclear entry
into mitosis in wild-type embryos ( n = 6 for each concentration). Aph and
CHX (Sigma-Aldrich) were used at fi nal concentrations of 300 μ M and
1 mg/ml, respectively, in 2% DMSO and injected either on their own or
mixed together during mitosis. CycB-R was injected at 13, 56, or 100 μ M.
Roscovitine and RO-3306 (EMD) were injected at 10 mM in 100% DMSO.
Injection of 100% DMSO was used as a control. Rhodamine-conjugated
tubulin (cytoskeleton) and Cy5-labeled histone (provided by E. Homola, Uni-
versity of Alberta, Edmonton, Canada) were injected at interphase of the
previous cycle. The quantifi cation of the RLC-GFP signal was performed by
drawing two regions of interest of the same size, one near the site of CycB
injection and one in a region not affected by CycB injection. The mean pixel
intensity for each region was calculated using the Leica software.
Single-embryo Western blot and determination of the amount of injected
CycB relative to the level of endogenous CycB
RLC-GFP embryos were prepared for microinjection as described in the
previous paragraph. They were monitored with the confocal microscope
Page 12
75COORDINATION OF CYTOPLASMIC AND NUCLEAR MITOTIC ENTRY • Royou et al.
Pines , J. , and T. Hunter . 1991 . Human cyclins A and B1 are differentially located
in the cell and undergo cell cycle-dependent nuclear transport. J. Cell
Biol. 115 : 1 – 17 .
Pines , J. , and T. Hunter . 1994 . The differential localization of human cyclins
A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J.
13 : 3772 – 3781 .
Price , D. , S. Rabinovitch , P.H. O ’ Farrell , and S.D. Campbell . 2000 . Drosophila
wee1 has an essential role in the nuclear divisions of early embryogen-
esis. Genetics . 155 : 159 – 166 .
Purdy , A. , L. Uyetake , M.G. Cordeiro , and T.T. Su . 2005 . Regulation of mitosis
in response to damaged or incompletely replicated DNA require different
levels of Grapes ( Drosophila Chk1). J. Cell Sci. 118 : 3305 – 3315 .
Riggs , B. , W. Rothwell , S. Mische , G.R. Hickson , J. Matheson , T.S. Hays , G.W.
Gould , and W. Sullivan . 2003 . Actin cytoskeleton remodeling during
early Drosophila furrow formation requires recycling endosomal compo-
nents Nuclear-fallout and Rab11. J. Cell Biol. 163 : 143 – 154 .
Riggs , B. , B. Fasulo , A. Royou , S. Mische , J. Cao , T.S. Hays , and W. Sullivan .
2007 . The concentration of Nuf, a Rab11 effector, at the microtubule-
organizing center is cell cycle-regulated, dynein-dependent, and coincides
with furrow formation. Mol. Biol. Cell . 18 : 3313 – 3322 .
Royou , A. , W. Sullivan , and R. Karess . 2002 . Cortical recruitment of nonmuscle
myosin II in early syncytial Drosophila embryos: its role in nuclear axial
expansion and its regulation by Cdc2 activity. J. Cell Biol. 158 : 127 – 137 .
Royou , A. , C. Field , J.C. Sisson , W. Sullivan , and R. Karess . 2004 . Reassessing
the role and dynamics of nonmuscle myosin II during furrow formation in
early Drosophila embryos. Mol. Biol. Cell . 15 : 838 – 850 .
Sanchez , Y. , C. Wong , R.S. Thoma , R. Richman , Z. Wu , H. Piwnica-Worms ,
and S.J. Elledge . 1997 . Conservation of the Chk1 checkpoint pathway
in mammals: linkage of DNA damage to Cdk regulation through Cdc25.
Science . 277 : 1497 – 1501 .
Schuh , M. , C.F. Lehner , and S. Heidmann . 2007 . Incorporation of Drosophila
CID/CENP-A and CENP-C into centromeres during early embryonic
anaphase. Curr. Biol. 17 : 237 – 243 .
Sibon , O.C. , V.A. Stevenson , and W.E. Theurkauf . 1997 . DNA-replication
checkpoint control at the Drosophila midblastula transition. Nature .
388 : 93 – 97 .
Sluder , G. , E.A. Thompson , C.L. Rieder , and F.J. Miller . 1995 . Nuclear enve-
lope breakdown is under nuclear not cytoplasmic control in sea urchin
zygotes. J. Cell Biol. 129 : 1447 – 1458 .
Smeets , M.F. , E.H. Mooren , and A.C. Begg . 1994 . The effect of radiation on
G2 blocks, cyclin B expression and cdc2 expression in human squamous
carcinoma cell lines with different radiosensitivities. Radiother. Oncol.
33 : 217 – 227 .
Stif er , L.A. , J.Y. Ji , S. Trautmann , C. Trusty , and G. Schubiger . 1999 . Cyclin
A and B functions in the early Drosophila embryo. Development .
126 : 5505 – 5513 .
Stumpff , J. , T. Duncan , E. Homola , S.D. Campbell , and T.T. Su . 2004 . Drosophila
Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis.
Curr. Biol. 14 : 2143 – 2148 .
Su , T.T. , F. Sprenger , P.J. DiGregorio , S.D. Campbell , and P.H. O ’ Farrell . 1998 .
Exit from mitosis in Drosophila syncytial embryos requires proteolysis
and cyclin degradation, and is associated with localized dephosphoryla-
tion. Genes Dev. 12 : 1495 – 1503 .
Takizawa , C.G. , and D.O. Morgan . 2000 . Control of mitosis by changes in the
subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr. Opin. Cell
Biol. 12 : 658 – 665 .
Vassilev , L.T. , C. Tovar , S. Chen , D. Knezevic , X. Zhao , H. Sun , D.C.
Heimbrook , and L. Chen . 2006 . Selective small-molecule inhibitor re-
veals critical mitotic functions of human CDK1. Proc. Natl. Acad. Sci.
USA . 103 : 10660 – 10665 .
Walworth , N.C. 2001 . DNA damage: Chk1 and Cdc25, more than meets the eye.
Curr. Opin. Genet. Dev. 11 : 78 – 82 .
Wasserman , W.J. , and L.D. Smith . 1978 . The cyclic behavior of a cytoplasmic fac-
tor controlling nuclear membrane breakdown. J. Cell Biol. 78 : R15 – R22 .
Yang , J. , E.S. Bardes , J.D. Moore , J. Brennan , M.A. Powers , and S. Kornbluth .
1998 . Control of cyclin B1 localization through regulated binding of the
nuclear export factor CRM1. Genes Dev. 12 : 2131 – 2143 .
Enoch , T. , M. Peter , P. Nurse , and E.A. Nigg . 1991 . p34cdc2 acts as a lamin ki-
nase in  ssion yeast. J. Cell Biol. 112 : 797 – 807 .
Ferrell , J.E., Jr . 2002 . Self-perpetuating states in signal transduction: positive
feedback, double-negative feedback and bistability. Curr. Opin. Cell Biol.
14 : 140 – 148 .
Fogarty , P. , R.F. Kalpin , and W. Sullivan . 1994 . The Drosophila maternal-
effect mutation grapes causes a metaphase arrest at nuclear cycle 13.
Development . 120 : 2131 – 2142 .
Fogarty , P. , S.D. Campbell , R. Abu-Shumays , B.S. Phalle , K.R. Yu , G.L. Uy ,
M.L. Goldberg , and W. Sullivan . 1997 . The Drosophila grapes gene is
related to checkpoint gene chk1/rad27 and is required for late syncytial
division  delity. Curr. Biol. 7 : 418 – 426 .
Furnari , B. , N. Rhind , and P. Russell . 1997 . Cdc25 mitotic inducer targeted by
chk1 DNA damage checkpoint kinase. Science . 277 : 1495 – 1497 .
Hagting , A. , C. Karlsson , P. Clute , M. Jackman , and J. Pines . 1998 . MPF local-
ization is controlled by nuclear export. EMBO J. 17 : 4127 – 4138 .
Hagting , A. , M. Jackman , K. Simpson , and J. Pines . 1999 . Translocation of cy-
clin B1 to the nucleus at prophase requires a phosphorylation-dependent
nuclear import signal. Curr. Biol. 9 : 680 – 689 .
Heald , R. , M. McLoughlin , and F. McKeon . 1993 . Human wee1 maintains mi-
totic timing by protecting the nucleus from cytoplasmically activated
Cdc2 kinase. Cell . 74 : 463 – 474 .
Huang , J. , and J.W. Raff . 1999 . The disappearance of cyclin B at the end of mito-
sis is regulated spatially in Drosophila cells. EMBO J. 18 : 2184 – 2195 .
Jackman , M. , C. Lindon , E.A. Nigg , and J. Pines . 2003 . Active cyclin B1-Cdk1
rst appears on centrosomes in prophase. Nat. Cell Biol. 5 : 143 – 148 .
Jacobs , H.W. , J.A. Knoblich , and C.F. Lehner . 1998 . Drosophila Cyclin B3 is
required for female fertility and is dispensable for mitosis like Cyclin B.
Genes Dev. 12 : 3741 – 3751 .
Ji , J.Y. , J.M. Squirrell , and G. Schubiger . 2004 . Both cyclin B levels and DNA-
replication checkpoint control the early embryonic mitoses in Drosophila .
Development . 131 : 401 – 411 .
Jin , P. , Y. Gu , and D.O. Morgan . 1996 . Role of inhibitory CDC2 phosphorylation
in radiation-induced G2 arrest in human cells. J. Cell Biol. 134 : 963 – 970 .
Jin , P. , S. Hardy , and D.O. Morgan . 1998 . Nuclear localization of cyclin B1 con-
trols mitotic entry after DNA damage. J. Cell Biol. 141 : 875 – 885 .
Jin , Z. , E.M. Homola , P. Goldbach , Y. Choi , J.A. Brill , and S.D. Campbell .
2005 . Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates mul-
tiple aspects of cell cycle behavior during gametogenesis. Development .
132 : 4075 – 4085 .
Lamb , N.J. , A. Fernandez , A. Watrin , J.C. Labbe , and J.C. Cavadore . 1990 .
Microinjection of p34cdc2 kinase induces marked changes in cell shape,
cytoskeletal organization, and chromatin structure in mammalian  bro-
blasts. Cell . 60 : 151 – 165 .
Lee , J. , A. Kumagai , and W.G. Dunphy . 2001 . Positive regulation of Wee1 by
Chk1 and 14-3-3 proteins. Mol. Biol. Cell . 12 : 551 – 563 .
Li , J. , A.N. Meyer , and D.J. Donoghue . 1997 . Nuclear localization of cyclin B1
mediates its biological activity and is regulated by phosphorylation. Proc.
Natl. Acad. Sci. USA . 94 : 502 – 507 .
McCleland , M.L. , and P.H. O ’ Farrell . 2008 . RNAi of mitotic cyclins in Drosophila
uncouples the nuclear and centrosome cycle. Curr. Biol. 18 : 245 – 254 .
Meijer , L. , A. Borgne , O. Mulner , J.P. Chong , J.J. Blow , N. Inagaki , M. Inagaki ,
J.G. Delcros , and J.P. Moulinoux . 1997 . Biochemical and cellular effects
of roscovitine, a potent and selective inhibitor of the cyclin-dependent
kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243 : 527 – 536 .
Melo , J. , and D. Toczyski . 2002 . A uni ed view of the DNA-damage checkpoint.
Curr. Opin. Cell Biol. 14 : 237 – 245 .
Morgan , D.O. 2006 . The Cell Cycle: Principles of Control.
New Science Press
Ltd, London . 297 pp .
Moutinho-Santos , T. , P. Sampaio , I. Amorim , M. Costa , and C.E. Sunkel . 1999 .
In vivo localisation of the mitotic POLO kinase shows a highly dynamic
association with the mitotic apparatus during early embryogenesis in
Drosophila . Biol. Cell . 91 : 585 – 596 .
Onischenko , E.A. , N.V. Gubanova , E.V. Kiseleva , and E. Hallberg . 2005 . Cdk1
and okadaic acid-sensitive phosphatases control assembly of nuclear pore
complexes in Drosophila embryos. Mol. Biol. Cell . 16 : 5152 – 5162 .
Ookata , K. , S. Hisanaga , E. Okumura , and T. Kishimoto . 1993 . Association of
p34cdc2/cyclin B complex with microtubules in star sh oocytes. J. Cell
Sci. 105 : 873 – 881 .
Peng , C.Y. , P.R. Graves , R.S. Thoma , Z. Wu , A.S. Shaw , and H. Piwnica-
Worms . 1997 . Mitotic and G2 checkpoint control: regulation of 14-3-3
protein binding by phosphorylation of Cdc25C on serine-216. Science .
277 : 1501 – 1505 .
Peter , M. , J. Nakagawa , M. Doree , J.C. Labbe , and E.A. Nigg . 1990 . In vitro dis-
assembly of the nuclear lamina and M phase-speci c phosphorylation of
lamins by cdc2 kinase. Cell . 61 : 591 – 602 .
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