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 interphase 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 defined 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 μM GST or 65 μM 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 (, 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 (, CycB, white outlines). Premature NEB occurs as a wave probably caused by the slow diffusion of CycB from the injection site. These observations indicate that CycB injection at 65 μM is sufficient 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 nuclear 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 (, 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 (, bottom row).
Figure 1. CycB drives premature NEB and overrides CHX-induced interphase arrest. (A) Injection of CycB induces premature NEB. GFP-H2Av (cyan) embryos were injected with rhodamine-tubulin (red) and with either GST or CycB at the onset of cycle 13 interphase. The (more ...)
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 (, left). Exogenous CycB locally overcame the CHX-induced interphase arrest as indicated by NEB and rapid spindle formation (, 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 μM of exogenous CycB at the center of the gradient was less than fivefold the level of endogenous CycB at mitosis (see Materials and methods).
Injection of CycB prematurely drives the cytoplasm into mitosis
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; ; and see Fig. S1 for quantification 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; , blue outlines). Interestingly, in both areas, RLC-GFP disappearance always preceded NEB (, orange outlines). A previous study has shown that RLC-GFP dispersion is driven by CDK1 activity (Royou et al., 2002
). Therefore, our results indicate 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 μm below the plasma membrane, only furrows that have extended to this depth are visualized (). In control and CycB-injected regions, F-actin furrows progressed to this depth at 11.5 min. and 3.5 min after injection, respectively (, blue outlines). These experiments indicate that exogenous CycB drives premature reorganization of the actin cytoskeleton.
To confirm that the effect of CycB was not confined 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; ; Riggs et al., 2003
; Cao et al., 2008
). We find 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 (, blue outlines). Collectively, these observations indicate that high levels of CycB induce 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 raises the possibility that cytoplasmic and nuclear CDK1 activities 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 concentration of 32 μM (). In six out of eight injected embryos, 32-μM CycB dilution induced cytoplasmic and nuclear CDK1 activation at a similar rate as 65 μM CycB. However, in two embryos, although CycB induced premature myosin dispersion, it did not induce premature NEB (). This uncoupling between 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 interphase 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 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 different 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 (). 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 (, blue and orange outlines, respectively; and Video 4, available 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; ), NEB was delayed and occurred 5.5 min after myosin had dispersed (10.1 ± 2.1, n
= 5; ). This uncoupling between the cytoplasmic and nuclear cycle was even more dramatic when CycB was injected 6–9 min after the onset of cellularization. Injection of CycB at this later time failed to induce NEB (), yet the cytoplasm entered mitosis rapidly after CycB injection (, blue outlines; and Fig. S2). CycB also induced the reorganization of GFP-moesin into furrows and the dispersion of Nuf from the MTOC (, 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 experiments show that increasing the level of CycB is sufficient to induce 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.
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 (more ...)
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 mutants and normal embryos, we injected the embryos simultaneously 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 , CHX and Aph were injected together at mitosis of cycle 12 in embryos expressing RLC-GFP, which allowed us to simultaneously monitor cytoplasmic 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 . We injected CycB at the onset of interphase (early injection) and 10 min later (late injection). Injection of CycB during early interphase overcame the CHX + Aph–induced interphase arrest (). The timing of cytoplasmic and nuclear CDK1 activation was similar to embryos in which only CycB was injected (). 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 (; 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 (; and Fig. S1). Finally, in 5 out of 15 embryos, neither cytoplasmic nor nuclear CDK1 activity was detected ().
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 (more ...)
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, sufficient time after NEF is required to establish a functional S-phase checkpoint. To test this hypothesis, we performed the same experiments in the S-phase checkpoint–compromised mutant grp
(). 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 consistent premature cytoplasmic and nuclear entry into mitosis at the site of injection ().
These results support our previous conclusion that cytoplasmic 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 analyzed the subcellular localization of CycB in living embryos by injecting rhodamine-labeled CycB (CycB-R).
We injected a low concentration of 13 μM CycB-R at the onset of interphase of cycles 12 and 13 in embryos expressing RLC-GFP (), the kinase Polo fused with GFP (GFP-Polo; ; Moutinho-Santos et al., 1999
), or the centromere-associated protein Cid fused with GFP (GFP-Cid; ; Schuh et al., 2007
). At this concentration, CycB-R did not induce premature entry into mitosis in five 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 accumulates in discrete puncta within the nucleus, indicating that the recombinant CycB used in these experiments is efficiently imported into the nucleus (, arrows). After NEB, CycB-R is primarily associated with the bipolar spindle, consistent with observations in other cell types (Pines and Hunter, 1991
; Yang et al., 1998
). In addition, a strong signal was also detected on the metaphase plate (). CycB-R colocalized with kinetochore GFP-Polo before NEB and throughout mitosis (, arrows). The CycB-R puncta in the nucleus before NEB colocalized with GFP-Cid (, 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 before NEB. The specific localization of CycB-R into kinetochores before NEB provides a means to monitor CycB nuclear import.
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 μM CycB-R (red) at interphase of cycle 12 (A) or 13 (B and C). Arrows highlight the punctate nuclear localization (more ...)
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, we injected CycB-R after CHX + Aph treatment into wild-type and grp
mutant embryos expressing RLC-GFP (). We assayed three different CycB-R concentrations (100, 56, and 13 μM). 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 (, schematic). In wild-type embryos injected with 100 μM CycB-R, the cytoplasm entered mitosis, as indicated by the disappearance of the RLC-GFP signal from the cortex (, blue outlines; 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 localization in the nucleus (; 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 (, blue outlines; and Video 7) and was immediately followed by NEB (, orange outlines; and Video 7).
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 (more ...)
Injection of 56 or 13 μM 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 (). The first 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 (, red arrow). Once in the nucleus, the CycB-R signal at the kinetochore increased progressively. This observation rules out the possibility that the S-phase checkpoint is preventing CycB nuclear accumulation by increasing the rate of nuclear CycB degradation (, compare the second row with the third row). In contrast, injection of 56 or 13 μM CycB-R in the grp mutant triggered cytoplasmic and nuclear entry into mitosis (, blue and orange outlines, respectively). The first sign of CycB-R nuclear accumulation before NEB was detected at 2.2 min and 3.7 min after injection of 56 μM and 13 μM CycB-R, respectively (, red arrow).
These data indicate that the Grp S-phase checkpoint prevents 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. Support for this idea comes from experiments in Xenopus laevis
, demonstrating that Chk1 phosphorylates and activates Wee1, a CDK1 inhibitor (Lee et al., 2001
). Moreover, in mammalian cultured cells, Wee1 can protect the nucleus from active cytoplasmic 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 μM CycB-R provoked a rapid cytoplasmic and nuclear CDK1 activation (, 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 ().
Collectively, these data reveal that high nuclear CDK1 activity correlates with rapid CycB nuclear import, and low nuclear CDK1 activity correlates with delayed CycB nuclear import (). The S-phase checkpoint, acting through Grp and Wee1, inhibits nuclear CDK1 activity and prevents CycB nuclear accumulation.
Figure 7. Grp prevents CycB nuclear accumulation by a Wee1-independent mechanism. (A–C) Wild-type (A), grp (B), and wee1 (C) mutant embryos expressing RLC-GFP (cyan) were injected with a mix of Aph and CHX (CHX + Aph) at metaphase of cycle 12. 5–8 (more ...)
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 influences 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 distinct, small molecule CDK1 inhibitors RO-3306 (Vassilev et al., 2006
) and Roscovitine (Meijer et al., 1997
). We performed the same experiments as described in , monitoring CycB nuclear import in wild-type, grp
, and wee1
embryos but with CDK1 maintained inactive through injection of CDK1 inhibitors (). In grp
mutant embryos treated with CHX + Aph, CycB-R triggered rapid myosin dispersion from the cortex and NEB (). In contrast, the addition of Roscovitine or RO-3306 before CycB-R injection prevented both myosin dispersion and NEB (). This demonstrates that these compounds efficiently 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 first signs of CycB-R in the nuclei were detected at a mean of 12.9 min (n
= 7; ). 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 five injected embryos, CycB-R started accumulating in the nucleus at a mean of 12.2 min (). In grp
mutant embryos injected with CHX + 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 first 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 (; and Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200801153/DC1
). In contrast, in the wee1
mutant, no nuclear CycB-R was detected in any Roscovitine- or RO-3306–injected embryos for up to 15 min (; and Video 10). These data reveal that in addition to its role in maintaining nuclear CDK1 in an inactive state, Grp prevents nuclear accumulation of CycB through a separate mechanism not shared by Wee1 ().