Here we began by addressing the question of which cyclins are required for an early mitotic event, chromatin condensation. Using diced siRNA pools and fluorescence video microscopy, we found that knocking down cyclin A2 delays chromatin condensation (), indicating that cyclin A2 directly or indirectly regulates the process and corroborating previous microinjection studies with cyclin A2 antibodies and p21-derived cyclin A2 inhibitors (Pagano et al., 1992
; Furuno et al., 1999
). The delay in chromatin condensation was also seen with a second d-siRNA pool, derived from the cyclin A2 3′-UTR, and was partially rescued by expressing an RNAi-resistant cyclin A2 construct (), arguing that the delay is due to cyclin A2 knockdown rather than an off-target effect. Cyclin A2 knockdown also delayed the onset of histone H3 phosphorylation, an event that correlates with chromatin condensation.
In principle the delay in chromatin condensation seen in cyclin A2 knockdown cells could be due to a delay in DNA replication and a consequent activation of the DNA replication/damage checkpoint response. However, we found no evidence for a delay in DNA replication or for prolonged activation of the DNA replication checkpoint in cyclin A2 knockdown cells (). The simplest interpretation of these results is that cyclin A2 is a mitotic regulator, as previously proposed (Swenson et al., 1986
; Pagano et al., 1992
; Guadagno and Newport, 1996
; Furuno et al., 1999
; Fung et al., 2007
; Gong et al., 2007
; Deibler and Kirschner, 2010
). The alternative possibility is that even though cyclin A2 is dispensable for the replication of most of the DNA, a small proportion of the DNA remains unreplicated and brings about enough activation of the DNA replication checkpoint to block mitotic entry but not enough checkpoint activation to detect by phospho-Chk1 immunoblotting.
We then assessed whether cyclin A2's role in chromatin condensation might be mediated by cyclin B1-Cdk1, whose nuclear accumulation and activation is regulated by cyclin A2 (Fung et al., 2007
; Gong et al., 2007
; De Boer et al., 2008
). Knocking down cyclin B1 alone caused a small delay in chromatin condensation, and it caused a marked delay in a cyclin A2-knockdown background (), consistent with the idea that both cyclin A2 and cyclin B1 can support chromatin condensation. By examining individual cells we found that chromatin condensation commences when cyclin B1 begins to accumulate in the nucleus (), putting cyclin B1 in the proper location to bring about chromatin condensation and demonstrating that the cyclin B1 nuclear translocation and chromatin condensation are tightly linked. This finding agrees with recent results from Gavet and Pines showing that cyclin B1-Cdk1 activation, as monitored with a FRET reporter, is tightly associated with cyclin B1-Cdk1 nuclear translocation and chromatin condensation (Gavet and Pines, 2010a
). Expression of a constitutively-nuclear form of cyclin B1 rescues chromatin condensation in cyclin A2 knockdown cells (), indicating that nuclear cyclin B1-Cdk1 complexes can support chromatin condensation in the absence or near-absence of cyclin A2.
There are at least three plausible models consistent with these observations. The first is that cyclin B1-Cdk1 is the normal initiator of chromatin condensation, with cyclin A2 being required because it is necessary for the activation and nuclear accumulation of cyclin B1-Cdk1 complexes. That is, cyclin A2-Cdk is exclusively a primer kinase, and cyclin B1-Cdk1 is an effector kinase. This model is compatible with recent live cell imaging studies of Cdk1 activation that emphasize the tight temporal correlation between cyclin B1-Cdk1 activation and chromatin condensation (Gavet and Pines, 2010a
) and with the evidence shown here of the tight correlation between cyclin B1-Cdk1 translocation and chromatin condensation. It also fits well with the biochemical identification of cyclin B1-Cdk1 as M-phase promoting factor in frog eggs and marine invertebrate eggs (Dunphy et al., 1988
; Lohka et al., 1988
; Labbe et al., 1989
). The main evidence against this model is the relatively modest delay in chromatin condensation seen in cyclin B1 knockdown cells. However, there are several possible ways to rationalize this modest delay: the small residual levels of cyclin B1 present in knockdown cells could be sufficient to mediate chromatin condensation, or the cyclin A2-Cdk complexes could provide a back-up function.
The second model is that cyclin A2 normally triggers both cyclin B1-Cdk1 activation/translocation and chromatin condensation, but the former is not required for the latter. In this model, cyclin A2 is both a priming kinase and an effector kinase for early mitotic events, whereas cyclin B1 is an effector kinase only for late mitotic events. Consistent with this model, we did see some cells where chromatin condensation began before any detectable cyclin B1 translocation (), but there was too much uncertainty in the experimental data to rule out the possibility that some small amount of cyclin B1 was always present in the nucleus at the time of chromatin condensation.
The third model, which we favor (), is a hybrid of the first two. Cyclin A2 triggers cyclin B1-Cdk1 activation/translocation, and the nuclear cyclin B1-Cdk1 and cyclin A2 complexes cooperate to bring about early mitotic events like chromatin condensation and NEB. This model accounts for all of our experimental observations, including the relatively mild delays in chromatin condensation (this work) and NEB (Gong et al., 2007
) seen in cyclin B1-knockdown cells, and the near-abolition of both events in cyclin A2/B1-double knockdown cells. This model is also supported by recent studies of HeLa cell extracts (Deibler and Kirschner, 2010
). These extracts carry out Cdk activation and NEB in vitro (Deibler and Kirschner, 2010
) and are more amenable to biochemical dissection than intact cells. Deibler and Kirschner have found that in HeLa cell extracts, cyclin A2 synergizes with fully-active cyclin B1-Cdk1 to promote NEB (Deibler and Kirschner, 2010
), in strong support of the hybrid model.
Figure 9. Schematic view of the hypothesized roles of cyclin A2 and cyclin B1 in the regulation of early and late mitotic events. Based on recent studies of HeLa cell extracts (Deibler and Kirschner, 2010 ), we assume that Cdk1 is the relevant cyclin A2-associated (more ...)
Notable motif-level features of this hybrid model () include feed-forward regulation (cyclin A2 promotes chromatin condensation and NEB both directly and through the intermediacy of cyclin B1-Cdk1), interlinked feedback loops, and reciprocal regulation (Cdk1 activates Cdc25 and inactivates Wee1; DNA damage activates Wee1 and inactivates Cdc25). Modeling studies and quantitative experimental studies in Xenopus
egg extracts argue that these features help generate a robust, bistable mitotic trigger (Novak and Tyson, 1993
; Pomerening et al., 2003
; Sha et al., 2003
; Ferrell, 2008
). In many ways, the triggering of mitosis in HeLa cells is more complicated than it is in Xenopus
extract. For example, the timing of mitosis is determined by the rate of cyclin B synthesis in Xenopus
extracts and embryo (Hartley et al., 1996
), but not in somatic cells (Jin et al., 1996
; Jin et al., 1998
). In addition, Cdk1 appears to increase in activity more gradually in HeLa cell extracts than it does in Xenopus
extracts (Deibler and Kirschner, 2010
). Finally, there are checkpoints present in somatic cells that are inoperative in Xenopus
extracts and embryos (Dasso and Newport, 1990
; Minshull et al., 1994
). The systems-level motifs shown in may contribute to the robustness of the more complicated somatic cell mitotic trigger, or, alternatively, they may be vestiges of the simpler embryonic cell cycle oscillator circuit.
We also addressed the question of whether cyclins B1 and B2 might be more critical for later steps in mitosis. Knocking down cyclin B1 and B2 has been reported to cause qualitative defects in mitotic progression (Gong et al., 2007
). However, mitosis is normally fairly brief in HeLa cells, and there is only a short time between when cyclin A2 is degraded (in prometaphase) and when cyclins B1 and B2 are degraded (in metaphase). This may explain why cyclin A2 can cover for cyclins B1 and B2 fairly well in an unperturbed mitosis. Mitosis can be lengthened or arrested by adding microtubule-disrupting agents like nocodazole. It seemed plausible that a requirement for B1/B2 function might be more apparent in the spindle assembly checkpoint arrest than it is in an unperturbed mitosis. Indeed, this was the case: nocodazole-treated cyclin B1 knockdown cells were defective in maintaining the mitotic state, and cyclin B1/B2 double knockdown cells were more severely defective ( and ). This result was not unexpected; nevertheless, given how unexpectedly subtle the early mitotic phenotypes were in cyclin B1/B2 knockdown cells, it was nice to obtain a result that conformed to expectations.
The cyclin B1 knockdown phenotypes seen here and in our previous work (Gong et al., 2007
) are weaker than those reported by Fung and coworkers (Fung et al., 2007
). There are several possible explanations for this discrepancy. It is possible that Fung et al.
achieved a more complete cyclin B1 knockdown than we did, although from the cyclin B1 blots shown in the three papers this did not appear to be the case. It is possible that the shRNA constructs used by Fung and coworkers produced off-target effects or caused a general saturation of normal miRNA processing and export pathways (Grimm et al., 2006
), and that this contributed to the stronger phenotype they reported. It is also possible that cyclin A2 can cover the functions of cyclin B1 and B2 to differing extents in different cell lines (even in different HeLa cell lines). Whatever the explanation, our work shows that cyclin A2 is the single most vulnerable cyclin target for the disruption of M-phase, and that knocking down cyclins A2 and B1 together synergistically disrupts M-phase. These findings could be of interest in developing antimitotic therapeutic agents.
The present studies point toward the importance of cyclin A2 in early mitotic events and underscore the many unanswered questions about the regulation of cyclin A2 complexes. When, exactly, are the cyclin A2 complexes activated? And what brings about their activation? Is their activation triggered simply by the accumulation of cyclin A2 to a threshold level (probably not, because cyclin A2 overexpression does not accelerate mitotic entry ), or the release of the CDK regulators Wee1 and Cdc25 from control by the DNA replication checkpoint, or something else? Answers to these questions will be important for understanding how the initiation of mitosis is tied into the earlier events of the cell cycle.