Role for cyclin A in the dependence of mitosis on completion of DNA replication.

THE cyclins were first identified by their cell-cycle-dependent synthesis and destruction and have a key role in the control of mitosis in Xenopus embryonic cell cycles. All higher eukaryotes have at least two types of cyclins, the A- and B-type, which can be distinguished by sequence motifs and the timing of their destruction in the cell cycle. The degradation of both cyclins is required for exit from mitosis, but the activation and destruction of cyclin A occur earlier in the cell cycle than with the B-type cyclins. This suggests that cyclin A has a distinct role in cell-cycle progression. We have used an antisense oligodeoxy-nucleotide directed against cyclin A to investigate this role. Ablation of cyclin A messenger RNA in cytostatic factor/metaphase-arrested extracts of Xenopus eggs, followed by in vitro progression into interphase, resulted in the premature appearance of cyclin B-cdc2-associated H1 kinase activity and premature entry into mitosis. Although cyclin A-ablated extracts could initiate DNA synthesis during interphase, S phase was not completed before entry into mitosis. The effects of cyclin A ablation were reversed by the addition of cyclin A mRNA or cyclin A protein to the extracts.     

Cyclin is degraded by the ubiquitin pathway.

Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.     

Cyclin synthesis drives the early embryonic cell cycle

We have produced extracts of frog eggs that can perform multiple cell cycles in vitro. Destruction of the endogenous messenger RNA arrests the extracts in interphase. The addition of exogenous cyclin mRNA is sufficient to produce multiple cell cycles. The newly synthesized cyclin protein accumulates during each interphase and is degraded at the end of each mitosis.     

Triggering of cyclin degradation in interphase extracts of amphibian eggs by cdc2 kinase

The cell cycles of early Xenopus embryos consist of a rapid succession of alternating S and M phases. These cycles are controlled by the activity of a protein kinase complex (cdc2 kinase) which contains two subunits. One subunit is encoded by the frog homologue of the fission yeast cdc2+ gene, p34cdc2 and the other is a cyclin. The concentration of cyclins follows a sawtooth oscillation because they accumulate in interphase and are destroyed abruptly during mitosis. The association of cyclin and p34cdc2 is not sufficient for activation of cdc2 kinase, however; dephosphorylation of key tyrosine and threonine residues of p34cdc2 is necessary to turn on its kinase activity. The activity of cdc2 kinase is thus regulated by a combination of translational and post-translational mechanisms. The loss of cdc2 kinase activity at the end of mitosis depends on the destruction of the cyclin subunits. It has been suggested that this destruction is induced by cdc2 kinase itself, thereby providing a negative feedback loop to terminate mitosis. Here we report direct experimental evidence for this idea by showing that cyclin proteolysis can be triggered by adding cdc2 kinase to a cell-free extract of interphase Xenopus eggs.     

APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)). Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7). We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref. 4). Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.     

Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

Impact of nuclear actin on the prophase chromosome construction of Physarum polycephalum

A cell-free system efficiently promoting mitosis has been developed using the precise natural synchronous plasmodium of Physarum polycephalum. The content changes of nuclear cyclin B were exploited to represent the prophase process of Physarum polycephalum. The possible function of nuclear actin on chromosome construction was investigated by detecting the content changes of nuclear cyclin B in the late G2 phase nuclei treated with cytochalasin B and incubated in the cell-free system. Our results showed that nuclear actin plays an important role in the process of the chromosome construction.     

The reversibility of mitotic exit in vertebrate cells

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.     

Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry

Oscillations in cyclin-dependent kinase (CDK) activity drive the somatic cell cycle. After entry into mitosis, CDKs activate the anaphase-promoting complex (APC), which then promotes cyclin degradation and mitotic exit. The re-accumulation of cyclin A causes the inactivation of APC and entry into S phase, but how cyclin A can accumulate in the presence of active APC has remained unclear. Here we show that, during G1, APC autonomously switches to a state permissive for cyclin A accumulation. Crucial to this transition is the APC(Cdh1)-dependent autoubiquitination and proteasomal degradation of the ubiquitin-conjugating enzyme (E2) UbcH10. Because APC substrates inhibit the autoubiquitination of UbcH10, but not its E2 function, APC activity is maintained as long as G1 substrates are present. Thus, through UbcH10 degradation and cyclin A stabilization, APC autonomously downregulates its activity. This indicates that the core of the metazoan cell cycle could be described as a self-perpetuating but highly regulated oscillator composed of alternating CDK and APC activities.     

Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae.

Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis.     

Mechanism limiting centrosome duplication to once per cell cycle

The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the 'once-only' control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability.     

Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.

In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed. In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator. Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae. We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression. As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells. Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics. We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.     

The Rae1-Nup98 complex prevents aneuploidy by inhibiting securin degradation

Cdc20 and Cdh1 are the activating subunits of the anaphase-promoting complex (APC), an E3 ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation, APC activity directed against these mitotic regulators must be inhibited until all chromosomes are properly attached to the mitotic spindle. Here we show that in mitosis timely destruction of securin by APC is regulated by the nucleocytoplasmic transport factors Rae1 and Nup98. We show that combined Rae1 and Nup98 haploinsufficiency in mice results in premature separation of sister chromatids, severe aneuploidy and untimely degradation of securin. We find that Rae1 and Nup98 form a complex with Cdh1-activated APC (APC(Cdh1)) in early mitosis and specifically inhibit APC(Cdh1)-mediated ubiquitination of securin. Dissociation of Rae1 and Nup98 from APC(Cdh1) coincides with the release of the mitotic checkpoint protein BubR1 from Cdc20-activated APC (APC(Cdc20)) at the metaphase to anaphase transition. Together, our results suggest that Rae1 and Nup98 are temporal regulators of APC(Cdh1) that maintain euploidy by preventing unscheduled degradation of securin.     

cdc2 links the Drosophila cell cycle and asymmetric division machineries

Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb and/or Prospero during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Here we show that neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. Our findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence.